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K ATP channels as molecular sensors of cellular metabolism
Abstract
In responding to cytoplasmic nucleotide levels, ATP-sensitive potassium (K(ATP)) channel activity provides a unique link between cellular energetics and electrical excitability. Over the past ten years, a steady drumbeat of crystallographic and electrophysiological studies has led to detailed structural and kinetic models that define the molecular basis of channel activity. In parallel, the uncovering of disease-causing mutations of K(ATP) has led to an explanation of the molecular basis of disease and, in turn, to a better understanding of the structural basis of channel function.
... ATP-sensitive potassium channels (K ATP ) couple increased glucose level with insulin release (Ashcroft & Rorsman, 1989;Cook & Hales, 1984;Nichols, 2006;Rorsman & Trube, 1985). Many observations have supported this view: K ATP is the dominant resting conductance of the beta cell (Ashcroft & Rorsman, 1989; 0 Jeremías Corradi is an Assistant Professor of Pharmacology at the National University of the South, in Argentina, where he carried out his PhD and postdoctoral studies with Cecilia Bouzat. ...
... His work focuses on islet ion channels, cell fuel metabolism, calcium signalling and diabetes. Nichols, 2006;Satin et al., 2015), mutations in the channel are strongly linked to metabolic diseases (Ashcroft et al., 2017;Pipatpolkai et al., 2020) and drugs targeting K ATP are oral anti-diabetic agents (Ashcroft et al., 2017). K ATP is the main link connecting glucose metabolism to the membrane depolarization that activates voltage-gated calcium channels. ...
... K ATP is the main link connecting glucose metabolism to the membrane depolarization that activates voltage-gated calcium channels. This increases the intracellular calcium concentration, which in turn evokes the release of insulin (Nichols, 2006). K ATP is spontaneously active, and its activity is directly blocked by ATP and increased by ADP, as shown by recordings of channel activity in inside-out membrane patches (Bokvist et al., 1991;Cook & Hales, 1984;Rorsman & Trube, 1985). ...
Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP‐sensitive potassium channel (KATP) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell KATP locally produces the ATP that inhibits KATP activity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside‐out patches inhibited KATP. To test the relative contributions of local vs. mitochondrial ATP production, we recorded KATP activity using mouse beta cells and INS‐1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of KATP activity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well‐known action of protons to inhibit KATP. In cell‐attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on KATP channel closure by glucose, further suggesting that PK is not an important regulator of KATP. In contrast, addition of mitochondrial inhibitors robustly increased KATP activity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of KATP in pancreatic beta cells. image
Key points
Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit KATP activity in excised patches. PEP solutions only inhibit KATP activity if the pH is unbalanced.
Modulating pyruvate kinase has minimal effects on KATP activity.
Mitochondrial inhibition, in contrast, robustly potentiates KATP activity in cell‐attached patches.
Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close KATP.
Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the KATP activity of pancreatic beta cells.
... Structural basis of PIP 2 and ATP antagonism PIP 2 and ATP functionally compete to open and close K ATP channels, respectively [36,61]. Comparison of the PIP 2 -bound open structure and the ATP-bound closed structure reveals that PIP 2 binding disrupts ATP binding at two levels, first by directly competing for shared binding residues and second by causing a conformational change that disfavors ATP binding [21] (Figure 3c). ...
... SUR has two, but although both bind Mgnucleotides, only NBD2 is capable of hydrolyzing MgATP. It is hypothesized that Mg-nucleotide binding at the two NBDs of SUR causes dimerization of the NBDs in a head-to-tail fashion [2,61,65,66], as it does in other ABC proteins [30], and that NBD dimerization regulates the Kir6 subunit to decrease ATP inhibition and open the channel. Whether MgATP hydrolysis is involved in K ATP channel stimulation has been investigated by several approaches. ...
KATP channels are ligand-gated potassium channels that couple cellular energetics with membrane potential to regulate cell activity. Each channel is an eight subunit complex comprising four central pore-forming Kir6 inward rectifier potassium channel subunits surrounded by four regulatory subunits known as the sulfonylurea receptor, SUR, which confer homeostatic metabolic control of KATP gating. SUR is an ATP binding cassette (ABC) protein family homolog that lacks membrane transport activity but is essential for KATP expression and function. For more than four decades, understanding the structure-function relationship of Kir6 and SUR has remained a central objective of clinical significance. Here, we review progress in correlating the wealth of functional data in the literature with recent KATP cryoEM structures.
... ATP-sensitive potassium channels (K ATP ) are regulated by intracellular nucleotide concentrations and thus serve to couple metabolic state to membrane excitability in diverse cell types (Davis et al., 2022;McClenaghan and Nichols, 2022;Nichols, 2006). K ATP channels are hetero-octameric complexes composed of four pore-forming inward rectifier potassium (Kir) channel subunits, Kir6.1 or Kir6.2, and four regulatory sulfonylurea receptor (SUR) subunits, SUR1, SUR2A, or SUR2B (Aguilar-Bryan and Bryan, 1999;Inagaki et al., 1997;Martin et al., 2017;Shyng and Nichols, 1997 Different combinations of Kir and SUR subunits give rise to functionally and pharmacologically distinct K ATP channel subtypes that are expressed in a cell typespecific manner, creating opportunities for developing selective therapeutics targeting different organ systems (Gribble et al., 1997;Gribble et al., 1998;Inagaki et al., 1996;Inagaki et al., 1995c;Nichols, 2023). ...
... In the fasting state, Kir6.2/SUR1 channels are kept open by the low concentration of intracellular ATP relative to ADP. However, the influx of glucose following a meal and ensuing stimulation of ATP production leads to inhibition of Kir6.2/SUR1, membrane potential depolarization, opening of voltage-gated calcium channel and calcium influx, exocytotic release of insulin into the circulation, and lowering of blood glucose levels back toward pre-prandial levels (Nichols, 2006). Sulfonylurea drugs such as glibenclamide that inhibit Kir6.2/SUR1 have been used clinically for decades to treat TDM due to their ability to stimulate insulin secretion and lower blood glucose (Kharade et al., 2016;Nichols, 2023). ...
Vascular smooth muscle KATP channels critically regulate blood flow and blood pressure by modulating vascular tone and therefore represent attractive drug targets for treating several cardiovascular disorders. However, the lack of potent inhibitors that can selectively inhibit Kir6.1/SUR2B (vascular KATP) over Kir6.2/SUR1 (pancreatic KATP) has eluded discovery despite decades of intensive research. We therefore screened 47,872 chemically diverse compounds for novel inhibitors of heterologously expressed Kir6.1/SUR2B channels. The most potent inhibitor identified in the screen was an N-aryl-N'-benzyl urea compound termed VU0542270. VU0542270 inhibits Kir6.1/SUR2B with an IC50 of approximately 100 nM but has no apparent activity toward Kir6.2/SUR1 or several other members of the Kir channel family at doses up to 30 µM (>300-fold selectivity). By expressing different combinations of Kir6.1 or Kir6.2 with SUR1, SUR2A, or SUR2B, the VU0542270 binding site was localized to SUR2. Initial structure-activity relationship exploration around VU0542270 revealed basic texture related to structural elements that are required for Kir6.1/SUR2B inhibition. Analysis of the pharmacokinetic properties of VU0542270 showed that it has a short in vivo half-life due to extensive metabolism. In pressure myography experiments on isolated mouse ductus arteriosus vessels, VU0542270 induced ductus arteriosus constriction in a dose-dependent manner similar to that of the nonspecific KATP channel inhibitor glibenclamide. The discovery of VU0542270 provides conceptual proof that SUR2-specific KATP channel inhibitors can be developed using a molecular target-based approach and offers hope for developing cardiovascular therapeutics targeting Kir6.1/SUR2B. SIGNIFICANCE STATEMENT: Small-molecule inhibitors of vascular smooth muscle KATP channels might represent novel therapeutics for patent ductus arteriosus, migraine headache, and sepsis; however, the lack of selective channel inhibitors has slowed progress in these therapeutic areas. Here, this study describes the discovery and characterization of the first vascular-specific KATP channel inhibitor, VU0542270.
... K ATP channels are nucleotide-regulated potassium channels, comprising poreforming K ir 6 subunits co-assembled with regulatory SUR subunits, that couple cellular metabolism and diverse cellular signalling pathways to the membrane potential. 2 The two mammalian K ir 6 isoforms, K ir 6.1 (KCNJ8) and K ir 6.2 (KCNJ11) and two SUR isoforms, SUR1 (ABCC8) and SUR2 (ABCC9), show distinct properties and tissue expression patterns. SUR proteins are members of the ABC-transporter family and share core structural features of two transmembrane domains (TMD1 and TMD2) and two nucleotide binding domains (NBD1 and NBD2) with family members such as CFTR (ABCC7) and the multidrug resistance protein (MRP-1; ABCC1). ...
... SURs have no recognized transporter function, but instead, regulate K ATP channel complexes, conferring Mg-nucleotide activation and pharmacological sensitivity and modulating ATP inhibition. 2 Functional K ATP channel expression at the plasma membrane requires co-assembly of four K ir 6 subunits with four SUR subunits. [3][4][5][6][7][8][9] Extensive study has shown that truncation of SUR proteins impairs or abolishes surface expression of K ATP channels 10,11 and truncations of SUR1 are associated with congenital hyperinsulinism due to loss of pancreatic K ATP function. ...
Loss-of-function mutation of ABCC9, the gene encoding the SUR2 subunit of ATP sensitive-potassium (KATP) channels, was recently associated with autosomal recessive ABCC9-related intellectual disability and myopathy syndrome (AIMS).
Here we identify nine additional subjects, from seven unrelated families, harboring different homozygous LoF variants in ABCC9 and presenting with a conserved range of clinical features. All variants are predicted to result in severe truncations or in-frame deletions within SUR2, leading to the generation of non-functional SUR2-dependent KATP channels.
Affected individuals show psychomotor delay and intellectual disability of variable severity, microcephaly, corpus callosum and white matter abnormalities, seizures, spasticity, short stature, muscle fatigability, and weakness. Heterozygous parents do not show any conserved clinical pathology but report multiple incidences of intrauterine fetal death, which were also observed in an eighth family included in this study. In vivo studies of abcc9 LoF in zebrafish revealed an exacerbated motor response to pentylenetetrazole, a pro-convulsive drug, consistent with impaired neurodevelopment associated with an increased seizure susceptibility.
Our findings define an ABCC9 LoF related phenotype, expanding the genotypic and phenotypic spectrum of AIMS and reveal novel human pathologies arising from KATP channel dysfunction.
... Thus, KATP channels are open during states of low metabolic activity (e.g. low levels of ATP/high levels of ADP) resulting in plasma membrane hyperpolarization [87]. The ability to couple cellular metabolic state (ATP/ADP ratio) to the electrical activity of the cell membrane is critical in numerous physiological processes and is a key feature of KATP channels [88]. ...
... BKCa channel openers such as andolast and cilostazol have been investigated for the treatment of bronchial asthma and intermittent claudication, respectively and both were associated with frequent adverse events, including headache [87]. Specifically, cilostazol is known to induce headaches in healthy volunteers as well as migraine-like attacks in patients with migraine [100,101]. ...
Migraine is a debilitating neurological condition affecting millions of people worldwide. Until few years ago, preventive migraine treatments were based on molecules with pleiotropic targets, developed for other indications and discovered by serendipity to be effective in migraine prevention although often burdened by tolerability issues leading to low adherence. However, the progresses in unravelling the migraine pathophysiology allowed to identify novel putative targets as calcitonin gene-related peptide (CGRP). Nevertheless, despite the revolution brought by CGRP monoclonal antibodies and gepants, a significant percentage of patients still remains burdened by an unsatisfactory response suggesting that other pathways may play a critical role with an extent of involvement varying among different migraine patients. Specifically, neuropeptides of the CGRP family as adrenomedullin and amylin, molecules of the secretin family as pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP), receptors as transient receptor potential (TRP) channels, intracellular downstream determinants as potassium channels but also the opioid system and the purinergic pathway have been suggested to be involved in migraine pathophysiology. The present review provides an overview of these pathways highlighting, based on preclinical and clinical evidences as well as provocative studies, their potential role as future targets for migraine preventive treatment.
... ATP-sensitive potassium (K ATP ) channels, activated by intracellular ADP and inhibited by intracellular ATP, are present in many tissues, coupling the metabolic state of the cell to electrical activity. 1 The channels are formed as octameric complexes of pore-forming (Kir6) and modulatory sulphonylurea receptor (SUR) subunits, encoded by two Kir6 genes, KCNJ8 (Kir6.1) and KCNJ11 (Kir6.2), ...
... Given the prior findings with transgenic K ATP GOF animals, and the prominence of Kir6.1 and SUR2 in lymphatic smooth muscle, we sought to understand the extent to which lymphatic contractile function might be compromised in mice with CSassociated mutations, with the potential to explain the susceptibility of CS patients to lymphedema, using both ex vivo and in vivo assays in heterozygous Kir6. 1 ...
Cantú Syndrome (CS) is an autosomal dominant disorder caused by gain-of-function (GoF) mutations in the Kir6.1 and SUR2 subunits of KATP channels. KATP overactivity results in a chronic reduction in arterial tone and hypotension, leading to other systemic cardiovascular complications. However, the underlying mechanism of lymphedema, developed by >50% of CS patients, is unknown. We investigated whether lymphatic contractile dysfunction occurs in mice expressing CS mutations in Kir6.1 (Kir6.1[V65M]) or SUR2 (SUR2[A478V], SUR2[R1154Q]). Pressure myograph tests of contractile function of popliteal lymphatic vessels over the physiological pressure range revealed significantly impaired contractile strength and reduced frequency of spontaneous contractions at all pressures in heterozygous Kir6.1[V65M] vessels, compared to control littermates. Contractile dysfunction of intact popliteal lymphatics in vivo was confirmed using near-infrared fluorescence microscopy. Homozygous SUR2[A478V] vessels exhibited profound contractile dysfunction ex vivo, but heterozygous SUR2[A478V] vessels showed essentially normal contractile function. However, further investigation of vessels from all three GoF mouse strains revealed significant disruption in contraction wave entrainment, decreased conduction speed and distance, multiple pacemaker sites, and reversing wave direction. Tests of 2-valve lymphatic vessels forced to pump against an adverse pressure gradient revealed that all CS-associated genotypes were essentially incapable of pumping under an imposed outflow load. Our results show that varying degrees of lymphatic contractile dysfunction occur in proportion to the degree of molecular GoF in Kir6.1 or SUR2. This is the first example of lymphatic contractile dysfunction caused by a smooth muscle ion channel mutation and potentially explains the susceptibility of CS patients to lymphedema.
... Another important subfamily of Kir channels, the ATP-sensitive potassium (K ATP ) channels, were first identified in cardiac muscle, but are predominantly expressed in the nervous system (Noma, 1983;Thomzig et al., 2005;Thomzig et al., 2001). Expression of different types of K ATP channels was found in various brain regions, including, but not limited to the , 2008;Drain et al., 1998;Nichols, 2006;Nichols et al., 1996;Trapp et al., 2003;Zingman et al., 2001), which has been clearly demonstrated by the recently solved three-dimensional structures of K ATP channels (Lee et al., 2017;Martin et al., 2017;Puljung, 2018;Wu et al., 2018). ...
... Consequently, K ATP acts as a cellular sensor and modulates membrane excitability based on the intracellular metabolic state: a high ATP/ADP ratio closes the K ATP channel pore, increasing the excitability, while a low ATP/ADP ratio reduces excitability by opening it(Craig et al., 2008; Gribble et al., 2000;Zingman et al., 2001).Furthermore, K ATP channel activity is also regulated by a variety of other metabolic signals, NO), all of which contribute to the essential properties of K ATP channels as metabolic sensors(Dragicevic et al., 2015; Fan and Makielski, 1997;Yang et al., 2020). Such a multifactorial regulatory mechanism is particularly important for neurons, where K ATP channels function as an energy control valve that integrates the ATP/ADP ratio with electroactivity to enable sophisticated neuronal functions(Lawson, 2000; Liss et al., 2005; Liss and Roeper, 2001;Nichols, 2006). ...
Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) region of the midbrain. The loss of neurons results in a subsequent reduction of dopamine in the striatum, which underlies the core motor symptoms of PD. To date, there are no effective treatments to stop, slow, or reverse the pathological progression of dopaminergic neurodegeneration. This unfortunate predicament is because of the current early stages in understanding the biological targets and pathways involved in PD pathogenesis. Ion channels have become emerging targets for new therapeutic development for PD due to their essential roles in neuronal function and neuroinflammation. Potassium channels are the most prominent ion channel family and have been shown to be critically important in PD pathology because of their roles in modulating neuronal excitability, neurotransmitter release, synaptic transmission, and neuroinflammation. In this review, members of the subfamilies of voltage-gated K+ channels, inward rectifying K+ channels, and Ca2+-activated potassium channels are described. Evidence of the role of these channels in PD aetiology is discussed together with the latest views on related pathological mechanisms and their potential as biological targets for developing neuroprotective drugs for PD. Significance Statement Parkinson's disease (PD) is the second most common neurodegenerative disorder, featuring progressive degeneration of dopaminergic neurons in the midbrain. It is a multifactorial disease involving multiple risk factors and complex pathobiological mechanisms. Mounting evidence suggests that ion channels play vital roles in the pathogenesis and progression of PD by regulating neuronal excitability and immune cell function. Therefore, they have become "hot" biological targets for PD, as demonstrated by multiple clinical trials of drug candidates targeting ion channels for PD therapy.
... tions, but open in response to severe ischaemia to protect the heart from damage. These integral membrane proteins are inhibited by ATP and activated by Mg 2+ -bound nucleotides and thus translate changes in metabolism to changes in membrane K + permeability and cellular excitability (reviewed by Nichols, 2006;Tinker et al., 2018). Functional K ATP channels form as octamers of four K ir 6 pore-forming K + channel subunits and four sulphonylurea receptor (SUR) subunits Martin et al., 2017). ...
Background and Purpose
The canonical Kir6.2/SUR2A ventricular KATP channel is highly ATP‐sensitive and remains closed under normal physiological conditions. These channels activate only when prolonged metabolic compromise causes significant ATP depletion and then shortens the action potential to reduce contractile activity. Pharmacological activation of KATP channels is cardioprotective, but physiologically, it is difficult to understand how these channels protect the heart if they only open under extreme metabolic stress. The presence of a second KATP channel population could help explain this. Here, we characterise the biophysical and pharmacological behaviours of a constitutively active Kir6.1‐containing KATP channel in ventricular cardiomyocytes.
Experimental Approach
Patch‐clamp recordings from rat ventricular myocytes in combination with well‐defined pharmacological modulators was used to characterise these newly identified K⁺ channels. Action potential recording, calcium (Fluo‐4) fluorescence measurements and video edge detection of contractile function were used to assess functional consequences of channel modulation.
Key Results
Our data show a ventricular K⁺ conductance whose biophysical characteristics and response to pharmacological modulation were consistent with Kir6.1‐containing channels. These Kir6.1‐containing channels lack the ATP‐sensitivity of the canonical channels and are constitutively active.
Conclusion and Implications
We conclude there are two functionally distinct populations of ventricular KATP channels: constitutively active Kir6.1‐containing channels that play an important role in fine‐tuning the action potential and Kir6.2/SUR2A channels that activate with prolonged ischaemia to impart late‐stage protection against catastrophic ATP depletion. Further research is required to determine whether Kir6.1 is an overlooked target in Comprehensive in vitro Proarrhythmia Assay (CiPA) cardiac safety screens.
... glycolysis, the citrate cycle, and oxidative phosphorylation, starting from intracellular glucose 23 . The cell membrane features Na-K ATPase and ATP-sensitive K channels, with an increase in intracellular ATP levels, leading to elevated intracellular potassium concentrations 22,24 . Generally, stimulation of β2 adrenergic receptors by ritodrine involves the activation of Na-K ATPase pumps on the cell membrane, facilitating the intracellular transition of potassium 25 . ...
In the management of pregnancy, ritodrine has been used to prevent preterm birth, and magnesium sulfate (MgSO 4 ) has been used to prevent preterm labor and preeclampsia. Neonates born to mothers receiving these medications occasionally show an increase in serum potassium concentration. Recently, an elevated risk of neonatal hyperkalemia has been reported, particularly when ritodrine and MgSO 4 are co-administered; however, the underlying mechanisms remain unclear. We investigated the association between potassium levels and metabolites in the serum of umbilical cord blood of infants with antenatal exposure to antenatal ritodrine and MgSO 4 using a metabolomic analysis. Our findings revealed a significant elevation in serum potassium concentration associated with metabolomic findings of activation of glycolysis and the derived metabolic routes in preterm neonates exposed to both ritodrine and MgSO 4 . Our data indicate that the concurrent administration of ritodrine and MgSO 4 caused distinctive metabolic alterations, potentially leading to an additional increase in the intracellular potassium concentration in the fetus. Consequently, this mechanism may imply an elevation in serum potassium concentration postnatally through the redistribution of potassium.
... This mutation is located in the NBD. Additionally, it is known that ADP and other diphosphate nucleosides inhibit KATP channels in the absence of Mg 2+ , so the presence of Mg 2+ is critical for its activation [30,55]. KATP channel regulation relies on changes in both ATP and Mg-ADP concentrations [56]. ...
Ionic channels are present in eucaryotic plasmatic and intracellular membranes. They coordinate and control several functions. One of the most diverse ionic channels family are the potassium (K) channels. Within this family are the ATP-dependent potassium channels (KATP), which belong to the potassium rectifier channel family. These channels were initially described in heart muscle, yet they are also present in different types of tissues such as pancreatic, skeletal muscle, brain, and vascular and non-vascular smooth muscle. In ß-pancreatic cells, KATP are primarily responsible for maintaining membrane potential and for the depolarization mediated insulin release. KATP density/activity seems to be determinant in physiological the outcome, in pancreatic cells decrease of this density/activity may be related to Insulin resistance (IR). In extra pancreatic tissues KATP relation with IR is beginning to be explored. It is possible that in skeletal muscle, the KATP are involved in insulin dependent glucose recapture and that KATP activation leads to IR. In adipose tissue, KATP containing Kir6.2, could be related to increase in free fatty acids and insulin resistance, therefore, pathological processes leading to a prolonged adipocyte KATP inhibition might promote obesity related to insulin resistance. In central nervous system KATP activation can regulate peripheric glycemia and lead to brain IR which could be a much earlier manifestation of peripheral alterations that lead to the development of pathologies such as obesity and type 2 diabetes mellitus. In this review we aim to discuss the KATP channel characteristics, its relationship to known clinical disorders and the mechanisms and potential relations with peripheral and central insulin resistance.
... This mutation is located in the NBD. Additionally, it is known that ADP and other diphosphate nucleosides inhibit KATP channels in the absence of Mg 2+ , so the presence of Mg 2+ is critical for its activation [30,55]. KATP channel regulation relies on changes in both ATP and Mg-ADP concentrations [56]. ...
Ionic channels are present in eucaryotic plasma and intracellular membranes. They coordinate and control several functions. Potassium channels belong to the most diverse family of ionic channels that includes ATP-dependent potassium (KATP) channels in the potassium rectifier channel subfamily. These channels were initially described in heart muscle and then in other tissues such as pancreatic, skeletal muscle, brain, and vascular and non-vascular smooth muscle tissues. In pancreatic beta cells, KATP channels are primarily responsible for maintaining the membrane potential and for depolarization-mediated insulin release, and their decreased density and activity may be related to insulin resistance. KATP channels' relationship with insulin resistance is beginning to be explored in extra-pancreatic beta tissues like the skeletal muscle, where KATP channels are involved in insulin-dependent glucose recapture and their activation may lead to insulin resistance. In adipose tissues, KATP channels containing Kir6.2 protein subunits could be related to the increase in free fatty acids and insulin resistance; therefore, pathological processes that promote prolonged adipocyte KATP channel inhibition might lead to obesity due to insulin resistance. In the central nervous system, KATP channel activation can regulate peripheric glycemia and lead to brain insulin resistance, an early peripheral alteration that can lead to the development of pathologies such as obesity and Type 2 Diabetes Mellitus (T2DM). In this review, we aim to discuss the characteristics of KATP channels, their relationship with clinical disorders, and their mechanisms and potential associations with peripheral and central insulin resistance.
... If dietary therapy fails, the use of sulfonylureas is an excellent example of personalized medicine. These drugs bind to the receptor SUR1 (encoded by ABCC8) of the ATP-dependent potassium channel, closing it and stimulating insulin secretion [49]. As an analysis of the Diabetes Prospective Follow-Up (DPV) registry has shown, an early diagnosis is of great importance, as patients respond better to early sulfonylurea treatment, resulting in improved metabolic outcomes throughout the disease course [50]. ...
Introduction
Childhood obesity is increasing worldwide and presents as a global health issue due to multiple metabolic comorbidities. About 1% of adolescents with obesity develop type 2 diabetes (T2D); however, little is known about the genetic and pathophysiological background at young age. The objective of this study was to assess the prevalence of impaired glucose regulation (IGR) in a large cohort of children and adolescents with obesity and to characterize insulin sensitivity and insulin secretion. We also wanted to investigate adolescents with insulin secretion disorder more closely and analyze possible candidate genes of diabetes in a subcohort.
Methods
We included children and adolescents with obesity who completed an oral glucose tolerance test (OGTT, glucose + insulin) in the outpatient clinic. We calculated Matsuda index, the area under the curve (AUC (Ins/Glu)), and an oral disposition index (ISSI-2) to estimate insulin resistance and beta-cell function. We identified patients with IGR and low insulin secretion (maximum insulin during OGTT < 200 mU/l) and tested a subgroup using next generation sequencing to identify possible mutations in 103 candidate genes.
Results
The total group consisted of 903 children and adolescents with obesity. 4.5% showed impaired fasting glucose, 9.4% impaired glucose tolerance, and 1.2% T2D. Matsuda index and Total AUC (Ins/Glu) showed a hyperbolic relationship. Out of 39 patients with low insulin secretion, we performed genetic testing on 12 patients. We found five monogenetic defects (ABCC8 (n = 3), GCK (n = 1), and GLI2/PTF1A (n = 1)).
Conclusion
Using surrogate parameters of beta-cell function and insulin resistance can help identify patients with insulin secretion disorder. A prevalence of 40% mutations of known diabetes genes in the subgroup with low insulin secretion suggests that at least 1.7% of patients with adolescent obesity have monogenic diabetes. A successful molecular genetic diagnosis can help to improve individual therapy.
... Pinpointing the exact location of KATP channel blockade is challenging, but potential sites include the spinal, brain, and peripheral levels (Ocana, 2004). The absence of KATP current potentially contributes to neuropathic pain through various mechanisms like increased membrane excitability, amplification of neurotransmitter release, and modulation of neuronal excitability (Yamada and Inagaki, 2005;Miki and Seino, 2005;Nichols, 2006;Soundarapandian et al., 2007). This therefore suggests that FME might have caused decreased membrane excitability, decreased in neurotransmitter release and decrease neuronal excitability to abolish pain sensations at peripheral levels. ...
Pyrexia occurs as a result of the resetting of the hypothalamic set-point. Non steroidal anti-inflammatory drugs used for the treatment of fever and related illnesses are associated with side effects including gastrointestinal irritation, bleeding, ulcers and perforation. In traditional medicine, many plants have been found to possess antipyretic activity. Ficus asperifolia (Miq), family Moraceae has been traditionally used to treat pain and fevers. The aim of the study was thus to evaluate the antipyretic and antinociceptive activity of the methanol leaf extract of Ficus asperifolia (FME) in murine models. Preliminary phytochemical screening and oral acute toxicity studies were conducted using standard protocols. Antipyretic activity was evaluated using the Brewer’s yeast induced pyrexia model. Antinociceptive effects were investigated using the acetic acid and thermal induced models. The mechanism of action of the plant was also evaluated against several antagonists. FME significantly (p<0.05) and dose dependently reduced yeast induced pyrexia. FME at doses 125, 250 and 500 mg/kg significantly (p<0.05) decreased acetic acid induced abdominal writhes and increased the mean reaction time of mice to thermal-induced pain stimulus. Pre-treatment of mice with naloxone (a non-selective opioid receptor antagonist), glibenclamide (a potassium channel blocker) and L-NNA (a nitric oxide synthase inhibitor) significantly reversed the analgesic action of the extract suggesting that the activity is likely mediated via the involvement of these pathways.
... The process of alternative splicing of ABCC9 gives rise to unique SUR2A and SUR2B subunits, which differ in their carboxyl terminal amino acid sequences. The spatial relationship of KCNJ8 with ABCC9 on chromosome 12 and of KCNJ11 with ABCC8 on chromosome 11 indicates a likely co-regulation and gene duplication phenomenon [18]. Each Kir6 unit encompasses two membrane-spanning domains surrounding a pore loop, crucial for K + selectivity. ...
ATP-sensitive potassium (KATP) channels are found in plasma membranes and mitochondria. These channels are a type of ion channel that is regulated by the intracellular concentration of adenosine triphosphate (ATP) and other nucleotides. In cell membranes, they play a crucial role in linking metabolic activity to electrical activity, especially in tissues like the heart and pancreas. In mitochondria, KATP channels are involved in protecting cells against ischemic damage and regulating mitochondrial function. This review delves into the role of KATP channels in cancer biology, underscoring their critical function. Notably responsive to changes in cellular metabolism, KATP channels link metabolic states to electrical activity, a feature that becomes particularly significant in cancer cells. These cells, characterized by uncontrolled growth, necessitate unique metabolic and signaling pathways, differing fundamentally from normal cells. Our review explores the intricate roles of KATP channels in influencing the metabolic and ionic balance within cancerous cells, detailing their structural and operational mechanisms. We highlight the channels’ impact on cancer cell survival, proliferation, and the potential of KATP channels as therapeutic targets in oncology. This includes the challenges in targeting these channels due to their widespread presence in various tissues and the need for personalized treatment strategies. By integrating molecular biology, physiology, and pharmacology perspectives, the review aims to enhance the understanding of cancer as a complex metabolic disease and to open new research and treatment avenues by focusing on KATP channels. This comprehensive approach provides valuable insights into the potential of KATP channels in developing innovative cancer treatments.
... When the concentration of the blood glucose is high, the ratio of intracellular ATP to ADP is increased thus, promoting inhibition of the KATP channel, depolarizing Vm, and thereby, increasing the voltage-gated Ca2+ channel (CaV) activity. An increased CaV-mediated Ca2+ influx raises the concentration of intracellular free Ca2+ ([Ca2+]in), thus promoting the secretion of insulin (Ashcroft et al., 2013;Ashcroft et al., 2012;Nichols 2006). Among multiple genes that are associated with the development of T2D, the Potassium Inwardly Rectifying Channel Subfamily J Member 11 (KCNJ11) garnered the most attention. ...
The most common endocrine disease in the world is type 2 diabetes mellitus (DM2). The pathogenesis of this disease is complex and not fully investigated. This review represents the modern sight on the correlation between genetic, epigenetic and environmental factors in the formation of T2D. Keywords: Type 2 Diabetes mellitus (T2DM), pathogenesis, SNP (single nucleotide polymorphism)
... The significantly increased expression of ABCC9 transcript in MN-R might facilitate membrane-availability of K ATP channels: proteins coded by ABCC9 and ABCC8 are regulatory subunits that form functional K ATP channels in association with Kir6 proteins, 76-78 modulate K ATP sensitivity to ATP, and are mandatory for membrane trafficking of the channel. [78][79][80] Single-cell sequencing also revealed a higher expression of RNA coding for the partially voltage-sensitive 81,82 sodium leak channel NALCN in MN-L. As NALCN determines neuron excitability and increases the excitability of neural networks, [83][84][85] an increased availability of voltage-dependent NALCN sodium flow that masks charges lost by K ATP and depolarization-triggered M-type potassium outflow could diminish the influence of KV7 2/3 and K ATP on shaping the MN-L sub-threshold inputoutput function. ...
