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Pore-forming subunits of K-ATP channels, Kir6.1 and Kir6.2, display prominent differences in regional and cellular distribution in the rat brain
Abstract
K-ATP channels consist of two structurally different subunits: a pore-forming subunit of the Kir6.0-family (Kir6.1 or Kir6.2) and a sulfonylurea receptor (SUR1, SUR2, SUR2A, SUR2B) with regulatory activity. The functional diversity of K-ATP channels in brain is broad and of fundamental importance for neuronal activity. Here, using immunocytochemistry with monospecific antibodies against the Kir6.1 and Kir6.2 subunits, we analyze the regional and cellular distribution of both proteins in the adult rat brain. We find Kir6.2 to be widely expressed in all brain regions, suggesting that the Kir6.2 subunit forms the pore of the K-ATP channels in most neurons, presumably protecting the cells during cellular stress conditions such as hypoglycemia or ischemia. Especially in hypothalamic nuclei, in particular the ventromedial and arcuate nucleus, neurons display Kir6.2 immunoreactivity only, suggesting that Kir6.2 is the pore-forming subunit of the K-ATP channels in the glucose-responsive neurons of the hypothalamus. In contrast, Kir6.1-like immunolabeling is restricted to astrocytes (Thomzig et al. [2001] Mol Cell Neurosci 18:671-690) in most areas of the rat brain and very weak or absent in neurons. Only in distinct nuclei or neuronal subpopulations is a moderate or even strong Kir6.1 staining detected. The biological functions of these K-ATP channels still need to be elucidated.
... Research on KATP channels mostly focus on the peripheral tissues, whereas the precise physiological roles of KATP channels in the central nervous system (CNS) are poorly elucidated. Kir6.2, a KATP channel pore-forming subunit, is mainly expressed in neuronal populations in the brain [2]. Several studies have been conducted on the involvement of astrocytic kir6.2 in regulating brain functions [3,4], raising concerns about the cell-specific distribution of kir6.2 in the brain. ...
... Kir6.2 is widely distributed in the CNS but mainly resides in neurons [2,31,32]. As the most abundant cells in the brain, astrocytes only express kir6.2 ...
... Our previous study has found that aberrantly-expressed kir6.2 in A1 astrocytes is a neurodegeneration propagator under inflammatory stress [21]. We confirm in the current study that kir6.2 is expressed specifically in neurons under physiological conditions, both in vivo and in vitro, which is consistent with former report [2]. ...
Kir6.2, a pore-forming subunit of the ATP-sensitive potassium (KATP) channels, regulates the functions of metabolically active tissues and acts as an ideal therapeutic target for multiple diseases. Previous studies have been conducted on peripheral kir6.2, but its precise physiological roles in the central nervous system (CNS) have rarely been revealed. In the current study, we evaluated the neurophenotypes and neuroethology of kir6.2 knockout (kir6.2−/−) mice. We demonstrated the beneficial effects of kir6.2 on maintaining the morphology of mesencephalic neurons and controlling the motor coordination of mice. The mechanisms underlying the abnormal neurological features of kir6.2 deficiency were analyzed by RNA sequencing (RNA-seq). Pm20d1, a gene encoding PM20D1 secretase that promotes the generation of endogenous mitochondria uncouplers in vivo, was dramatically upregulated in the midbrain of kir6.2−/− mice. Further investigations verified that PM20D1-induced increase of N-acyl amino acids (N-AAAs) from circulating fatty acids and amino acids promoted mitochondrial impairments and cut down the ATP generation, which mediated the morphological defects of the mesencephalic neurons and thus led to the behavioral impairments of kir6.2 knockout mice. This study is the first evidence to demonstrate the roles of kir6.2 in the morphological maintenance of neurite and motor coordination control of mice, which extends our understanding of kir6.2/KATP channels in regulating the neurophysiological function.
... these subunits in each region contribute to the diversity of K ATP channels [23][24][25][26]. Kir6.2, one of the pore-forming subunits of K ATP channels, is widely distributed throughout rat brain neurons and glial cells [27,28]. Especially, Kir6.2 is highly expressed in regions containing monoamine neurons such as the substantia nigra (SN), ventral tegmental area (VTA), striatum, locus coeruleus (LC), and dorsal raphe nucleus (DRN). ...