... ABCC8 and ABCC9 are vital components of the ATP-sensitive potassium (K ATP ) channels in the heart [39][40][41]. These K ATP channels serve as unique cellular energy sensors, safeguarding cardiomyocytes, particularly during conditions such as ischemia [42]. Atherosclerosis; Alzheimer's disease [35,36] ABCA5 Macrophages in (coronary) arteries A macrophage cholesterol efflux. ...
Adenosine triphosphate binding cassette (ABC) transporters are a broad family of membrane protein complexes that use energy to transport molecules across cells and/or intracellular organelle lipid membranes. Many drugs used to treat cardiac diseases have an affinity for these transporters. Among others, P-glycoprotein (P-gp) plays an essential role in regulating drug concentrations that reach cardiac tissue and therefore contribute to cardiotoxicity. As a molecular imaging modality, positron emission tomography (PET) has emerged as a viable technique to investigate the function of P-gp in organs and tissues. Using PET imaging to evaluate cardiac P-gp function provides new insights for drug development and improves the precise use of medications. Nevertheless, information in this field is limited. In this review, we aim to examine the current applications of ABC transporter PET imaging and its tracers in the heart, with a specific emphasis on P-gp. Furthermore, the opportunities and challenges in this novel field will be discussed.
... It was initially reported that reduced glucose availability interferes with the membrane conductance of neurons, primarily via ATP-sensitive potassium (K ATP ) channels that act as metabolic sensors coupling neuronal excitability to ATP levels [63,71,72]. Indeed, a reduction of cytoplasmatic ATP concentration has a hyperpolarizing effect mediated by K ATP channel opening [73]. More recently, it was shown that the transcriptional repressor REST/NRSF is activated by the reduced intracellular concentrations of NADH induced by the inhibition of glycolysis [15]. ...
Dietary restriction, such as low glycemic index diet (LGID), have been successfully used to treat drug-resistant epilepsy. However, if such diet could also counteract antiepileptogenesis is still unclear. Here, we investigated whether the administration of LGID during the latent pre-epileptic period, prevents or delays the appearance of the overt epileptic phenotype. To this aim, we used the Synapsin II knockout (SynIIKO) mouse, a model of temporal lobe epilepsy in which seizures manifest 2–3 months after birth, offering a temporal window in which LGID may affect epileptogenesis. Pregnant SynIIKO mice were fed with either LGID or standard diet during gestation and lactation. Both diets were maintained in weaned mice up to 5 months of age. LGID delayed the seizure onset and induced a reduction of seizures severity only in female SynIIKO mice. In parallel with the epileptic phenotype, high-density multielectrode array recordings revealed a reduction of frequency, amplitude, duration, velocity of propagation and spread of interictal events by LGID in the hippocampus of SynIIKO females, but not mutant males, confirming the gender-specific effect. ELISA-based analysis revealed that LGID increased cortico-hippocampal allopregnanolone (ALLO) levels only in females, while it was unable to affect ALLO plasma concentrations in either sex. The results indicate that the gender-specific interference of LGID with the epileptogenic process can be ascribed to a gender-specific increase in cortical ALLO, a neurosteroid known to strengthen GABAergic transmission. The study highlights the possibility of developing a personalized gender-based therapy for temporal lobe epilepsy.
... 1,2 ATP-sensitive potassium (K ATP ) channels are a powerful defense system and the cell's central metabolic sensor in terms of its energy supply. 3,4 Such highly ATP-sensitive molecular structures play an exceptional role in the synchronization of metabolism and electrical activity of cells, regulation of potential-dependent membrane functions, and maintenance of hormonal homeostasis, while their dysfunction may promote pathogenesis. Conversely, activation of these channels has a protective effect, including cerebro-, neuro-, and cardioprotective, which consists in the suppression of metabolism and excitability, as well as the normalization of bioenergetic processes in the cell, while maintaining a high ATP content. ...
Background: The aging process is accompanied by the weakening of the protective systems of the organism, in particular by the decrease in the expression of ATP-sensitive potassium (KATP) channels and in the synthesis of H2S. The aim of our work was to investigate the role of KATP channels in the cardioprotection induced by pyridoxal-5-phosphate (PLP) in aging. Methods: Experiments were performed on adult and old (aged 24 months) male Wistar rats, which were divided into 3 groups: adults, old, and old PLP-treated rats. PLP was administered orally once a day for 14 days at a dose of 0.7 mg/kg. The levels of mRNA expression of subunits KATP channels were determined by reverse transcription and real-time polymerase chain reaction analysis. Protein expression levels were determined by the Western blot. Cardiac tissue morphology was determined using transverse 6 μm deparaffinized sections stained with picrosirius red staining. Vasorelaxation responses of isolated aortic rings and the function of Langendorff-perfused isolated hearts during ischemia-reperfusion, H2S levels, and markers of oxidative stress were also studied. Results: Administration of PLP to old rats reduces cardiac fibrosis and improves cardiac function during ischemia-reperfusion and vasorelaxation responses to KATP channels opening. At the same time, there was a significant increase in mRNA and protein expression of SUR2 and Kir6.1 subunits of KATP channels, H2S production, and reduced markers of oxidative stress. The specific KATP channel inhibitor—glibenclamide prevented the enhancement of vasodilator responses and anti-ischemic protection in PLP-treated animals. Conclusions: We suggest that this potential therapeutic effect of PLP in old animals may be a result of increased expression of KATP channels and H2S production.
... Rapid changes in membrane potential initiate neurotransmitter release in neurons,a prompt muscle contraction in cardiomyocytes, [2][3] and evoke insulin secretion in pancreatic β cells. 4 In a related fashion, the value of the membrane potentialset largely by the difference in internal and external potassium ion concentration and the selective permeability of the plasma membrane to K + ions plays an outsized role in the physiology of these cell types, controlling their excitability by setting the distance to action threshold. Further, values of membrane potentials in nonelectrically excitable cells may play important roles in signaling, differentiation, and cell cycle progression. ...
Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes.
Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes, but also measure values of membrane potentials. This study discloses a new fluorescent indicator which can address both.
We describe the synthesis of a new sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence.
Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials with greater signal-to-noise than state-of-the-art BeRST (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of action potentials in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.
... These channels have various functions in GI tissues: regulation of insulin secretion, protection against cellular stress, and adaptation to metabolic changes [205,206]. ROS can either decrease or increase K ATP channel activity, depending on the tissue and the specific isoforms expressed. Redox regulation has been linked to the N-terminus of the Kir6.2 subunit, particularly Cys42. ...
The pathogenesis of various gastrointestinal (GI) disorders, including gastritis, ulcers, inflammatory bowel disease (IBD) and cancer, can be linked to oxidative stress. It is known that reactive species carry out a crucial role in the genesis and progression of these pathologies; however, the contribution of ionic channels in their development is still under discussion. The function of ion channels in the gastrointestinal tract influences a variety of cellular processes. Acid-base balance, mucus layer, microbiota and mucosal blood flow are only some of the essential features for maintaining the mucosal integrity of the cellular barrier in the intestine, allowing for the preservation of proper permeability and ensuring tissue homeostasis. As the functional modulation of several ion channels is altered during oxidative stress conditions associated with gastrointestinal inflammation, this review focuses on contributing new insight into the roles of and the relationship between ion channels and oxidative stress in GI diseases. The association between ion channels and oxidative stress conditions could be used in diagnostics and the development of new pharmacological treatments for major gastrointestinal diseases.
... Two major SUR2 splice variants, SUR2A and SUR2B, differ in the last 42 amino acids (Chutkow et al., 1996;Shindo et al., 1998). ATP inhibition through binding to Kir6 subunits and MgATP or MgADP activation through induction of nucleotide binding domain (NBD) dimerization in the SUR subunits (Nichols et al., 1996;Nichols, 2006;Tinker et al., 2018) dominate channel regulation. ...
Gain-of-function (GOF) of KATP channels, resulting from mutations in either KCNJ8 (encoding Kir6.1) or ABCC9 (encoding SUR2), cause Cantú syndrome (CS), a channelopathy characterized by excess hair growth, coarse facial appearance, cardiomegaly, and lymphedema. Here, we established a pipeline for rapid analysis of CS mutation consequences in Landing pad HEK 293 cell lines stably expressing wild type and mutant human Kir6.1 and SUR2B. Thallium-influx and cell membrane potential, reported by fluorescent Tl-sensitive Fluozin-2 and voltage-sensitive DiBAC4(3) dyes, respectively, were used to assess channel activity. In the Tl-influx assay, CS-associated Kir6.1 mutations increased sensitivity to the KATP channel activator, pinacidil, but there was strikingly little effect of pinacidil for any SUR2B mutations, reflecting unexpected differences in the molecular mechanisms of Kir6.1 versus SUR2B mutations. Compared to the Tl-influx assay, the DiBAC4(3) assay presents more significant signal changes in response to subtle KATP channel activity changes, and all CS mutants (both Kir6.1 and SUR2B), but not WT channels, caused marked hyperpolarization, demonstrating that all mutants were activated under ambient conditions in intact cells. Most SUR2 CS mutations were markedly inhibited by <100nM glibenclamide, but sensitivity to inhibition by glibenclamide, repaglinide, and PNU37883A, was markedly reduced for Kir6.1 CS mutations. Understanding functional consequences of mutations can help with disease diagnosis and treatment. The analysis pipeline we have developed has the potential to rapidly identify mutational consequences, aiding future CS diagnosis, drug discovery, and individualization of treatment. Significance Statement We have developed new fluorescence-based assays of channel activities and drug sensitivities of Cantú syndrome (CS) mutations in human Kir6.1/SUR2B-dependent KATP channels, showing that Kir6.1 mutations increase sensitivity to activation by potassium channel openers, while SUR2B mutations markedly reduce KCO sensitivity. However, both Kir6.1 and SUR2B CS mutations are both more hyperpolarized than WT cells under basal conditions, confirming pathophysiologically relevant gain-of-function, validating DiBAC4(3) fluorescence to characterize hyperpolarization induced by KATP channel activity under basal, non KCO-activated conditions.
... The KATP channels are activated by Mg2+bound nucleotides and adenosine diphosphate (ADP, which act on the SUR subunit, and inhibited upon binding of intracellular ATP to the Kir6 subunit. Thus, KATP channels are open during states of low metabolic activity (e.g., low levels of ATP/high levels of ADP) resulting in plasma membrane hyperpolarization [91]. The ability to couple cellular metabolic state (ATP/ADP ratio) to the electrical activity of the cell membrane is critical in numerous physiological processes and is a key feature of KATP channels [92]. ...
Migraine is a debilitating neurological condition affecting millions of people worldwide. Until a few years ago, preventive migraine treatments were based on molecules with pleiotropic targets, developed for other indications, and discovered by serendipity to be effective in migraine prevention, although often burdened by tolerability issues leading to low adherence. However, the progresses in unravelling the migraine pathophysiology allowed identifying novel putative targets as calcitonin gene-related peptide (CGRP). Nevertheless, despite the revolution brought by CGRP monoclonal antibodies and gepants, a significant percentage of patients still remains burdened by an unsatisfactory response, suggesting that other pathways may play a critical role, with an extent of involvement varying among different migraine patients. Specifically, neuropeptides of the CGRP family, such as adrenomedullin and amylin; molecules of the secretin family, such as pituitary ade-nylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP); receptors, such as transient receptor potential (TRP) channels; intracellular downstream determinants, such as potassium channels, but also the opioid system and the purinergic pathway, have been suggested to be involved in migraine pathophysiology. The present review provides an overview of these pathways , highlighting, based on preclinical and clinical evidence, as well as provocative studies, their potential role as future targets for migraine preventive treatment.
... Magnesium affects ATP-induced changes in ORIC dynamics. Next, we tested if Mg 2+ ion, known to form a complex with ATP and influence interaction of ATP with some ATP-regulated channels 23 , has an effect on ORIC in the presence of ATP. As shown in Fig. 2a,b, ATP-mediated stabilization of ORIC is mildly negatively regulated by Mg 2+ , since time course of Ip70mV, normalized to maximal value, shows that ORIC activation is slowed down in (Mg 2+ + ATP) pip . ...
Ion channels are vital components of filamentous fungi signaling in communication with their environment. We exploited the ability of the apical region of growing sporangiophores of Phycomyces blakesleeanus to form membrane-enveloped cytoplasmic droplets (CDs), to examine ion currents in the filamentous fungi native plasma membrane. In hypoosmotic conditions, the dominant current in the CDs is ORIC, an osmotically activated, anionic, outwardly rectified, fast inactivating instantaneous current that we have previously characterized. Here, we examined the effect of ATP on ORIC. We show that CDs contain active mitochondria, and that respiration inhibition by azide accelerates ORIC inactivation. ATP, added intracellularly, reduced ORIC run-down and shifted the voltage dependence of inactivation toward depolarized potentials, in a manner that did not require hydrolysis. Notably, ATP led to slowing down of ORIC inactivation, as evidenced by an increased time constant of inactivation, τin, and slower decline of τin during prolonged recordings. Flavonoids (genistein and quercetin) had the effect on ORIC opposite to ATP, acting as current inhibitors, possibly by disrupting the stabilizing effect of ATP on ORIC. The integration of osmotic sensing with ATP dependence of the anionic current, typical of vertebrate cells, is described here for the first time in filamentous fungi.
... Gain-of-function SUR1 mutations lead to neonatal diabetes. 260 Sulfhydration in diabetes. S-sulfhydration is similar to S-nitrosylation in some chemical features. ...
The ever-increasing prevalence of noncommunicable diseases (NCDs) represents a major public health burden worldwide. The most common form of NCD is metabolic diseases, which affect people of all ages and usually manifest their pathobiology through life-threatening cardiovascular complications. A comprehensive understanding of the pathobiology of metabolic diseases will generate novel targets for improved therapies across the common metabolic spectrum. Protein posttranslational modification (PTM) is an important term that refers to biochemical modification of specific amino acid residues in target proteins, which immensely increases the functional diversity of the proteome. The range of PTMs includes phosphorylation, acetylation, methylation, ubiquitination, SUMOylation, neddylation, glycosylation, palmitoylation, myristoylation, prenylation, cholesterylation, glutathionylation, S-nitrosylation, sulfhydration, citrullination, ADP ribosylation, and several novel PTMs. Here, we offer a comprehensive review of PTMs and their roles in common metabolic diseases and pathological consequences, including diabetes, obesity, fatty liver diseases, hyperlipidemia, and atherosclerosis. Building upon this framework, we afford a through description of proteins and pathways involved in metabolic diseases by focusing on PTM-based protein modifications, showcase the pharmaceutical intervention of PTMs in preclinical studies and clinical trials, and offer future perspectives. Fundamental research defining the mechanisms whereby PTMs of proteins regulate metabolic diseases will open new avenues for therapeutic intervention.
... K ATP channels are one of the main endogenous mechanisms of cell protection when its energy resources are reduced. They are considered to be the central metabolic sensor of the cell regarding its energy supply [4]. These channels play a unique role in the synchronization of cellular metabolism and electrical activity, regulation of potential-dependent membrane functions, and maintenance of hormonal homeostasis, and dysfunction of K ATP channels may contribute to the pathogenesis [5]. ...
Background:
In the present work, we investigated the effect of exogenous glutathione in old rats on the expression of ATP-sensitive potassium (KATP) channels, the mitochondrial permeability transition pore (mPTP) opening in the heart, and the vasorelaxation responses of isolated aortic rings to activation of KATP channels.
Methods:
Experiments were performed on adult (6 months) and old (24 months) male Wistar rats, which were divided into three groups: adult, old, and glutathione-treated old rats. Glutathione was injected intraperitoneally at a dose of 52 mg/kg 1 hour before the studies. The mRNA expression of KATP channels was determined using reverse transcription and real-time polymerase chain reaction analysis. The effect of glutathione administration on mPTP opening, relaxation responses of isolated aortic rings, and oxidative stress markers was studied.