... Especially, Kir6.2 is highly expressed in regions containing monoamine neurons such as the substantia nigra (SN), ventral tegmental area (VTA), striatum, locus coeruleus (LC), and dorsal raphe nucleus (DRN). Moreover, Kir6.2 is also observed in the synaptic neuropil in the hippocampus, amygdala, basal ganglia and cerebral cortex, indicating that Kir6.2 is localized in dendrites and axons of monoamine neurons [28]. Therefore, it is inferred that Kir6.2 may be involved in monoamine neurotransmission. ...
... It has been widely accepted that brain monoamine neurons play a critical role in several emotional behaviors. As mentioned above, Kir6.2 is highly expressed in brain regions containing monoamine neurons [28]. Although it is likely that brain Kir6.2 affects emotional behaviors and plays a role in psychiatric disorders associated with monoamine neurotransmission, the mechanisms that underlie the impact of Kir6.2 on emotional behavior are not yet understood. ...
... Expression of these components varies between different tissues. In pancreatic β-cells, predominantly Kir6.2 and SUR1 form the channel, whereas Kir6.1 and Kir6.2 and SUR1 and SUR2 are all expressed in the brain (Sakura et al., 1995;Davis-Taber et al., 2000;Li et al., 2003;Thomzig et al., 2005). Kir6.2 is predominantly expressed in neurons, whereas Kir6.1 is found only in a small population of neurons, and is primarily present on astrocytes (Thomzig et al., 2001(Thomzig et al., , 2005. ...
... In pancreatic β-cells, predominantly Kir6.2 and SUR1 form the channel, whereas Kir6.1 and Kir6.2 and SUR1 and SUR2 are all expressed in the brain (Sakura et al., 1995;Davis-Taber et al., 2000;Li et al., 2003;Thomzig et al., 2005). Kir6.2 is predominantly expressed in neurons, whereas Kir6.1 is found only in a small population of neurons, and is primarily present on astrocytes (Thomzig et al., 2001(Thomzig et al., , 2005. Specifically Kir6.2 has shown to be important for glucose-sensing in the VMH, as VMH neurons from mice lacking Kir6.2 no longer respond to increases in glucose (Miki et al., 2001). ...
... Specifically Kir6.2 has shown to be important for glucose-sensing in the VMH, as VMH neurons from mice lacking Kir6.2 no longer respond to increases in glucose (Miki et al., 2001). Both Kir6.2 and Kir6.1 are expressed in the NAc, whereas SUR1 appears not to be expressed (Karschin et al., 1997;Thomzig et al., 2005). No studies have currently reported on SUR2 expression in the NAc. ...
Glucose-sensing neurons are neurons that alter their activity in response to changes in extracellular glucose. These neurons, which are an important mechanism the brain uses to monitor changes in glycaemia, are present in the hypothalamus, where they have been thoroughly investigated. Recently, glucose-sensing neurons have also been identified in brain nuclei which are part of the reward system. However, little is known about the molecular mechanisms by which they function, and their role in the reward system. We therefore aim to provide an overview of molecular mechanisms that have been studied in the hypothalamic glucose-sensing neurons, and investigate which of these transporters, enzymes and channels are present in the reward system. Furthermore, we speculate about the role of glucose-sensing neurons in the reward system.
... ATP-sensitive potassium (K-ATP) channels are the unique potassium channels coupling cell metabolism to cell membrane potential [19]. They are composed of pore-forming Kir6.x (6.1 or 6.2) subunits and sulfonylurea receptor subunits, regulated by intracellular ATP and ADP concentrations. ...
... The most important finding presented here is that Kir6.1/K-ATP channel in astrocytes plays a crucial role in the pathogenesis of depression. As a metabolic gatekeeper, the K-ATP channel is extensively distributed in brain neurons and glial cells, specifically located at the membranes of cell and mitochondrial organelle [19,22]. Our previous studies showed that K-ATP channel opener could improve depressive behavior via inhibition of inflammation in mouse hypothalamus [32], while Kir6.2 knockout aggravated depressive behavior by promoting CA3 neuron death [21]. ...
Rationale: Astrocyte dysfunction is one of the important pathological mechanisms of depression. Stress contributes to the onset of depression. As metabolic stress sensor, Kir6.1-contaning K-ATP channel (Kir6.1/K-ATP) is prominently expressed in astrocytes. However, the involvement of Kir6.1/K-ATP channel in depression remains obscure.
Methods: Astrocyte-specific Kir6.1 knockout mice were used to prepare two mouse models of depression to explore the role of astrocytic Kir6.1/K-ATP channel in depression. Primary astrocytes were cultured to reveal the underlying mechanism for Kir6.1-regulated astrocytic pyroptosis.
Results: We identified that chronic stress reduced the astrocytic Kir6.1 expression in hippocampus of mice. We further observed astrocyte-specific knockout of Kir6.1 induced depressive-like behaviors in mice. Moreover, we found that astrocytic Kir6.1 deletion increased NLRP3-mediated astrocytic pyroptosis in response to stress. Mechanistically, Kir6.1 associated with NLRP3, and this interaction prevented the assembly and activation of NLRP3 inflammasome, thereby inhibition of astrocytic pyroptosis. More importantly, VX-765, an effective and selective inhibitor for NLRP3 inflammasome, could reverse the astrocytic pyroptosis and rescue the deterioration of behaviors in astrocytic Kir6.1 knockout mice.
Conclusions: Our findings illustrate that Kir6.1/K-ATP channel in astrocytes is an essential negative modulator of astrocytic pyroptosis and plays a crucial role in depression and suggest that astrocytic Kir6.1/K-ATP channel may be a promising therapeutic target for depression.
... or SUR2-Kir6.2 in hippocampal pyramidal cells and SUR1-Kir6.2 in hippocampal interneurons [63]. The Kir6.2 channel is predominantly expressed in neurons and plays an essential role in glucose sensing and neuronal excitability in the metabolic cycle [59]. In contrast, the Kir6.1 channel is mainly expressed in glial cells (astrocytes or microglia) and plays a critical role in adult neurogenesis [42,57]. ...
... We also confirmed that repeated administration of propolis for 8 weeks further improved the rescue of cognitive deficits by memantine in APP-KI mice and restored injured hippocampal LTP in APP-KI mice ( Fig. 4 and Fig. 5). The Kir6.2 channel is enriched and abundantly expressed in hippocampal neurons [57,59]. We demonstrated that Kir6.2 was co-localized with postsynaptic density neuron 95, a dendritic spine marker in hippocampal neurons [41]. ...
Propolis is a complex resinous substance that is relevant as a therapeutic target for Alzheimer’s disease (AD) and other neurodegenerative diseases. In this study, we confirmed that propolis (Brazilian green propolis) further enhances the rescue of cognitive deficits by the novel AD drug memantine in APP-KI mice. In memory-related behavior tasks, administration of a single dose of propolis at 1–100 mg/kg p.o. significantly enhanced the rescue of cognitive deficits by memantine at 1 mg/kg p.o. in APP-KI mice. In in vitro studies, propolis significantly increased intracellular Ca²⁺ concentration and calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation in Kir6.2-overexpressed N2A cells treated with memantine. Propolis also significantly increased adenosine 5′-triphosphate (ATP) contents and CaMKII autophosphorylation, which was impaired in Aβ-treated Kir6.2-overexpressed N2A cells. Similarly, repeated administration of propolis at 100 mg/kg p.o. for 8 weeks further enhanced the rescue of cognitive deficits by memantine in APP-KI mice. Consistent with the rescued cognitive deficits in APP-KI mice, repeated administration of propolis markedly ameliorated memantine-dependent rescue of injured long-term potentiation (LTP) in APP-KI mice, concomitant with increased CaMKII autophosphorylation and calcium/calmodulin-dependent protein kinase IV (CaMKIV) phosphorylation in the hippocampal CA1 region. Furthermore, repeated administration of both memantine and propolis significantly restored the decreased ATP contents in the CA1 region of APP-KI mice. Finally, we confirmed that repeated administration of memantine at 1 mg/kg p.o. and propolis at 100 mg/kg p.o. for 8 weeks failed to restore the cognitive deficits in Kir6.2−/− mice. Our study demonstrates that propolis increases ATP contents and promotes the amelioration of cognitive deficits rescued by memantine via Kir6.2 channel inhibition in the CA1 region.