Results:
It was shown that the expression levels of Kir6.1, Kir6.2, and SUR1 subunits of KATP channels and levels of reduced glutathione were significantly increased in glutathione-treated old rats (by 8.3, 2.8, 13.1, and 1.5-fold, respectively), whereas the levels of oxidative stress markers (hydrogen peroxide, diene conjugates, malondialdehyde, and rate of superoxide generation) in heart mitochondria and mPTP opening were significantly reduced. Relaxation of aortic rings was significantly increased in response to the actions of KATP channel openers flocalin and pinacidil in glutathione-treated animals, which was prevented by glibenclamide.
Conclusions:
Thus, the administration of exogenous glutathione to old rats resulted in a significant increase in the expression levels of the Kir6.1, Kir6.2, and SUR1 subunits of KATP channels and a decrease in oxidative stress. This was accompanied by inhibition of mPTP opening and enhancement of vasorelaxation responses to activation of KATP channels.
... SUR1 blockers sulfonylureas were shown to inhibit SUR1-TRPM4 function [24]. SUR1 is an auxiliary subunit of the K ATP channel, which senses ATP levels in pancreatic β cells [25]. Sulfonylureas such as glibenclamide are widely used to control blood glucose levels in diabetic patients by regulating insulin secretion. ...
Reperfusion therapy for acute ischemic stroke aims to restore the blood flow of occluded blood vessels. However, successful recanalization is often associated with disruption of the blood-brain barrier, leading to reperfusion injury. Delayed recanalization increases the risk of severe reperfusion injury, including severe cerebral edema and hemorrhagic transformation. The TRPM4-blocking antibody M4P has been shown to alleviate reperfusion injury and improve functional outcomes in animal models of early stroke reperfusion. In this study, we examined the role of M4P in a clinically relevant rat model of delayed stroke reperfusion in which the left middle cerebral artery was occluded for 7 h. To mimic the clinical scenario, M4P or control IgG was administered 1 h before recanalization. Immunostaining showed that M4P treatment improved vascular morphology after stroke. Evans blue extravasation demonstrated attenuated vascular leakage following M4P treatment. With better vascular integrity, cerebral perfusion was improved, leading to a reduction of infarct volume and animal mortality rate. Functional outcome was evaluated by the Rotarod test. As more animals with severe injuries died during the test in the control IgG group, we observed no difference in functional outcomes in the surviving animals. In conclusion, we identified the potential of TRPM4 blocking antibody M4P to ameliorate vascular injury during delayed stroke reperfusion. If combined with reperfusion therapy, M4P has the potential to improve current stroke management.
... ATP-sensitive potassium (K ATP ) channels, widely expressed in various tissues, couple the cellular metabolic state, and multiple signaling pathways, to the membrane potential and cellular excitability (Nichols, 2006). K ATP channels assemble as octameric complexes of four pore-forming Kir6 subunits and four regulatory SUR (sulfonylurea receptor) subunits (Shyng & Nichols, 1997;Martin et al, 2017;Sung et al, 2021), encoded by two pairs of paralogous genes: ABCC8 (SUR1) and KCNJ11 (Kir6.2) on human chromosome 11 and ABCC9 (SUR2) and KCNJ8 (Kir6.1) on chromosome 12. ...
ABCC9-related intellectual disability and myopathy syndrome (AIMS) arises from loss-of-function (LoF) mutations in the ABCC9 gene, which encodes the SUR2 subunit of ATP-sensitive potassium (KATP ) channels. KATP channels are found throughout the cardiovascular system and skeletal muscle and couple cellular metabolism to excitability. AIMS individuals show fatigability, muscle spasms, and cardiac dysfunction. We found reduced exercise performance in mouse models of AIMS harboring premature stop codons in ABCC9. Given the roles of KATP channels in all muscles, we sought to determine how myopathy arises using tissue-selective suppression of KATP and found that LoF in skeletal muscle, specifically, underlies myopathy. In isolated muscle, SUR2 LoF results in abnormal generation of unstimulated forces, potentially explaining painful spasms in AIMS. We sought to determine whether excessive Ca2+ influx through CaV 1.1 channels was responsible for myopathology but found that the Ca2+ channel blocker verapamil unexpectedly resulted in premature death of AIMS mice and that rendering CaV 1.1 channels nonpermeable by mutation failed to reverse pathology; results which caution against the use of calcium channel blockers in AIMS.
... Next, we tested if Mg 2+ ion, known to form a complex with ATP and in uence interaction of ATP with some ATP-regulated channels 23 ...
Ion channels are vital components of filamentous fungi signaling in communication with their environment. We exploited the ability of the apical region of growing sporangiophores of Phycomyces blakesleeanus to form membrane-enveloped cytoplasmic droplets (CDs), to examine ion currents in the filamentous fungi native plasma membrane. In hypoosmotic conditions, the dominant current in the CDs is ORIC, an osmotically activated, anionic, outwardly rectified, fast inactivating instantaneous current that we have previously characterized. Here, we examined the effect of ATP on ORIC. We show that CDs contain active mitochondria, and that respiration inhibition by azide accelerates ORIC inactivation. ATP added intracellularly, reduced ORIC run-down and shifted the voltage dependence of inactivation toward depolarized potentials, in a manner that did not require hydrolysis. Notably, ATP led to slowing down of ORIC inactivation, as evidenced by an increased time constant of inactivation, τ in , and diminished decreasing of τ in during prolonged recordings. Flavonoids (genistein and quercetin) had the effect on ORIC opposite to ATP, acting as current inhibitors, possibly by disrupting the stabilizing effect of ATP on ORIC. The integration of osmotic sensing with ATP dependence of the anionic current, typical of vertebrate cells, is described here for the first time in filamentous fungi.
... We found that improved surface trafficking of K ATP channels occurs after IPC protocols through a PKC-dependent mechanism that correlated with improved cardioprotection (26), suggesting that maintenance of the K ATP channel surface expression may be key to cardioprotection. K ATP channel trafficking defects are also implicated in genetic variations associated with hypoglycemia, diabetes mellitus, atrial fibrillation, and dilated cardiomyopathy (27,28) and it is therefore of paramount importance to elucidate underlying pathways that maintain the K ATP channel surface density. Improved K ATP channel surface trafficking contributes to the cardioprotective effects of CaMKII inhibitors (25,29) and specific trafficking proteins such as EHD2 that maintain surface expression (30), but other mechanisms remain unexplored. ...
Sirtuins are NAD+-dependent deacetylases with beneficial roles in conditions relevant to human health, including metabolic disease, type II diabetes, obesity, cancer, aging, neurodegenerative diseases and cardiac ischemia. Since ATP-sensitive K+ (KATP) channels have cardioprotective roles, we investigated whether they are regulated by sirtuins. Nicotinamide mononucleotide (NMN) was used to increase cytosolic NAD+ levels and to activate sirtuins in cell lines, isolated rat and mouse cardiomyocytes or insulin-secreting INS-1 cells. KATP channels were studied with patch clamping, biochemistry techniques and antibody uptake experiments. NMN led to an increase in intracellular NAD+ levels and an increase in the KATP channel current, without significant changes in the unitary current amplitude or open probability. An increased surface expression was confirmed using surface biotinylation approaches. The rate of KATP channel internalization was diminished by NMN, which may be a partial explanation for the increased surface expression. We show that NMN acts via sirtuins since the increased KATP channel surface expression was prevented by blockers of SIRT1 and SIRT2 (Ex527 and AGK2), and mimicked by SIRT1 activation (SRT1720). The pathophysiological relevance of this finding was studied using a cardioprotection assay with isolated ventricular myocytes, in which NMN protected against simulated ischemia or hypoxia in a KATP channel-dependent manner. Overall, our data draws a link between intracellular NAD+, sirtuin activation, KATP channel surface expression and cardiac protection against ischemic damage.
Cardiovascular disease is a leading cause of morbidity and mortality, and exercise-training (TRN) is known to reduce risk factors and protect the heart from ischemia and reperfusion injury. Though the cardioprotective effects of exercise are well-documented, underlying mechanisms are not well understood. This review highlights recent findings and focuses on cardiac factors with emphasis on K+ channel control of the action potential duration (APD), β-adrenergic and adenosine regulation of cardiomyocyte function, and mitochondrial Ca2+ regulation. TRN-induced prolongation and shortening of the APD at low and high activation rates, respectively, is discussed in the context of a reduced response of the sarcolemma delayed rectifier potassium channel (IK) and increased content and activation of the sarcolemma KATP channel. A proposed mechanism underlying the latter is presented, including the phosphatidylinositol-3kinase/protein kinase B pathway. TRN induced increases in cardiomyocyte contractility and the response to adrenergic agonists are discussed. The TRN-induced protection from reperfusion injury is highlighted by the increased content and activation of the sarcolemma KATP channel and the increased phosphorylated glycogen synthase kinase-3β, which aid in preventing mitochondrial Ca2+ overload and mitochondria-triggered apoptosis. Finally, a brief section is presented on the increased incidences of atrial fibrillation associated with age and in life-long exercisers.
Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes but also measure values of membrane potentials. This study discloses a fluorescent indicator which can address both. We describe the synthesis of a sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials (APs) with greater signal-to-noise than state-of-the-art BeRST 1 (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of APs in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.
ATP-sensitive potassium (KATP) channels, composed of four pore-lining Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1) subunits, control insulin secretion in pancreatic β-cells. KATP channel opening is stimulated by PIP2 and inhibited by ATP. Mutations that increase channel opening by PIP2 reduce ATP inhibition and cause neonatal diabetes. Although considerable evidence has implicated a role for PIP2 in KATP channel function, previously solved open-channel structures have lacked bound PIP2, and mechanisms by which PIP2 regulates KATP channels remain unresolved. Here, we report the cryoEM structure of a KATP channel harboring the neonatal diabetes mutation Kir6.2-Q52R, in the open conformation, bound to amphipathic molecules consistent with natural C18:0/C20:4 long-chain PI(4,5)P2 at two adjacent binding sites between SUR1 and Kir6.2. The canonical PIP2 binding site is conserved among PIP2-gated Kir channels. The non-canonical PIP2 binding site forms at the interface of Kir6.2 and SUR1. Functional studies demonstrate both binding sites determine channel activity. Kir6.2 pore opening is associated with a twist of the Kir6.2 cytoplasmic domain and a rotation of the N-terminal transmembrane domain of SUR1, which widens the inhibitory ATP binding pocket to disfavor ATP binding. The open conformation is particularly stabilized by the Kir6.2-Q52R residue through cation-π bonding with SUR1-W51. Together, these results uncover the cooperation between SUR1 and Kir6.2 in PIP2 binding and gating, explain the antagonistic regulation of KATP channels by PIP2 and ATP, and provide a putative mechanism by which Kir6.2-Q52R stabilizes an open channel to cause neonatal diabetes.
SUR2, similar to SUR1, is a regulatory subunit of the ATP‐sensitive potassium channel (KATP), which plays a key role in numerous important physiological processes and is implicated in various diseases. Recent structural studies have revealed that, like SUR1, SUR2 can undergo ligand‐dependent dynamic conformational changes, transitioning between an inhibitory inward‐facing conformation and an activating occluded conformation. In addition, SUR2 possesses a unique inhibitory Regulatory helix (R helix) that is absent in SUR1. The binding of the activating Mg‐ADP to NBD2 of SUR2 competes with the inhibitory Mg‐ATP, thereby promoting the release of the R helix and initiating the activation process. Moreover, the signal generated by Mg‐ADP binding to NBD2 might be directly transmitted to the TMD of SUR2, prior to NBD dimerization. Furthermore, the C‐terminal 42 residues (C42) of SUR2 might allosterically regulate the kinetics of Mg‐nucleotide binding on NBD2. These distinctive properties render SUR2 intricate sensors for intracellular Mg‐nucleotides.
Precise matching of energy substrate delivery to local metabolic needs is essential for the health and function of all tissues. Here, we outline a mechanistic framework for understanding this critical process, which we refer to as electro-metabolic signaling (EMS). All tissues exhibit changes in metabolism over varying spatiotemporal scales and have widely varying energetic needs and reserves. We propose that across tissues, common signatures of elevated metabolism or increases in energy substrate usage that exceed key local thresholds rapidly engage mechanisms that generate hyperpolarizing electrical signals in capillaries that then relax contractile elements throughout the vasculature to quickly adjust blood flow to meet changing needs. The attendant increase in energy substrate delivery serves to meet local metabolic requirements and thus avoids a mismatch in supply and demand and prevents metabolic stress. We discuss in detail key examples of EMS that our laboratories have discovered in the brain and the heart, and we outline potential further EMS mechanisms operating in tissues such as skeletal muscle, pancreas, and kidney. We suggest that the energy imbalance evoked by EMS uncoupling may be central to cellular dysfunction from which the hallmarks of aging and metabolic diseases emerge and may lead to generalized organ failure states—such as diverse flavors of heart failure and dementia. Understanding and manipulating EMS may be key to preventing or reversing these dysfunctions.
With the growing demand for next-generation health care, the integration of electronic components into implantable medical devices (IMDs) has become a vital factor in achieving sophisticated healthcare functionalities such as electrophysiological monitoring and electroceuticals worldwide. However, these devices confront technological challenges concerning a noninvasive power supply and biosafe device removal. Addressing these challenges is crucial to ensure continuous operation and patient comfort and minimize the physical and economic burden on the patient and the healthcare system. This Review highlights the promising capabilities of bioresorbable triboelectric nanogenerators (B-TENGs) as temporary self-clearing power sources and self-powered IMDs. First, we present an overview of and progress in bioresorbable triboelectric energy harvesting devices, focusing on their working principles, materials development, and biodegradation mechanisms. Next, we examine the current state of on-demand transient implants and their biomedical applications. Finally, we address the current challenges and future perspectives of B-TENGs, aimed at expanding their technological scope and developing innovative solutions. This Review discusses advancements in materials science, chemistry, and microfabrication that can advance the scope of energy solutions available for IMDs. These innovations can potentially change the current health paradigm, contribute to enhanced longevity, and reshape the healthcare landscape soon.
Sarcolemmal ATP-sensitive potassium (K ATP ) channels play a vital role in cardioprotection. Cardiac K ATP channels are enriched in caveolae and physically interact with the caveolae structural protein caveolin-3 (Cav3). Disrupting caveolae impairs the regulation of K ATP channels through several signaling pathways. However, the direct functional effect of Cav3 on K ATP channels is still poorly understood. Here we used the cardiac K ATP channel subtype, Kir6.2/SUR2A, and showed that Cav3 greatly reduced K ATP channel surface density and current amplitude in a caveolae-independent manner. A screen of Cav3 functional domains revealed that a 25 amino acid region in the membrane attachment domain of Cav3 is the minimal effective segment (MAD1). The peptide corresponding to the MAD1 segment decreased K ATP channel current in a concentration-dependent manner with an IC50 of ~5 μM. The MAD1 segment prevented K ATP channel recycling, thus decreased K ATP channel surface density and abolished the cardioprotective effect of ischemic preconditioning. Our research identified the Cav3 MAD1 segment as a novel negative regulator of K ATP channel recycling, providing pharmacological potentials in the treatment of diseases with K ATP channel trafficking defects.
Understanding the physiological and molecular regulation of tick feeding is necessary for developing intervention strategies to curb disease transmission by ticks. Pharmacological activation of ATP-gated inward rectifier potassium (KATP) channels reduced fluid secretion from isolated salivary gland and blood feeding in the lone star tick, Amblyomma americanum, yet the temporal expression pattern of KATP channel proteins remained unknown. KATP channels were highly expressed in type II and III acini in off-host stage and early feeding phase ticks, yet expression was reduced in later stages of feeding. We next assessed KATP channel regulation of the secreted proteome of tick saliva. LC-MS/MS analysis identified 40 differentially secreted tick saliva proteins after exposure to KATP activators or inhibitors. Secretion of previously validated tick saliva proteins that promote tick feeding, AV422, AAS27, and AAS41 were significantly reduced by upwards of 8 log units in ticks exposed to KATP channel activators when compared to untreated ticks. Importantly, activation of KATP channels inhibited tick feeding and vice versa for KATP channel inhibitors. Data indicate KATP channels regulate tick feeding biology by controlling secretion of pro-feeding proteins that are essential during early feeding phases, which provides insights into physiological and molecular regulation of tick feeding behavior.