... ABBC8) subunits (Aguilar-Bryan et al., 1992;Aguilar-Bryan et al., 1995;Inagaki et al., 1995b;Martin et al., 2017;Shyng and Nichols, 1997). Kir6.2/SUR1 channels are also expressed in various brain regions ( Karschin et al., 1997;Thomzig et al., 2005), where their physiological roles are not as well understood. ...
... Kir6.2 and SUR1 mRNA expression appears to be widespread and overlapping in the rodent brain, with higher levels of expression in the hippocampus, neocortex, olfactory bulb, cerebellum, midbrain, and brainstem (Karschin et al., 1997). In contrast, Kir6.1 appears to be weakly expressed in the brain (Thomzig et al., 2005). The SUR2 splice variants SUR2A and SUR2B exhibit distinct expression patterns, with SUR2A mRNA expression highest in neurons and SUR2B mRNA expression highest in certain neuronal populations, astrocytes, and oligodendrocytes (Zhou et al., 2012). ...
Glucose-stimulated insulin secretion from pancreatic beta-cells is controlled by ATP-regulated potassium (KATP) channels comprised of Kir6.2 and SUR1 subunits. The KATP channel opener diazoxide is FDA approved for treating hyperinsulinism and hypoglycemia but suffers from off-target effects on vascular KATP channels and other ion channels. The development of more specific openers would provide critically needed tool compounds for probing the therapeutic potential of Kir6.2/SUR1 activation. Here, we characterize a novel-scaffold activator of Kir6.2/SUR1 that our group recently discovered in a high-throughput screen. Optimization efforts with medicinal chemistry identified key structural elements that are essential for VU0071063-dependent opening Kir6.2/SUR1. VU0071063 has no effects on heterologously expressed Kir6.1/SUR2B channels or ductus arteriole tone, indicating it does not open vascular KATP channels. VU0071063 induces hyperpolarization of beta-cell membrane potential and inhibits insulin secretion with greater potency than diazoxide. VU0071063 exhibits metabolic and pharmacokinetic properties that are favorable for an in vivo probe and is brain penetrant. Administration of VU0071063 inhibits glucose-stimulated insulin secretion and glucose lowering in mice. Taken together, these studies indicate that VU0071063 is a more potent and specific opener of Kir6.2/SUR1 than diazoxide and should be useful as an in vitro and in vivo tool compound for investigating the therapeutic potential of Kir6.2/SUR1 expressed in the pancreas and brain. SIGNIFICANCE STATEMENT: N/A.
... In pancreatic beta cells Kir6.2/SUR1 are the major subunits expressed, in cardiac myocytes Kir6.2/ SUR2A subunits, in smooth muscles SUR2B, in adipose tissue Kir6.1/SUR2B, and in the brain neurons mostly Kir6.2/SUR1 while in astrocytes only Kir6.1/SUR1 and 2 [71][72][73][74][75] . K ATP channels were first described in isolated ventricular myocytes of the guinea pig [76] , and have been studied for their role in diseases from diabetes and hyperinsulinemia to cardiac arrhythmias and cardiovascular disease. ...
... Neuronal K ATP channels K ATP pore forming subunits Kir6.1 and Kir6.2, as well as their regulatory subunits SUR1 and 2B, are expressed at high levels in the brain (cortical and hippocampal areas) [73,74,81,94,95] . Neuronal K ATP channels play an important role in regulating neuronal excitability and spontaneous firing in neurons including: cholinergic basal forebrain neurons, expiratory neurons, entorhinal layer 3 cortical neurons, substantia nigra neurons, thalamocortical neurons [96][97][98][99][100] . ...