Pheochromocytomas/paragangliomas (PPGLs) are neuroendocrine tumours, mostly resulting from mutations in predisposing genes. Mutations of succinate dehydrogenase (SDH) subunit B (SDHB) are associated with high probability of metastatic disease. Since bioelectrical properties and signalling in cancer are an emerging field, we investigated the metabolic, functional and electrophysiological characteristics in human SDHB knockout pheochromocytoma cells. These cells exhibited reduced SDH function with elevated succinate-to-fumarate ratio and reduced intracellular ATP levels. The analysis of membrane passive properties revealed a more hyperpolarized membrane potential and a lower cell capacitance of SDHB deficient cells compared to parental ones. These bioelectrical changes were associated with reduced proliferation and adhesion capacity of SDHB deficient cells. In SDHB deficient cells only, we also observed an increased amplitude of potassium currents suggesting an activation of ATP-sensitive potassium channels (KATP). Indeed, exposure of the SDHB deficient cells to glibenclamide, a specific KATP inhibitor, or to ATP caused normalization of potassium current features. In this work, we show for the first time that reduced intracellular ATP levels in SDHB deficient chromaffin cells impaired cell bioelectrical properties, which in turn, increased cell aggressiveness. Moreover, we first ever demonstrated that glibenclamide, not only reduced the outward potassium currents in SDHB deficient cells, but increased their growth capacity, reduced their ability to migrate, and shifted their phenotype towards one more similar to that of parental one.
ATP-sensitive potassium ion channels (KATP) are transmembrane proteins that modulate insulin release and muscle contraction. KATP channels are composed of two types of subunit, Kir6 and SUR, which exist in two and three isoforms respectively with different tissue distribution. In this work, we identify a previously undescribed ancestral vertebrate gene encoding a Kir6-related protein that we have named Kir6.3, which may not have a SUR binding partner, unlike the other two Kir6 proteins. Whereas Kir6.3 was lost in amniotes including mammals, it is still present in several early-diverging vertebrate lineages such as frogs, coelacanth, and rayfinned fishes. Molecular dynamics (MD) simulations using homology models of Kir6.1, Kir6.2, and Kir6.3 from the coelacanth Latimeria chalumnae showed that the three proteins exhibit subtle differences in their dynamics. Steered MD simulations of Kir6-SUR pairs suggest that Kir6.3 has a lower binding affinity for the SUR proteins than either Kir6.1 or Kir6.2. As we found no additional SUR gene in the genome of the species that have Kir6.3, it most likely forms a lone tetramer. These findings invite studies of the tissue distribution of Kir6.3 in relation to the other Kir6 as well as SUR proteins to determine the functional roles of Kir6.3.
KATP channels are metabolic sensors for intracellular ATP/ADP ratios, play essential roles in many physiological processes, and are implicated in a spectrum of pathological conditions. SUR2A-containing KATP channels differ from other subtypes in their sensitivity to Mg-ADP activation. However, the underlying structural mechanism remains poorly understood. Here we present a series of cryo-EM structures of SUR2A in the presence of different combinations of Mg-nucleotides and the allosteric inhibitor repaglinide. These structures uncover regulatory helix (R helix) on the NBD1-TMD2 linker, which wedges between NBD1 and NBD2. R helix stabilizes SUR2A in the NBD-separated conformation to inhibit channel activation. The competitive binding of Mg-ADP with Mg-ATP to NBD2 mobilizes the R helix to relieve such inhibition, allowing channel activation. The structures of SUR2B in similar conditions suggest that the C-terminal 42 residues of SUR2B enhance the structural dynamics of NBD2 and facilitate the dissociation of the R helix and the binding of Mg-ADP to NBD2, promoting NBD dimerization and subsequent channel activation.
The genetic basis of the human vocal system is largely unknown, as are the sequence variants that give rise to individual differences in voice and speech. Here, we couple data on diversity in the sequence of the genome with voice and vowel acoustics in speech recordings from 12,901 Icelanders. We show how voice pitch and vowel acoustics vary across the life span and correlate with anthropometric, physiological, and cognitive traits. We found that voice pitch and vowel acoustics have a heritable component and discovered correlated common variants in ABCC9 that associate with voice pitch. The ABCC9 variants also associate with adrenal gene expression and cardiovascular traits. By showing that voice and vowel acoustics are influenced by genetics, we have taken important steps toward understanding the genetics and evolution of the human vocal system.
Elevated blood glucose levels, or hyperglycemia, can increase brain excitability and amyloid-beta (Aβ) release offering a mechanistic link between type-2-diabetes and Alzheimer's disease (AD). Since the cellular mechanisms governing this relationship are poorly understood, we explored whether ATP-sensitive potassium (KATP) channels, which couple changes in energy availability with cellular excitability, play a role in AD pathogenesis. First, we demonstrate that KATP channel subunits, Kir6.2/KCNJ11 and SUR1/ABCC8 are expressed on excitatory and inhibitory neurons in the human brain and cortical expression of KCNJ11 and ABCC8 changes with Alzheimer's pathology in humans and mice. Next, we explored whether eliminating neuronal KATP channel activity uncoupled the relationship between metabolism, excitability, and Aβ pathology in a novel mouse model of cerebral amyloidosis and neuronal KATP channel ablation (e.g. APP/PS1, Kir6.2-/- mouse). Using both acute and chronic paradigms, we demonstrate that Kir6.2-KATP channels are metabolic sensors that regulate hyperglycemic-dependent increases in interstitial fluid levels of Aβ, amyloidogenic processing of APP, and amyloid plaque formation, which may be dependent on lactate release. These studies identify a new role for Kir6.2-KATP channels in Alzheimer's disease and suggest that pharmacological manipulation of Kir6.2-KATP channels holds therapeutic promise in reducing Aβ pathology in diabetic or prediabetic patients.
Congenital hyperinsulinism (CHI) is the most common cause of persistent hypoglycemia in infancy/childhood and is a serious condition associated with severe recurrent attacks of hypoglycemia due to dysregulated insulin secretion. Timely diagnosis and effective treatment are crucial to prevent severe hypoglycemia that may lead to life-long neurological complications. In pancreatic β-cells, adenosine triphosphate (ATP)-sensitive K+ (KATP) channels are a central regulator of insulin secretion vital for glucose homeostasis. Genetic defects that lead to loss of expression or function of KATP channels are the most common cause of HI (KATP-HI). Much progress has been made in our understanding of the molecular genetics and pathophysiology of KATP-HI in the past decades; however, treatment remains challenging, in particular for patients with diffuse disease who do not respond to the KATP channel activator diazoxide. In this review, we discuss current approaches and limitations on the diagnosis and treatment of KATP-HI, and offer perspectives on alternative therapeutic strategies.
Sur1 knockout mouse β-cells lack KATP channels and show spontaneous Ca²⁺ action potentials equivalent to those seen in patients with persistent hyperinsulinemic hypoglycemia of infancy, but the mice are normoglycemic unless stressed. Sur1 –/– islets lack first phase insulin secretion and exhibit an attenuated glucose-stimulated second phase secretion. Loss of the first phase leads to mild glucose intolerance, whereas reduced insulin output is consistent with observed neonatal hyperglycemia. Loss of KATP channels impairs the rate of return to a basal secretory level after a fall in glucose concentration. This leads to increased hypoglycemia upon fasting and contributes to a very early, transient neonatal hypoglycemia. Whereas persistent hyperinsulinemic hypoglycemia of infancy underscores the importance of the KATP-dependent ionic pathway in control of insulin release, the Sur1 –/– animals provide a novel model for study of KATP-independent pathways that regulate insulin secretion.
A member of the inwardly rectifying potassium channel family was cloned here. The channel, called BIR (Kir6.2), was expressed in large amounts in rat pancreatic islets and glucose-responsive insulin-secreting cell lines. Coexpression with the sulfonylurea receptor SUR reconstituted an inwardly rectifying potassium conductance of 76 picosiemens that was sensitive to adenosine triphosphate (ATP) (IKATP) and was inhibited by sulfonylureas and activated by diazoxide. The data indicate that these pancreatic beta cell potassium channels are a complex composed of at least two subunits--BIR, a member of the inward rectifier potassium channel family, and SUR, a member of the ATP-binding cassette superfamily. Gene mapping data show that these two potassium channel subunit genes are clustered on human chromosome 11 at position 11p15.1.
Closure of ATP-sensitive K(+) channels (K(ATP) channels) in response to metabolically generated ATP or binding of sulfonylurea drugs stimulates insulin release from pancreatic beta-cells. Heterozygous gain-of-function mutations in the KCJN11 gene encoding the Kir6.2 subunit of this channel are found in approximately 47% of patients diagnosed with permanent diabetes at <6 months of age. There is a striking genotype-phenotype relationship with specific Kir6.2 mutations being associated with transient neonatal diabetes, permanent neonatal diabetes alone, and a novel syndrome characterized by developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome. All mutations appear to cause neonatal diabetes by reducing K(ATP) channel ATP sensitivity and increasing the K(ATP) current, which inhibits beta-cell electrical activity and insulin secretion. The severity of the clinical symptoms is reflected in the ATP sensitivity of heterozygous channels in vitro with wild type > transient neonatal diabetes > permanent neonatal diabetes > DEND syndrome channels. Sulfonylureas still close mutated K(ATP) channels, and many patients can discontinue insulin injections and show improved glycemic control when treated with high-dose sulfonylurea tablets. In conclusion, the finding that Kir6.2 mutations can cause neonatal diabetes has enabled a new therapeutic approach and shed new light on the structure and function of the Kir6.2 subunit of the K(ATP) channel.
ATP-sensitive K+ (KATP) channels regulate many cellular functions by linking cell metabolism to membrane potential. We have generated KATP channel-deficient mice by genetic disruption of Kir6.2, which forms the K+ ion-selective pore of the channel. The homozygous mice (Kir6.2−/−) lack KATP channel activity. Although the resting membrane potential and basal intracellular calcium concentrations ([Ca2+]i) of pancreatic beta cells in Kir6.2−/− are significantly higher than those in control mice (Kir6.2+/+), neither glucose at high concentrations nor the sulfonylurea tolbutamide elicits a rise in [Ca2+]i, and no significant insulin secretion in response to either glucose or tolbutamide is found in Kir6.2−/−, as assessed by perifusion and batch incubation of pancreatic islets. Despite the defect in glucose-induced insulin secretion,
Kir6.2−/− show only mild impairment in glucose tolerance. The glucose-lowering effect of insulin, as assessed by an insulin tolerance
test, is increased significantly in Kir6.2−/−, which could protect Kir6.2−/− from developing hyperglycemia. Our data indicate that the KATP channel in pancreatic beta cells is a key regulator of both glucose- and sulfonylurea-induced insulin secretion and suggest
also that the KATP channel in skeletal muscle might be involved in insulin action.
Adenosine triphosphate (ATP)–sensitive potassium (KATP) channels couple electrical activity to cellular metabolism through their inhibition by intracellular ATP. ATP inhibition
of KATP channels varies among tissues and is affected by the metabolic and regulatory state of individual cells, suggesting involvement
of endogenous factors. It is reported here that phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylinositol-4-phosphate (PIP) controlled ATP inhibition of cloned KATP channels (Kir6.2 and SUR1). These phospholipids acted on the Kir6.2 subunit and shifted ATP sensitivity by several orders of magnitude. Receptor-mediated activation of phospholipase C resulted
in inhibition of KATP-mediated currents. These results represent a mechanism for control of excitability through phospholipids.
The potassium channel from Streptomyces lividans is an integral membrane protein with sequence similarity to all known K+ channels, particularly in the pore region. X-ray analysis with data to 3.2 angstroms reveals that four identical subunits
create an inverted teepee, or cone, cradling the selectivity filter of the pore in its outer end. The narrow selectivity filter
is only 12 angstroms long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large water-filled
cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at the center
of the bilayer. Main chain carbonyl oxygen atoms from the K+ channel signature sequence line the selectivity filter, which is held open by structural constraints to coordinate K+ ions but not smaller Na+ ions. The selectivity filter contains two K+ ions about 7.5 angstroms apart. This configuration promotes ion conduction by exploiting electrostatic repulsive forces to
overcome attractive forces between K+ ions and the selectivity filter. The architecture of the pore establishes the physical principles underlying selective K+ conduction.
The ATP-sensitive potassium channel (KATP) regulates insulin secretion in pancreatic β cells. Loss of functional KATP channels because of mutations in either the SUR1 or Kir6.2 channel subunit causes persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We investigated the molecular mechanism by which a single phenylalanine deletion in SUR1 (ΔF1388) causes PHHI. Previous studies have shown that coexpression of ΔF1388 SUR1 with Kir6.2 results in no channel activity. We demonstrate here that the lack of functional expression is due to failure of the mutant channel to traffic to the cell surface. Trafficking of KATP channels requires that the endoplasmic reticulum-retention signal, RKR, present in both SUR1 and Kir6.2, be shielded during channel assembly. To ask whether ΔF1388 SUR1 forms functional channels with Kir6.2, we inactivated the RKR signal in ΔF1388 SUR1 by mutation to AAA (ΔF1388 SUR1AAA). Inactivation of similar endoplasmic reticulum-retention signals in the cystic fibrosis transmembrane conductance regulator has been shown to partially overcome the trafficking defect of a cystic fibrosis transmembrane conductance regulator mutation, ΔF508. We found that coexpression of ΔF1388 SUR1AAA with Kir6.2 led to partial surface expression of the mutant channel. Moreover, mutant channels were active. Compared with wild-type channels, the mutant channels have reduced ATP sensitivity and do not respond to stimulation by MgADP or diazoxide. The RKR → AAA mutation alone has no effect on channel properties. Our results establish defective trafficking of KATP channels as a molecular basis of PHHI and show that F1388 in SUR1 is critical for normal trafficking and function of KATP channels.
The ATP-sensitive potassium channel (KATP) controls insulin release in pancreatic β-cells and also modulates important functions
in other cell types. In this study we report that anionic phospholipids activated KATP in pancreatic β-cells, cardiac myocytes,
skeletal muscle cells, and a cloned KATP composed of two subunits (SUR/Kir6.2) stably expressed in a mammalian cell line.
The effectiveness was proportional to the number of negative charges on the head group of the anionic phospholipid. Screening
negative charges with polyvalent cations antagonized the effect. Enzymatic treatment with phospholipases that reduced charge
on the lipids also reduced or eliminated the effect. These results suggest that intact phospholipids with negative charges
are the critical requirement for activation of KATP, in distinction from the usual cell signaling pathway through phospholipids
that requires cleavage. Mutations of two positively charged amino acid residues at the C terminus of Kir6.2 accelerated loss
of channel activity and reduced the activating effects of phospholipids, suggesting involvement of this region in the activation.
Metabolism of anionic phospholipids in plasmalemmal membrane may be a novel and general mechanism for regulation of KATP and
perhaps other ion channels in the family of inward rectifiers.
The ATP-sensitive K-channel (K-ATP channel) plays a key role in insulin secretion from pancreatic beta-cells. It is closed by glucose metabolism, which stimulates insulin secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and Mg-ADP, which inhibit and potentiate channel activity, respectively. The beta-cell K-ATP channel consists of a pore-forming subunit, Kir6.2, and a regulatory subunit, SUR1. We have mutated (independently or together) two lysine residues in the Walker A (W(A)) motifs of the first (K719A) and second (K1384M) nucleotide-binding domains (NBDs) of SUR1. These mutations are expected to inhibit nucleotide hydrolysis. Our results indicate that the W(A) lysine of NBD1 (but not NBD2) is essential for activation of K-ATP currents by diazoxide. The potentiatory effects of Mg-ADP required the presence of the W(A) lysines in both NBDs. Mutant currents were slightly more sensitive to ATP than wild-type currents. Metabolic inhibition led to activation of wild-type and K1384M currents, but not K719A or K719A/K1384M currents, suggesting that there may be a factor in addition to ATP and ADP which regulates K-ATP channel activity.
ATP-sensitive potassium channels (K-ATP channels) couple cell metabolism to electrical activity and are important in the physiology and pathophysiology of many tissues. In pancreatic beta-cells, K-ATP channels link changes in blood glucose concentration to insulin secretion. They are also the target for clinically important drugs such as sulphonylureas, which stimulate secretion, and the K+ channel opener diazoxide, which inhibits insulin release. Metabolic regulation of K-ATP channels is mediated by changes in intracellular ATP and Mg-ADP levels, which inhibit and activate the channel, respectively. The beta-cell K-ATP channel is a complex of two proteins: an inward-rectifier K+ channel subunit, Kir6.2, and the sulphonylurea receptor, SUR1. We show here that the primary site at which ATP acts to mediate K-ATP channel inhibition is located on Kir6.2, and that SUR1 is required for sensitivity to sulphonylureas and diazoxide and for activation by Mg-ADP.
ATP-sensitive potassium (KATP) channels link cellular metabolism to electrical activity in nerve, muscle, and endocrine tissues. They are formed as a functional complex of two unrelated subunits-a member of the Kir inward rectifier potassium channel family, and a sulfonylurea receptor (SUR), a member of the ATP-binding cassette transporter family, which includes cystic fibrosis transmembrane conductance regulators and multidrug resistance protein, regulators of chloride channel activity. This recent discovery has brought together proteins from two very distinct superfamilies in a novel functional complex. The pancreatic KATP channel is probably formed specifically of Kir6.2 and SUR1 isoforms. The relationship between SUR1 and Kir6.2 must be determined to understand how SUR1 and Kir6.2 interact to form this unique channel. We have used mutant Kir6.2 subunits and dimeric (SUR1-Kir6.2) constructs to examine the functional stoichiometry of the KATP channel. The data indicate that the KATP channel pore is lined by four Kir6.2 subunits, and that each Kir6.2 subunit requires one SUR1 subunit to generate a functional channel in an octameric or tetradimeric structure.
KATP channels were reconstituted in COSm6 cells by coexpression of the sulfonylurea receptor SUR1 and the inward rectifier potassium channel Kir6.2. The role of the two nucleotide binding folds of SUR1 in regulation of KATP channel activity by nucleotides and diazoxide was investigated. Mutations in the linker region and the Walker B motif (Walker, J.E., M.J. Saraste, M.J. Runswick, and N.J. Gay. 1982. EMBO [Eur. Mol. Biol. Organ.] J. 1:945-951) of the second nucleotide binding fold, including G1479D, G1479R, G1485D, G1485R, Q1486H, and D1506A, all abolished stimulation by MgADP and diazoxide, with the exception of G1479R, which showed a small stimulatory response to diazoxide. Analogous mutations in the first nucleotide binding fold, including G827D, G827R, and Q834H, were still stimulated by diazoxide and MgADP, but with altered kinetics compared with the wild-type channel. None of the mutations altered the sensitivity of the channel to inhibition by ATP4-. We propose a model in which SUR1 sensitizes the KATP channel to ATP inhibition, and nucleotide hydrolysis at the nucleotide binding folds blocks this effect. MgADP and diazoxide are proposed to stabilize this desensitized state of the channel, and mutations at the nucleotide binding folds alter the response of channels to MgADP and diazoxide by altering nucleotide hydrolysis rates or the coupling of hydrolysis to channel activation.
Familial hyperinsulinism (HI) is a disorder characterized by dysregulation of insulin secretion and profound hypoglycemia. Mutations in both the Kir6.2 and sulfonylurea receptor (SUR1) genes have been associated with the autosomal recessive form of this disorder. In this study, the spectrum and frequency of SUR1 mutations in HI and their significance to clinical manifestations of the disease were investigated by screening 45 HI probands of various ethnic origins for mutations in the SUR1 gene. Single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses of genomic DNA revealed a total of 17 novel and three previously described mutations in SUR1 . The novel mutations comprised one nonsense and 10 missense mutations, two deletions, three mutations in consensus splice-site sequences and an in-frame insertion of six nucleotides. One mutation occurred in the first nucleotide binding domain (NBF-1) of the SUR1 molecule and another eight mutations were located in the second nucleotide binding domain (NBF-2), including two at highly conserved amino acid residues within the Walker A sequence motif. The majority of the remaining mutations was distributed throughout the three putative transmembrane domains of the SUR1 protein. With the exception of the 3993-9G-->A mutation, which was detected on 4.5% (4/88) disease chromosomes, allelic frequencies for the identified mutations varied between 1.1 and 2.3% for HI chromosomes, indicating that each mutation was rare within the patient cohort. The clinical manifestations of HI in those patients homozygous for mutations in the SUR1 gene are described. In contrast with the allelic homogeneity of HI previously described in Ashkenazi Jewish patients, these findings suggest that a large degree of allelic heterogeneity at the SUR1 locus exists in non-Ashkenazi HI patients. These data have important implications for genetic counseling and prenatal diagnosis of HI, and also provide a basis to further elucidate the molecular mechanisms underlying the pathophysiology of this disease.
ATP-sensitive K+ (KATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore-forming subunit, Kir6.2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg-nucleotides complicates analysis of nucleotide inhibition of Kir6. 2/SUR1 channels. We therefore used a truncated isoform of Kir6.2, that expresses ATP-sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6.2 is highly selective for ATP, and that both the adenine moiety and the beta-phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6.2: the N-terminus preceding, and the C-terminus immediately following, the transmembrane domains. Some mutations in the C-terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single-channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C-terminus in KATP channel gating.
The ATP-sensitive potassium channel, K(ATP) channel, a functional complex of the sulfonylurea receptor 1, SUR1, and an inward rectifier potassium channel subunit, Kir6.2, regulates insulin secretion in the pancreas. Mutations in both the Kir6.2 and SUR1 genes are associated with persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a disorder of pancreatic beta-cell function characterized by excess insulin secretion and hypoglycemia. We have studied the functional properties of novel SUR1 mutations identified in PHHI patients, including H125Q, N188S, F591L, T1139M, R1215Q, G1382S, and R1394H. R1394H and deltaF1388 SUR1, a previously identified PHHI mutation, resulted in no functional channels when coexpressed with Kir6.2 in COS cells, while H125Q, N188S, F591L, T1139M, R1215Q, and G1382S SUR1 generated functional channels in the absence of ATP. With the exception of N188S and H125Q, all mutants had reduced response to stimulation by MgADP. These results indicate that lack of, or reduction of, K(ATP) channel sensitivity to MgADP is a common molecular defect associated with the disease. The mutant channels also showed varied response to activation by the potassium channel opener diazoxide. Because these mutations are distributed throughout the molecule, our data have new implications for structure-function relationships of the K(ATP) channel, suggesting that structural elements in SUR1 outside of the two nucleotide-binding folds are also important in regulating channel activity.
1. ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. A truncated isoform of Kir6.2, Kir6.2DeltaC26, expresses ATP-sensitive channels in the absence of SUR1, suggesting the ATP-inhibitory site lies on the Kir6. 2 subunit. 2. We examined the effect on the channel ATP sensitivity of mutating the arginine residue at position 50 (R50) in the N-terminus of Kir6.2, by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2DeltaC26. 3. Substitution of R50 by serine, alanine or glycine reduced the Ki for ATP inhibition from 117 microM to 800 microM, 1.1 mM and 3.8 mM, respectively. The single-channel conductance and kinetics were unaffected by any of these mutations. Mutation to glutamate, lysine, asparagine, glutamine or leucine had a smaller effect (Ki, approximately 300-400 microM). The results indicate that the side chain of the arginine residue at position 50 is unlikely to contribute directly to the binding site for ATP, and suggest it may affect ATP inhibition by allosteric interactions. 4. Mutation of the isoleucine residue at position 49 to glycine (I49G) reduced the channel ATP sensitivity, while the mutation of the glutamate residue at position 51 to glycine (E51G) did not. 5. When a mutation in the N-terminus of Kir6.2DeltaC26 that alters ATP sensitivity (R50S; Ki, 800 microM) was combined with one in the C-terminus (E179Q; Ki, 300 microM), the Ki for the apparent ATP sensitivity was increased to 2.8 mM. The Hill coefficient was also increased. This suggests that the N- and C-termini of Kir6.2 may co-operate to influence channel closure by ATP.
ABC transporters (also known as traffic ATPases) form a large family of proteins responsible for the translocation of a variety of compounds across membranes of both prokaryotes and eukaryotes. The recently completed Escherichia coli genome sequence revealed that the largest family of paralogous E. coli proteins is composed of ABC transporters. Many eukaryotic proteins of medical significance belong to this family, such as the cystic fibrosis transmembrane conductance regulator (CFTR), the P-glycoprotein (or multidrug-resistance protein) and the heterodimeric transporter associated with antigen processing (Tap1-Tap2). Here we report the crystal structure at 1.5 A resolution of HisP, the ATP-binding subunit of the histidine permease, which is an ABC transporter from Salmonella typhimurium. We correlate the details of this structure with the biochemical, genetic and biophysical properties of the wild-type and several mutant HisP proteins. The structure provides a basis for understanding properties of ABC transporters and of defective CFTR proteins.
Inwardly rectifying K+ channels bind intracellular magnesium and polyamines to generate inward rectification. We have examined the architecture of the inner pore of Kir2.1 channels by covalently attaching a constrained number (from one to four) of positively charged moieties of different sizes to the channel. Our results indicate that the inner pore is formed solely by the second transmembrane segment and is unprecedentedly wide. At a position critical for inward rectification (D172), the pore is sufficiently wide to bind three Mg2+ ions or polyamine molecules simultaneously. Single-channel recordings directly demonstrate that partially modified channels exhibit distinct subconductance levels. Such a wide inner pore may greatly facilitate ion permeation and high-affinity binding of multiple pore blockers to generate strong inward rectification.
Pancreatic beta cell ATP-sensitive potassium (K(ATP)) channels regulate glucose-induced insulin secretion. The activity of the K(ATP) channel, composed of SUR1 and Kir6.2 subunits, is regulated by intracellular ATP and ADP, but the molecular mechanism is not clear. To distinguish the ATP binding properties of the two nucleotide-binding folds (NBFs) of SUR1, we prepared antibodies against NBF1 and NBF2, and the tryptic fragment of SUR1 was immunoprecipitated after photoaffinity labeling with 8-azido-[(32)P]ATP. The 35-kDa fragment was strongly labeled with 5 microM 8-azido-[(32)P]ATP even in the absence of Mg(2+) and was immunoprecipitated with the antibody against NBF1. The 65-kDa fragment labeled with 100 microM 8-azido-[alpha-(32)P]ATP in the presence of Mg(2+) was immunoprecipitated with anti-NBF2 and anti-C terminus antibodies. These results indicate that NBF1 of SUR1 binds 8-azido-ATP strongly in a magnesium-independent manner and that NBF2 binds 8-azido-ATP weakly in a magnesium-dependent manner. Furthermore, the 65-kDa tryptic fragment was not photoaffinity-labeled with 8-azido-[gamma-(32)P]ATP at 37 degrees C, whereas the 35-kDa tryptic fragment was, suggesting that NBF2 of SUR1 may have ATPase activity and that NBF1 has none or little.
We have investigated protein interactions involved in pancreatic beta-cell ATP-sensitive potassium channel assembly. These channels, which are of key importance for control of insulin release, are a hetero-oligomeric complex of pore-forming Kir6.2 subunits and sulfonylurea receptor (SUR1) subunits with two nucleotide-binding domains (NBD1 and NBD2). We divided SUR1 into two halves at Pro-1042. Expression of either the individual N- or C-terminal domain in a baculovirus expression system did not lead to glibenclamide binding activity, although studies with green fluorescent protein fusion proteins showed that both half-molecules were inserted into the plasma membrane. However, significant glibenclamide binding activity was observed when the half-molecules were co-expressed (even when NBD2 was deleted from the C-terminal half-molecule). Simultaneous expression of Kir6.2 resulted in enhanced glibenclamide binding activity. We conclude that the glibenclamide-binding site includes amino acid residues from both halves of the molecule, that there is strong interaction between different regions of SUR1, that NBD2 is not essential for glibenclamide binding, and that interactions between Kir6.2 and SUR1 participate in ATP-sensitive potassium channel assembly. Investigation of NBD1-green fluorescent protein fusion protein distribution inside insect cells expressing C-terminal halves of SUR1 demonstrated strong interaction between NBD1 and NBD2. We also expressed and purified NBD1 from Escherichia coli. Purified NBD1 was found to exist as a tetramer indicating strong homomeric attractions and a possible role for NBD1 in SUR1 assembly.
ATP-sensitive potassium (K(ATP)) channels, composed of sulfonylurea receptor (SURx) and Kir6.x, play important roles by linking cellular metabolic state to membrane potential in various tissues. Pancreatic, cardiac, and vascular smooth muscle K(ATP) channels, which consist of different subtypes of SURx, differ in their responses to cellular metabolic state. To explore the possibility that different interactions of SURx with nucleotides cause differential regulation of K(ATP) channels, we analyzed the properties of nucleotide-binding folds (NBFs) of SUR1, SUR2A, and SUR2B. SURx in crude membrane fractions was incubated with 8-azido-[alpha-(32)P]ATP or 8-azido-[gamma-(32)P]ATP under various conditions and was photoaffinity-labeled. Then, SURx was digested mildly with trypsin, and partial tryptic fragments were immunoprecipitated with antibodies against NBF1 and NBF2. Some nucleotide-binding properties were different among SUR subtypes as follows. 1) Mg(2+) dependence of nucleotide binding of NBF2 of SUR1 was high, whereas those of SUR2A and SUR2B were low. 2) The affinities of NBF1 of SUR1 for ATP and ADP, especially for ATP, were significantly higher than those of SUR2A and SUR2B. 3) The affinities of NBF2 of SUR2B for ATP and ADP were significantly higher than those of SUR2A. This is the first biochemical study to analyze and compare the nucleotide-binding properties of NBFs of three SUR subtypes, and our results suggest that their different properties may explain, in part, the differential regulation of K(ATP) channel subtypes. The high nucleotide-binding affinities of SUR1 may explain the high ability of SUR1 to stimulate pancreatic K(ATP) channels. It is also suggested that the C-terminal 42 amino acids affect the physiological roles of SUR2A and SUR2B by changing the nucleotide-binding properties of their NBFs.
ATP-sensitive K+ (KATP) channels are unique metabolic sensors formed by association of Kir6.2, an inwardly rectifying K+ channel, and the sulfonylurea receptor SUR, an ATP binding cassette protein. We identified an ATPase activity in immunoprecipitates of cardiac KATP channels and in purified fusion proteins containing nucleotide binding domains NBD1 and NBD2 of the cardiac SUR2A isoform. NBD2 hydrolyzed ATP with a twofold higher rate compared to NBD1. The ATPase required Mg2+ and was insensitive to ouabain, oligomycin, thapsigargin, or levamisole. K1348A and D1469N mutations in NBD2 reduced ATPase activity and produced channels with increased sensitivity to ATP. KATP channel openers, which bind to SUR, promoted ATPase activity in purified sarcolemma. At higher concentrations, openers reduced ATPase activity, possibly through stabilization of MgADP at the channel site. K1348A and D1469N mutations attenuated the effect of openers on KATP channel activity. Opener-induced channel activation was also inhibited by the creatine kinase/creatine phosphate system that removes ADP from the channel complex. Thus, the KATP channel complex functions not only as a K+ conductance, but also as an enzyme regulating nucleotide-dependent channel gating through an intrinsic ATPase activity of the SUR subunit. Modulation of the channel ATPase activity and/or scavenging the product of the ATPase reaction provide novel means to regulate cellular functions associated with KATP channel opening.