ATP-sensitive potassium (KATP) channels are ubiquitously expressed on the plasma membrane of cells in multiple organs, including the heart, pancreas and brain. KATP channels play important roles in controlling and regulating cellular functions in response to metabolic state, which are inhibited by ATP and activated by Mg-ADP, allowing the cell to couple cellular metabolic state (ATP/ADP ratio) to electrical activity of the cell membrane. KATP channels mediate insulin secretion in pancreatic islet beta cells, and controlling vascular tone. Under pathophysiological conditions, KATP channels play cytoprotective role in cardiac myocytes and neurons during ischemia and/or hypoxia. KATP channel is a hetero-octameric complex, consisting of four pore-forming Kir6.x and four regulatory sulfonylurea receptor SURx subunits. These subunits are differentially expressed in various cell types, thus determining the sensitivity of the cells to specific channel modifiers. Sulfonylurea class of antidiabetic drugs blocks KATP channels, which are neuroprotective in stroke, can be one of the high stoke risk factors for diabetic patients. In this review, we discussed the potential effects of KATP channel blockers when used under pathological conditions related to diabetics and cerebral ischemic stroke.
... Entretanto, o uso da própolis separadamente não foi suficiente para mitigar os prejuízos de memória para esses animais 28 . O canal Kir6.2 é principalmente encontrado em neurônios e possui uma função crucial na detecção de glicose e na regulação da excitabilidade neuronal dentro do ciclo metabólico 29 . ...
Introduction: Alzheimer's disease (AD) is an irreversible neurodegenerative disorder characterized by the deterioration of cognitive functions, for which the available pharmacotherapy is not capable of effecting a cure. Objectives: To investigate the neuroprotective properties of phytoconstituents from Jaborandi (Pilocarpus microphyllus) and propolis as potential agents in the therapy of AD. Methodology: This is an integrative literature review study, in which the PICO strategy was followed in formulating the guiding question. Five databases were used: PubMed, Web of Science, Scopus, Embase, and Lilacs. The analysis was conducted with the descriptors "epiisopiloturine", "episopilosin", "pilocarpine", "macaubine", "propolis", "alzheimer" extracted from the DeCS (Health Sciences Descriptors) and Medical Subject Headings (MeSH). Keywords were isolated and combined using boolean operators AND and OR. Results: The search resulted in 602 studies identified in the databases, 337 were duplicates, leaving 265 for title and abstract reading, of which 250 were excluded. In the end, 10 studies were read in full and 08 studies were included in the scope of the review. The research evidenced neuroprotective activity for propolis, through antioxidative, anti-inflammatory, and anticholinesterase action, configuring it with therapeutic potential in AD. For pilocarpine, a substance with various clinical applications, few studies demonstrated a correlation with AD, in cognitive and behavioral improvement. Conclusion: Therefore, benefits for the use of propolis in cognitive dysfunctions are concluded, while for pilocarpine, this relationship was not established. It is necessary to develop research that elucidates the possible functional roles for these substances.
... The functional K ATP channels are formed by Kir6x (Kir6.1 and Kir6.2) and SUR subunits (SUR1, SUR2A, and SUR2B) which are ATP-binding cassette subfamily members with regulatory activity. 19,57 The K ATP opening is dependent on ATP content that when ATP content decreases, the K ATP channels allow the passage of K + , thereby hyperpolarizing the membranes of microglia in the CNS, decreasing cell excitability and attenuating ATP assumption. 58 Opening of the K ATP channels can inhibit rotenone-induced neuroinflammation and glia activation. ...
Background
Parkinson's disease (PD) is a common neurodegenerative disorder. Microglia‐mediated neuroinflammation has emerged as an involving mechanism at the initiation and development of PD. Activation of adenosine triphosphate (ATP)‐sensitive potassium (K ATP ) channels can protect dopaminergic neurons from damage. Sodium butyrate (NaB) shows anti‐inflammatory and neuroprotective effects in some animal models of brain injury and regulates the K ATP channels in islet β cells. In this study, we aimed to verify the anti‐inflammatory effect of NaB on PD and further explored potential molecular mechanisms.