Phosphatidylinositol 4,5-bisphosphate (PIP2) activates KATP and other inward rectifier (Kir) channels. To determine residues important for PIP2 regulation, we have systematically mutated each positive charge in the COOH terminus of Kir6.2 to alanine. The effects of
these mutations on channel function were examined using 86Rb efflux assays on intact cells and inside-out patch-clamp methods. Both methods identify essentially the same basic residues
in two narrow regions (176–222 and 301–314) in the COOH terminus that are important for the maintenance of channel function
and interaction with PIP2. Only one residue (R201A) simultaneously affected ATP and PIP2 sensitivity, which is consistent with the notion that these ligands, while functionally competitive, are unlikely to bind
to identical sites. Strikingly, none of 13 basic residues in the terminal portion (residues 315–390) of the COOH terminus
affected channel function when neutralized. The data help to define the structural requirements for PIP2 sensitivity of KATP channels. Moreover, the regions and residues defined in this study parallel those uncovered in recent studies of PIP2 sensitivity in other inward rectifier channels, indicating a common structural basis for PIP2 regulation.
Interactions of sulfhydryl reagents with introduced cysteines in the pore-forming (Kir6.2) subunits of the K(ATP) channel were examined. 2-Aminoethyl methanethiosulfonate (MTSEA(+)) failed to modify Cd(2+)-insensitive control-Kir6.2 channels, but rapidly and irreversibly modified Kir6.2[L164C] (L164C) channels. Although a single Cd(2+) ion is coordinated by L164C, four MTSEA(+) "hits" can occur, each sequentially reducing the single-channel current. A dimeric fusion of control-Kir6.2 and L164C subunits generates Cd(2+)-insensitive channels, confirming that at least three cysteines are required for coordination, but MTSEA(+) modification of the dimer occurs in two hits. L164C channels were not modified by bromotrimethyl ammoniumbimane (qBBr(+)), even though qBBr(+) caused voltage-dependent block (as opposed to modification) that was comparable to that of MTSEA(+) or 3-(triethylammonium)propyl methanethiosulfonate (MTSPTrEA(+)), implying that qBBr(+) can also enter the inner cavity but does not modify L164C residues. The Kir channel pore structure was modeled by homology with the KcsA crystal structure. A stable conformation optimally places the four L164C side chains for coordination of a single Cd(2+) ion. Modification of these cysteines by up to four MTSEA(+) (or three MTSPTrEA(+), or two qBBr(+)) does not require widening of the cavity to accommodate the derivatives within it. However, like the KcsA crystal structure, the energy-minimized model shows a narrowing at the inner entrance, and in the Kir6.2 model this narrowing excludes all ions. To allow entry of ions as large as MTSPTrEA(+) or qBBr(+), the entrance must widen to >8 A, but this widening is readily accomplished by minimal M2 helix motion and side-chain rearrangement.
Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a genetic disorder characterized by excess secretion of insulin and hypoglycemia. In most patients, the disease is caused by mutations in sulfonylurea receptor-1 (SUR1), which, in association with Kir6.2, constitutes the functional ATP-sensitive potassium (K(ATP)) channel of the pancreatic beta-cell. Previous studies reported that coexpression of the PHHI mutant R1394H-SUR1 with Kir6.2 in COS cells produces no functional channels. To investigate if the loss of function could be due to impaired trafficking of mutant channels to the cell membrane, we have cotransfected wild-type and mutant SUR1 subunits with Kir6.2 into HEK293 cells and examined their cellular localization by immunofluorescent staining. Our results show that unlike the wild-type subunits, which showed fluorescence at the cell surface, the mutant subunits displayed fluorescence in punctate structures. Co-immunostaining with antibodies against organelle-specific marker proteins identified these structures as the trans-Golgi network. Limited localization in clathrin-positive, but transferrin receptor-negative vesicles was also observed. The post-endoplasmic reticulum localization suggests that the mutation does not impair the folding and assembly of the channels so as to cause its retention by the endoplasmic reticulum. Diazoxide, a K(ATP) channel opener drug that is used in the treatment of PHHI, restored the surface expression in a manner that could be prevented by the channel blocker glibenclamide. When expressed in Xenopus oocytes, R1394H-SUR1 formed functional channels with Kir6.2, indicating that the primary consequence of the mutation is impairment of trafficking rather than function. Thus, our data uncover a novel mechanism underlying the therapeutic action of diazoxide in the treatment of PHHI, i.e. its ability to recruit channels to the membrane. Furthermore, this is the first report to describe a trafficking disorder effecting retention of mutant proteins in the trans-Golgi network.
The ATP-sensitive potassium (K(ATP)) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 K(ATP) channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (tau(o)) and the short closed time (tauC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer tau(o), shorter tauC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the K(ATP) channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings.
Fundamental to the metabolic sensor function of ATP-sensitive K+ (KATP) channels is the sulfonylurea receptor. This ATP-binding cassette protein, which contains nucleotide binding domains (NBD1
and NBD2) with conserved Walker motifs, regulates the ATP sensitivity of the pore-forming Kir6.2 subunit. Although NBD2 hydrolyzes
ATP, a property essential in KATP channel gating, the role of NBD1, which has limited catalytic activity, if at all, remains less understood. Here, we provide
functional evidence that cooperative interaction, rather than the independent contribution of each NBD, is critical for KATP channel regulation. Gating of cardiac KATPchannels by distinct conformations in the NBD2 ATPase cycle, induced by γ-phosphate analogs, was disrupted by point mutation
not only of the Walker motif in NBD2 but also in NBD1. Cooling membrane patches to decelerate the intrinsic ATPase activity
counteracted ATP-induced KATP channel inhibition, an effect that mimicked stabilization of the MgADP-bound posthydrolytic state at NBD2 by the γ-phosphate
analog orthovanadate. Temperature-induced channel activation was abolished by mutations that either prevent stabilization
of MgADP at NBD2 or ATP at NBD1. These findings provide a paradigm of KATP channel gating based on integration of both NBDs into a functional unit within the multimeric channel complex.
KATP channels were reconstituted in COSm6 cells by coexpression of the sulfonylurea receptor SUR1 and the inward rectifier potassium channel Kir6.2. The role of the two nucleotide binding folds of SUR1 in regulation of KATP channel activity by nucleotides and diazoxide was investigated. Mutations in the linker region and the Walker B motif (Walker, J.E., M.J. Saraste, M.J. Runswick, and N.J. Gay. 1982. EMBO [Eur. Mol. Biol. Organ.] J. 1:945–951) of the second nucleotide binding fold, including G1479D, G1479R, G1485D, G1485R, Q1486H, and D1506A, all abolished stimulation by MgADP and diazoxide, with the exception of G1479R, which showed a small stimulatory response to diazoxide. Analogous mutations in the first nucleotide binding fold, including G827D, G827R, and Q834H, were still stimulated by diazoxide and MgADP, but with altered kinetics compared with the wild-type channel. None of the mutations altered the sensitivity of the channel to inhibition by ATP4−. We propose a model in which SUR1 sensitizes the KATP channel to ATP inhibition, and nucleotide hydrolysis at the nucleotide binding folds blocks this effect. MgADP and diazoxide are proposed to stabilize this desensitized state of the channel, and mutations at the nucleotide binding folds alter the response of channels to MgADP and diazoxide by altering nucleotide hydrolysis rates or the coupling of hydrolysis to channel activation.
KATP channels couple the intracellular energy state to membrane excitability and regulate a wide array of biologic activities. KATP channels contain a pore-forming inwardly rectifying potassium channel and a sulfonylurea receptor regulatory subunit (SUR1 or SUR2). To clarify the role of KATP channels in vascular smooth muscle, we studied Sur2 gene-targeted mice (Sur2–/–) and found significantly elevated resting blood pressures and sudden death. Using in vivo monitoring, we detected transient, repeated episodes of coronary artery vasospasm in Sur2–/– mice. Focal narrowings in the coronary arteries were present in Sur2–/– mice consistent with vascular spasm. We treated Sur2–/– mice with a calcium channel antagonist and successfully reduced vasospastic episodes.
Cardiovascular physiology in Sur2–/– mice. (a) Sur2–/– mice exhibit decreased survival. Survival of littermate control and heterozygous mice (Sur2+/+n = 15, 5 males and 10 females, and Sur2+/–, n = 41, 12 males and 29 females), homozygous mutant females (Sur2–/–, n = 18), and homozygous mutant males (Sur2–/–, n = 17) as a function of age. (b) Telemetric blood pressure recordings of Sur2–/– and normal littermate control mice. Representative blood pressure tracings from conscious, untethered control (Sur2+/+) and homozygous mutant (Sur2–/–) mice. (c) Systolic (white bars), diastolic (gray bars), and MAP (black bars) for littermate control and mutant mice (n = 6 and 4, respectively), recorded between days 2 and 4 after implantation. *P < 0.04, **P < 0.03, ***P < 0.02 versus normal littermate controls by two-tailed, unpaired Student t test. (d) Sur2–/– mice do not respond to KATP vasoactive agents. Representative MAP tracings from a littermate control mouse (dotted line) and a Sur2–/– mouse (solid line) during infusions of the agents indicated. Pinacidil was administered at 5 μg/kg/min and glibenclamide at 100 μg/kg/min. The interrupted line for glyburide infusion indicates the beginning of infusion for the normal control mouse (Sur2+/+), while the mutant mouse (Sur2–/–) infusion begins with the solid line.
Absent smooth muscle KATP channels. Electrophysiologic patch clamp recordings on isolated aortic smooth muscle cells from Sur2–/– and Sur2+/+ mice (Figure 2) demonstrated the absence of SUR2 KATP channels. The average cell capacitance was 8.03 ± 0.73 pF for Sur2+/+ mice and 7.74 ± 0.76 pF for Sur2–/– mice (P < 0.05). Immediately after membrane rupture, an increase of whole cell current was seen in Sur2+/+ cells, but was absent in Sur2–/– cells (Figure 2a). Extracellular application of 20 μM glibenclamide partially blocked this increase of whole cell current in Sur2+/+ smooth muscle cells, demonstrating that this current reflects SUR2 function (Figure 2a). At all voltages tested, typical voltage-dependent and glibenclamide-sensitive KATP current was seen in Sur2+/+ cells, but was absent in Sur2–/– cells. The current was outward under physiological potassium concentration (4out/140in) and reversed at –87 mV, near the theoretical value of –89 mV for K⁺ (Figure 2, b and c). The current density at a test pulse of –10 mV was 1.72 ± 0.64 pA/pF in Sur2+/+ cells and only 0.04 ± 0.04 pA/pF in Sur2–/– cells (P < 0.01). From these studies, we conclude that the glibenclamide-sensitive KATP current was indeed absent in the Sur2–/– vascular smooth muscle.
The ATP-inhibited potassium (K ATP) channel is assembled from four inward rectifier potassium (K ir 6.x) subunits and four sulfonylurea receptor (SURx) subunits. The inhibitory action of ATP is mediated by at least two distinct functional domains within the C-terminal cytoplasmic tail of K ir 6.2. The G334D mutation of K ir 6.2 virtually eliminates ATP-dependent gating with no effect on ligand-independent gating, suggesting a role in linkage of the site to the gate or in the ATP binding site, itself. The T171A mutation of K ir 6.2 strongly disrupts both ATP-dependent and ligand-independent gating, suggesting a role for T171 in the gating step. A neighboring mutation, I182Q, virtually eliminates ATP inhibition, but its effect on ligand-independent gating remained unknown. We have now characterized both the K i values for inhibition by ATP and the ligand-independent gating kinetics of 15 substitutions at position 182. All substitutions decreased ATP-dependent inhibition gating as measured by the K i , many profoundly so, yet had little or no effect on ligand-independent gating kinetics. Thus, substitutions at position 182 are unlikely to act by disrupting inhibition gate movement. Our results indicate an indispensable role for I182 in a step of the ATP binding mechanism, the linkage mechanism coupling the ATP binding site to the inhibition gate, or both.
Inwardly rectifying, ATP-sensitive K+ channels (K(ATP)) couple metabolism to either cell excitability (Kir6.x) or potassium secretion (Kir1.1). Phosphatidylinositol phospholipids, like PI(4,5)P2, antagonize nucleotide inhibition of K(ATP) channels enhancing the coupling of metabolic events to cell electrical or transport activity. The mechanism by which phospholipids relieve ATP block is unclear. We have shown that maltose-binding fusion proteins (MBP) containing the COOH termini of K(ATP) channels (Kir1.1, Kir6.1, and Kir6.2) form functional tetramers that directly bind at least two ATP molecules with negative cooperativity. Here we show that purified phosphatidylinositol phospholipids compete for 2,4,6,-trinitrophenyl (TNP)-ATP binding to the COOH termini of K(ATP) channels with EC50 values for PIP2 between 6-8 microM. The phospholipid potency profile was PIP3 > PIP2 = PIP > PI, suggesting that net phospholipid charge was important. A role for head group charge was supported by polycations (neomycin, spermine, and polylysine) reversing the effect of PIP2 on TNP-ATP binding to the Kir1.1 channel COOH terminal fusion protein. In contrast, the water-soluble charged hydrolytic product of PIP2, inositol(1,4,5)P3 (IP3), had no effect on TNP-ATP binding, suggesting that the acyl chain of PIP2 was also necessary for its effect on TNP-ATP binding. Indeed, neutral and charged lipids had weak, but significant, effects on TNP-ATP binding. Whereas microM concentrations of PIP2 could compete with TNP-ATP, we found that mM concentrations of MgATP were required to compete with PIP2 for binding to these K(ATP) channel COOH termini. Thus the COOH termini of K(ATP) channels form a nucleotide- and phospholipid-modulated channel gate on which ATP and phospholipids compete for binding.
Cation channel gating may occur either at or below the inner vestibule entrance or at the selectivity filter. To differentiate these possibilities in inward rectifier (Kir) channels, we examined cysteine accessibility in the ATP-gated Kir6.2 channel. MTSEA and MTSET both block channels and modify M2 cysteines with identical voltage dependence. If entry is restricted to open channels, modification rates will slow in ATP-closed channels, but because the reagent can be trapped in the pore following brief openings, this may not be apparent until open probability is extremely low (<0.01). When these conditions are met, modification does slow significantly, indicating gated access and highlighting an important caveat for interpretation of MTS-accessibility measurements: reagent "trapping" in nominally "closed" channels may obscure gated access.
Familial hyperinsulinism and pancreatic β-cell ATP-sensitive potassium channels. Familial hyperinsulinism, also known as persistent hyperinsulinemic hypoglycemia of infancy (PHHI), is a genetic disease characterized by mild to severe hypoglycemia in the presence of inappropriately high levels of insulin. The recessive form is caused by mutations in the adenosine 5′-triphosphate (ATP)-sensitive K+ channel (KATP channel) present in the plasma membrane of pancreatic β-cells. This channel is formed by two subunits, the high-affinity sulfonylurea receptor, SUR1, and KIR6.2, a member of the inwardly rectifying family of K+ channels. KATP channels regulate insulin secretion by linking membrane excitability with glucose metabolism. Approximately 50 mutations, in both channel subunits, that abolish or alter the regulation of β-cell KATP channels have been identified in patients with the recessive form of PHHI.Keywords: insulin; potassium channel; sulfonylurea receptor; pancreas
We have investigated protein interactions involved in pancreatic β-cell ATP-sensitive potassium channel assembly. These channels,
which are of key importance for control of insulin release, are a hetero-oligomeric complex of pore-forming Kir6.2 subunits
and sulfonylurea receptor (SUR1) subunits with two nucleotide-binding domains (NBD1 and NBD2). We divided SUR1 into two halves
at Pro-1042. Expression of either the individual N- or C-terminal domain in a baculovirus expression system did not lead to
glibenclamide binding activity, although studies with green fluorescent protein fusion proteins showed that both half-molecules
were inserted into the plasma membrane. However, significant glibenclamide binding activity was observed when the half-molecules
were co-expressed (even when NBD2 was deleted from the C-terminal half-molecule). Simultaneous expression of Kir6.2 resulted
in enhanced glibenclamide binding activity. We conclude that the glibenclamide-binding site includes amino acid residues from
both halves of the molecule, that there is strong interaction between different regions of SUR1, that NBD2 is not essential
for glibenclamide binding, and that interactions between Kir6.2 and SUR1 participate in ATP-sensitive potassium channel assembly.