Methods
We established an in vitro PD model in BV2 cells using 1‐methyl‐4‐phenylpyridinium (MPP ⁺ ). The effects of MPP ⁺ and NaB on BV2 cell viability were detected by cell counting kit‐8 assays. The morphology of BV2 cells with or without MPP ⁺ treatment was imaged via an optical microscope. The expression of Iba‐1 was examined by the immunofluorescence staining. The intracellular ATP content was estimated through the colorimetric method, and Griess assay was conducted to measure the nitric oxide production. The expression levels of pro‐inflammatory cytokines and K ATP channel subunits were evaluated by reverse transcription–quantitative polymerase chain reaction and western blot analysis.
Results
NaB (5 mM) activated the K ATP channels through elevating Kir6.1 and Kir6.1 expression in MPP ⁺ ‐challenged BV2 cells. Both NaB and pinacidil (a K ATP opener) suppressed the MPP ⁺ ‐induced activation of BV2 cells and reduced the production of nitrite and pro‐inflammatory cytokines in MPP ⁺ ‐challenged BV2 cells.
Conclusion
NaB treatment alleviates the MPP ⁺ ‐induced inflammatory responses in microglia via activation of K ATP channels.
... 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). ...
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.
... Previous studies showed a high expression of Kir6.2-K ATP channels in neurons [75] especially in the soma and dendrites [75][76][77]. Herein, immunolabeling of Kir6.2-K ATP channels revealed their presence in the presynaptic regions where synaptic vesicles were clustered. ...
Neurotransmitter release requires high energy demands, making the nerve terminals metabolically fragile and susceptible to oxidative stress. ATP-sensitive potassium (KATP) channels can be an important regulator orchestrating the influence of metabolic-related signals on exocytosis. Here, the relevance of ROS in KATP channel-dependent control of neurotransmitter release at the frog neuromuscular junction was studied.
Microelectrode recordings of end plate potentials at the distal and proximal compartments of nerve terminals as well as fluorescent techniques were used.
Activation of KATP channels in the proximal region suppressed evoked and spontaneous release in a lipid raft-dependent manner. Activation of KATP channels in the distal region reduced solely evoked release which was preserved after lipid raft disruption. Chelation of ROS potentiated the effects of KATP channel activation and unmasked the effects of KATP channel blocker on evoked exocytosis. Activation or inhibition of KATP channels suppressed or enhanced the depressant action of extracellular adenosine on evoked exocytosis. This was accompanied with an increase or decrease in adenosine-induced ROS production, respectively. KATP channel-dependent modulation of adenosine action was halted by antioxidant and NADPH-oxidase inhibitor. Also, activation of KATP channels led to an increase in ROS production suppressing the negative effects of extracellular ATP on evoked release in a ROS-dependent manner.
KATP channel-mediated modulation of release has specific features in distal and proximal compartments and depends on endogenous ROS levels and lipid raft integrity. Activation of KATP channels suppresses the action of extracellular adenosine and ATP on evoked release by increasing ROS production.
... Kir6) channels belong to a subfamily of the weak inward rectifier K+ channels that are classified into Kir6.1 and Kir6.2 subtypes (162). While Kir6.1 genes are mainly expressed in the mitochondria (163,164), Kir6.2 subunits are involved in most functional K ATP channels that are present in pancreatic b cells, cardiac muscle cells, smooth muscle cells, and all brain regions (162,165,166). These channels are negatively gated by ATP. ...
Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP), a pleiotropic neuropeptide, is widely distributed throughout the body. The abundance of PACAP expression in the central and peripheral nervous systems, and years of accompanying experimental evidence, indicates that PACAP plays crucial roles in diverse biological processes ranging from autonomic regulation to neuroprotection. In addition, PACAP is also abundantly expressed in the hypothalamic areas like the ventromedial and arcuate nuclei (VMN and ARC, respectively), as well as other brain regions such as the nucleus accumbens (NAc), bed nucleus of stria terminalis (BNST), and ventral tegmental area (VTA) – suggesting that PACAP is capable of regulating energy homeostasis via both the homeostatic and hedonic energy balance circuitries. The evidence gathered over the years has increased our appreciation for its function in controlling energy balance. Therefore, this review aims to further probe how the pleiotropic actions of PACAP in regulating energy homeostasis is influenced by sex and dynamic changes in energy status. We start with a general overview of energy homeostasis, and then introduce the integral components of the homeostatic and hedonic energy balance circuitries. Next, we discuss sex differences inherent to the regulation of energy homeostasis via these two circuitries, as well as the activational effects of sex steroid hormones that bring about these intrinsic disparities between males and females. Finally, we explore the multifaceted role of PACAP in regulating homeostatic and hedonic feeding through its actions in regions like the NAc, BNST, and in particular the ARC, VMN and VTA that occur in sex- and energy status-dependent ways.
... The KATP comprising of two subunits viz inwardly rectifying potassium channel Kir6.1 or 2 and the regulatory subunit sulfonylurea receptor SUR (SUR1 or SUR2) are strongly expressed in the hippocampus, prefrontal cortex, amygdala, and hypothalamus and are [24][25][26][27] implicated in the pathophysiology of depression. ...
Background: Depression is a mood disorder with poorly understood aetiology and treatment outcomes. Treatment with creatine, a nutraceutical associated with potassium sensitive adenosine triphosphate channels (KATP), produced improved results in preclinical studies of depression.
... The functional K ATP channels, which are hetero-octameric membrane protein complexes, are formed by four inward-rectifier potassium channel 6 (Kir6; either Kir6.1 or Kir6.2) subunits and four sulfonylurea receptor (SUR; as SUR1, SUR2A, or SUR2B) subunits. They are ATP-binding cassette subfamily members with regulatory activity (Li et al., 2017;Thomzig et al., 2005). ...
Accumulating evidence suggests that ATP‐sensitive potassium (KATP) channels play an important role in the selective degeneration of dopaminergic neurons in the substantia nigra (SN). Furthermore, the expression of the KATP channel subunit sulfonylurea receptor 1 (SUR1) is upregulated in the remaining nigral dopaminergic neurons in Parkinson's disease (PD). However, the mechanism underlying this selective upregulation of the SUR1 subunit and its subsequent roles in PD progression are largely unknown. In 3‐, 6‐, and 9‐month‐old A53T α‐synuclein transgenic (α‐SynA53T+/+) mice, only the SUR1 subunit and not SUR2B or Kir6.2 was upregulated, accompanied by neuronal damage. Moreover, the occurrence of burst firing in dopaminergic neurons was increased with the upregulation of the SUR1 subunit, whereas no changes in the firing rate were observed except in 9‐month‐old α‐SynA53T+/+ mice. After interference with SUR1 expression by injection of lentivirus into the SN, the progression of dopaminergic neuron degeneration was delayed. Further studies showed that elevated expression of the transcription factors FOXA1 and FOXA2 could cause the upregulation of the SUR1 subunit in α‐SynA53T+/+ mice. Our findings revealed the regulatory mechanism of the SUR1 subunit and the role of KATP channels in the progression of dopaminergic neuron degeneration, providing a new target for PD drug therapy. The elevated expression of SUR1 subunit of KATP channels in nigral dopaminergic neurons was regulated by the transcription factors FOXA1 and FOXA2 at the early stage of PD.The upregulated SUR1 promotes its transmembrane transport and then causes the membrane hyperpolarization, which may promote the occurrence of cluster discharges mediated by NMDA receptors and affect the activities of dopaminergic neurons.
... The SUR1/Kir6.1 or Kir6.2 and SUR2B/Kir6.2 K ATP channel subtypes are mainly expressed in the brain [44], whereas the Kir6.1 subunit is dominantly present in astrocytes, and the Kir6.2 subunit in neurons [45,46]. Given that ENSA has been proposed as a ligand of SUR1, we assessed whether SUR1 regulates NEP expression and/or activity in vivo. ...