Investigation of NBD1-green fluorescent protein fusion protein distribution inside insect cells expressing C-terminal halves
of SUR1 demonstrated strong interaction between NBD1 and NBD2. We also expressed and purified NBD1 from Escherichia coli. Purified NBD1 was found to exist as a tetramer indicating strong homomeric attractions and a possible role for NBD1 in SUR1
assembly.
We have cloned an isoform of the sulfonylurea receptor (SUR), designated SUR2. Coexpression of SUR2 and the inward rectifier K+ channel subunit Kir6.2 in COS1 cells reconstitutes the properties of K(ATP) channels described in cardiac and skeletal muscle. The SUR2/Kir6.2 channel is less sensitive than the SUR/Kir6.2 channel (the pancreatic beta cell KATP channel) to both ATP and the sulfonylurea glibenclamide and is activated by the cardiac K(ATP) channel openers, cromakalim and pinacidil, but not by diazoxide. In addition, SUR2 binds glibenclamide with lower affinity. The present study shows that the ATP sensitivity and pharmacological properties of K(ATP) channels are determined by a family of structurally related but functionally distinct sulfonylurea receptors.
Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple the cellular metabolic state to electrical activity and are a critical link between blood glucose concentration and pancreatic insulin secretion. A mutation in the second nucleotide-binding fold (NBF2) of the sulfonylurea receptor (SUR) of an individual diagnosed with persistent hyperinsulinemic hypoglycemia of infancy generated KATP channels that could be opened by diazoxide but not in response to metabolic inhibition. The hamster SUR, containing the analogous mutation, had normal ATP sensitivity, but unlike wild-type channels, inhibition by ATP was not antagonized by adenosine diphosphate (ADP). Additional mutations in NBF2 resulted in the same phenotype, whereas an equivalent mutation in NBF1 showed normal sensitivity to MgADP. Thus, by binding to SUR NBF2 and antagonizing ATP inhibition of KATP++ channels, intracellular MgADP may regulate insulin secretion.
1. We analysed the K+ channel composed of the sulphonylurea receptor 2B (SUR2B) and an inwardly rectifying K+ channel subunit Kir6.1 coexpressed in a mammalian cell line, HEK293T, with the patch clamp technique. 2. In the cell-attached configuration, K+ channel openers (pinacidil and nicorandil) activated approximately 33 pS K+ channels (approximately 145 mM external K+), which were inhibited by the sulphonylurea glibenclamide. 3. Although SUR2B forms an ATP-sensitive K+ channel with Kir6.2, whose amino acid sequence is approximately 70% homologous with that of Kir6.1, the K+ channel composed of SUR2B and Kir6.1 surprisingly did not spontaneously open on patch excision in the absence of intracellular ATP. 4. In inside-out patches, uridine diphosphate and guanosine diphosphate induced channel activity, which was inhibited by glibenclamide but not ATP. Intracellular ATP on its own activated the channels. K+ channel openers and intracellular nucleotides synergistically activated the channel. 5. Therefore, the K+ channel composed of SUR2B and Kir6.1 is not a classical ATP-sensitive K+ channel but closely resembles the nucleotide diphosphate-dependent K+ channel in vascular smooth muscle cells.
ATP-sensitive potassium channels (K(ATP) channels) are heteromultimers of sulfonylurea receptors (SUR) and inwardly rectifying potassium channel subunits (K(IR)6.x) with a (SUR-K(IR)6.x)4 stoichiometry. Association is specific for K(IR)6.x and affects receptor glycosylation and cophotolabeling of K(IR)6.x by 125I-azidoglibenclamide. Association produces digitonin stable complexes with an estimated mass of 950 kDa. These complexes can be purified by lectin chromatography or by using Ni2(+)-agarose and a his-tagged SUR1. Expression of SUR1 approximately (K(IR)6.2)i fusion constructs shows that a 1:1 SUR1:K(IR)6.2 stoichiometry is both necessary and sufficient for assembly of active K(ATP) channels. Coexpression of a mixture of strongly and weakly rectifying triple fusion proteins, rescued by SUR1, produced the three channel types expected of a tetrameric pore.
Adenosine triphosphate (ATP)–sensitive potassium (KATP) channels couple cell metabolism to electrical activity. Phosphatidylinositol phosphates (PIPs) profoundly antagonized ATP
inhibition of KATP channels when applied to inside-out membrane patches. It is proposed that membrane-incorporated PIPs can bind to positive
charges in the cytoplasmic region of the channel's Kir6.2 subunit, stabilizing the open state of the channel and antagonizing the inhibitory effect of ATP. The tremendous effect
of PIPs on ATP sensitivity suggests that in vivo alterations of membrane PIP levels will have substantial effects on KATP channel activity and hence on the gain of metabolism-excitation coupling.
ATP-sensitive potassium ("KATP") channels are rapidly inhibited by intracellular ATP. This inhibition plays a crucial role in the coupling of electrical activity to energy metabolism in a variety of cells. The KATP channel is formed from four each of a sulfonylurea receptor (SUR) regulatory subunit and an inwardly rectifying potassium (Kir6.2) pore-forming subunit. We used systematic chimeric and point mutagenesis, combined with patch-clamp recording, to investigate the molecular basis of ATP-dependent inhibition gating of mouse pancreatic beta cell KATP channels expressed in Xenopus oocytes. We identified distinct functional domains of the presumed cytoplasmic C-terminal segment of the Kir6.2 subunit that play an important role in this inhibition. Our results suggest that one domain is associated with inhibitory ATP binding and another with gate closure.
The ATP-sensitive potassium (KATP) channels in pancreatic beta cells are critical in the regulation of glucose-induced insulin secretion. Although electrophysiological studies provide clues to the complex control of KATP channels by ATP, MgADP, and pharmacological agents, the molecular mechanism of KATP-channel regulation remains unclear. The KATP channel is a heterooligomeric complex of SUR1 subunits of the ATP-binding-cassette superfamily with two nucleotide-binding folds (NBF1 and NBF2) and the pore-forming Kir6.2 subunits. Here, we report that MgATP and MgADP, but not the Mg salt of gamma-thio-ATP, stabilize the binding of prebound 8-azido-[alpha-32P]ATP to SUR1. Mutation in the Walker A and B motifs of NBF2 of SUR1 abolished this stabilizing effect of MgADP. These results suggest that SUR1 binds 8-azido-ATP strongly at NBF1 and that MgADP, either by direct binding to NBF2 or by hydrolysis of bound MgATP at NBF2, stabilizes prebound 8-azido-ATP binding at NBF1. The sulfonylurea glibenclamide caused release of prebound 8-azido-[alpha-32P]ATP from SUR1 in the presence of MgADP or MgATP in a concentration-dependent manner. This direct biochemical evidence of cooperative interaction in nucleotide binding of the two NBFs of SUR1 suggests that glibenclamide both blocks this cooperative binding of ATP and MgADP and, in cooperation with the MgADP bound at NBF2, causes ATP to be released from NBF1.
Sur1 knockout mouse beta-cells lack K(ATP) channels and show spontaneous Ca(2+) action potentials equivalent to those seen in patients with persistent hyperinsulinemic hypoglycemia of infancy, but the mice are normoglycemic unless stressed. Sur1(-/-) islets lack first phase insulin secretion and exhibit an attenuated glucose-stimulated second phase secretion. Loss of the first phase leads to mild glucose intolerance, whereas reduced insulin output is consistent with observed neonatal hyperglycemia. Loss of K(ATP) channels impairs the rate of return to a basal secretory level after a fall in glucose concentration. This leads to increased hypoglycemia upon fasting and contributes to a very early, transient neonatal hypoglycemia. Whereas persistent hyperinsulinemic hypoglycemia of infancy underscores the importance of the K(ATP)-dependent ionic pathway in control of insulin release, the Sur1(-/-) animals provide a novel model for study of K(ATP)-independent pathways that regulate insulin secretion.
K(ATP) channels can be formed from Kir6.2 subunits with or without SUR1. The open-state stability of K(ATP) channels can be increased or reduced by mutations throughout the Kir6.2 subunit, and is increased by application of PIP(2) to the cytoplasmic membrane. Increase of open-state stability is manifested as an increase in the channel open probability in the absence of ATP (Po(zero)) and a correlated decrease in sensitivity to inhibition by ATP. Single channel lifetime analyses were performed on wild-type and I154C mutant channels expressed with, and without, SUR1. Channel kinetics include a single, invariant, open duration; an invariant, brief, closed duration; and longer closed events consisting of a "mixture of exponentials," which are prolonged in ATP and shortened after PIP(2) treatment. The steady-state and kinetic data cannot be accounted for by assuming that ATP binds to the channel and causes a gate to close. Rather, we show that they can be explained by models that assume the following regarding the gating behavior: 1) the channel undergoes ATP-insensitive transitions from the open state to a short closed state (C(f)) and to a longer-lived closed state (C(0)); 2) the C(0) state is destabilized in the presence of SUR1; and 3) ATP can access this C(0) state, stabilizing it and thereby inhibiting macroscopic currents. The effect of PIP(2) and mutations that stabilize the open state is then to shift the equilibrium of the "critical transition" from the open state to the ATP-accessible C(0) state toward the O state, reducing accessibility of the C(0) state, and hence reducing ATP sensitivity.
To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins.
The ATP-inhibited potassium (K(ATP)) channel is assembled from four inward rectifier potassium (K(ir)6.x) subunits and four sulfonylurea receptor (SURx) subunits. The inhibitory action of ATP is mediated by at least two distinct functional domains within the C-terminal cytoplasmic tail of K(ir)6.2. The G334D mutation of K(ir)6.2 virtually eliminates ATP-dependent gating with no effect on ligand-independent gating, suggesting a role in linkage of the site to the gate or in the ATP binding site, itself. The T171A mutation of K(ir)6.2 strongly disrupts both ATP-dependent and ligand-independent gating, suggesting a role for T171 in the gating step. A neighboring mutation, I182Q, virtually eliminates ATP inhibition, but its effect on ligand-independent gating remained unknown. We have now characterized both the K(i) values for inhibition by ATP and the ligand-independent gating kinetics of 15 substitutions at position 182. All substitutions decreased ATP-dependent inhibition gating as measured by the K(i), many profoundly so, yet had little or no effect on ligand-independent gating kinetics. Thus, substitutions at position 182 are unlikely to act by disrupting inhibition gate movement. Our results indicate an indispensable role for I182 in a step of the ATP binding mechanism, the linkage mechanism coupling the ATP binding site to the inhibition gate, or both.
In pancreatic beta-cells, cytosolic [ATP(4-)] critically controls insulin secretion via inhibition of ATP-sensitive potassium (KATP) channels. These channels are heteromultimers composed with a 4:4 stoichiometry of an inwardly rectifying K+ channel subunit (Kir6.2) plus a regulatory sulfonylurea receptor. To elucidate stoichiometry of ATP(4-) action, we analyzed ATP(4-) sensitivity of channels coassembled from wild-type Kir6.2 and a loss of ATP(4-) sensitivity mutant (G334D). Concentration-inhibition curves for cDNA ratios of 1:1 or 1:10 resembled those for channel block resulting from interaction with 1 of 4 sites, whereas models for inhibition requiring occupation of 2, 3, or 4 sites were incongruous. Random assembly of wild-type Kir6.2 with the G334D mutant was confirmed by controls, which assessed the effect of an additional mutation that induced strong rectification (N160D). We conclude 4 identical noncooperative ATP(4-) sites to be grouped within 1 KATP channel complex, with occupation of 1 site being sufficient to induce channel closure. This architecture might facilitate coupling of [ATP(4-)] to insulin secretion and may protect against diabetic dysregulation resulting from heterozygous mutations in Kir6.2.
-ATP-sensitive potassium (K(ATP)) channels were discovered in ventricular cells, but their roles in the heart remain mysterious. K(ATP) channels have also been found in numerous other tissues, including vascular smooth muscle. Two pore-forming subunits, Kir6.1 and Kir6.2, contribute to the diversity of K(ATP) channels. To determine which subunits are operative in the cardiovascular system and their functional roles, we characterized the effects of pharmacological K(+) channel openers (KCOs, ie, pinacidil, P-1075, and diazoxide) in Kir6.2-deficient mice. Sarcolemmal K(ATP) channels could be recorded electrophysiologically in ventricular cells from Kir6.2(+/+) (wild-type [WT]) but not from Kir6.2(-/-) (knockout [KO]) mice. In WT ventricular cells, pinacidil induced an outward current and action potential shortening, effects that were blocked by glibenclamide, a K(ATP) channel blocker. KO ventricular cells exhibited no response to KCOs, but gene transfer of Kir6.2 into neonatal ventricular cells rescued the electrophysiological response to P-1075. In terms of contractile function, pinacidil decreased force generation in WT but not KO hearts. Pinacidil and diazoxide produced concentration-dependent relaxation in both WT and KO aortas precontracted with norepinephrine. In addition, pinacidil induced a glibenclamide-sensitive current of similar magnitude in WT and KO aortic smooth muscle cells and comparable levels of hypotension in anesthetized WT and KO mice. In both WT and KO aortas, only Kir6.1 mRNA was expressed. These findings indicate that the Kir6.2 subunit mediates the depression of cardiac excitability and contractility induced by KCOs; in contrast, Kir6.2 plays no discernible role in the arterial tree.
ATP-sensitive potassium (K(ATP)) channels are bifunctional multimers assembled by an ion conductor and a sulfonylurea receptor (SUR) ATPase. Sensitive to ATP/ADP, K(ATP) channels are vital metabolic sensors. However, channel regulation by competitive ATP/ADP binding would require oscillations in intracellular nucleotides incompatible with cell survival. We found that channel behavior is determined by the ATPase-driven engagement of SUR into discrete conformations. Capture of the SUR catalytic cycle in prehydrolytic states facilitated pore closure, while recruitment of posthydrolytic intermediates translated in pore opening. In the cell, channel openers stabilized posthydrolytic states promoting K(ATP) channel activation. Nucleotide exchange between intrinsic ATPase and ATP/ADP-scavenging systems defined the lifetimes of specific SUR conformations gating K(ATP) channels. Signal transduction through the catalytic module provides a paradigm for channel/enzyme operation and integrates membrane excitability with metabolic cascades.
ATP-sensitive potassium channels (K(ATP)) are involved in a diverse array of physiologic functions including protection of tissue against ischemic insult, regulation of vascular tone, and modulation of insulin secretion. To improve our understanding of the role of K(ATP) in these processes, we used a gene-targeting strategy to generate mice with a disruption in the muscle-specific K(ATP) regulatory subunit, SUR2. Insertional mutagenesis of the Sur2 locus generated homozygous null (Sur2(-/-)) mice and heterozygote (Sur2(+/-)) mice that are viable and phenotypically similar to their wild-type littermates to 6 weeks of age despite, respectively, half or no SUR2 mRNA expression or channel activity in skeletal muscle or heart. Sur2(-/-) animals had lower fasting and fed serum glucose, exhibited improved glucose tolerance during a glucose tolerance test, and demonstrated a more rapid and severe hypoglycemia after administration of insulin. Enhanced glucose use was also observed during in vivo hyperinsulinemic euglycemic clamp studies during which Sur2(-/-) mice required a greater glucose infusion rate to maintain a target blood glucose level. Enhanced insulin action was intrinsic to the skeletal muscle, as in vitro insulin-stimulated glucose transport was 1.5-fold greater in Sur2(-/-) muscle than in wild type. Thus, membrane excitability and K(ATP) activity, to our knowledge, seem to be new components of the insulin-stimulated glucose uptake mechanism, suggesting possible future therapeutic approaches for individuals suffering from diabetes mellitus.