Alzheimer’s disease (AD) is characterized by the deposition of amyloid β peptide (Aβ) in the brain. The neuropeptide somatostatin (SST) regulates Aβ catabolism by enhancing neprilysin (NEP)-catalyzed proteolytic degradation. However, the mechanism by which SST regulates NEP activity remains unclear. Here, we identified α-endosulfine (ENSA), an endogenous ligand of the ATP-sensitive potassium (KATP) channel, as a negative regulator of NEP downstream of SST signaling. The expression of ENSA is significantly increased in AD mouse models and in patients with AD. In addition, NEP directly contributes to the degradation of ENSA, suggesting a substrate-dependent feedback loop regulating NEP activity. We also discovered the specific KATP channel subtype that modulates NEP activity, resulting in the Aβ levels altered in the brain. Pharmacological intervention targeting the particular KATP channel attenuated Aβ deposition, with impaired memory function rescued via the NEP activation in our AD mouse model. Our findings provide a mechanism explaining the molecular link between KATP channel and NEP activation, and give new insights into alternative strategies to prevent AD.
... CQ and HCQ exert their anti-hyperglycaemic effects by inducing physiological changes in pancreatic beta cells. Previous study has demonstrated that CQ and HCQ are a known inhibitor for Kir6.2 [75], an adenosine triphosphatesensitive potassium channel expressed in many different tissues such as pancreatic beta cells, cardiomyocytes, neurons, and lymphocytes [76][77][78][79]. The downregulation of Kir6.2 was associated to an increase of insulin secretion in pancreatic beta cell lines [80]. ...
Chloroquine (CQ) and hydroxychloroquine (HCQ) are traditional anti-malarial drugs that have been repurposed for new therapeutic uses in many diseases due to their simple usage and cost-effectiveness. The pleiotropic effects of CQ and HCQ in regulating blood pressure, glucose homeostasis, lipid, and carbohydrate metabolism have been previously described in vivo and in humans, thus suggesting their role in metabolic syndrome (MetS) prevention. The anti-hyperglycaemic, anti-hyperlipidaemic, cardioprotective, anti-hypertensive, and anti-obesity effects of CQ and HCQ might be elicited through reduction of inflammatory response and oxidative stress, improvement of endothelial function, activation of insulin signalling pathway, inhibition of lipogenesis and autophagy, as well as regulation of adipokines and apoptosis. In conclusion, the current state of knowledge supported the repurposing of CQ and HCQ usage in the management of MetS.
... The Mito-KATP channel plays an important role in the physiology or pathological processes of mitochondria by regulating the volume and function of mitochondria to reflect the energy state of cells. Similar to Kir6.2 of KATP channel of plasma membrane, 42 MitoK as a functional subunit in Mito-KATP channel may exert critical roles on adapting neuronal activity to metabolic demands. To further elucidate whether Ipt mediated protective effects of mitochondrial morphology and function via Mito-KATP channel subunit MitoK, knocking down MitoK in N2a cells is necessary. ...
Synaptic plasticity damages play a crucial role in the onset and development of depression, especially in the hippocampus, which is more susceptible to stress and the most frequently studied brain region in depression. And, mitochondria have a major function in executing the complex processes of neurotransmission and plasticity. We have previously demonstrated that Iptakalim (Ipt), a new ATP‐sensitive potassium (K‐ATP) channel opener, could improve the depressive‐like behavior in mice. But the underlying mechanisms are not well understood. The present study demonstrated that Ipt reversed depressive‐like phenotype in vivo (chronic mild stress‐induced mice model of depression) and in vitro (corticosterone‐induced cellular model). Further study showed that Ipt could upregulate the synaptic‐related proteins postsynaptic density 95 (PSD 95) and synaptophysin (SYN), and alleviated the synaptic structure damage. Moreover, Ipt could reverse the abnormal mitochondrial fission and fusion, as well as the reduced mitochondrial ATP production and collapse of mitochondrial membrane potential in depressive models. Knocking down the mitochondrial ATP‐sensitive potassium (Mito‐KATP) channel subunit MitoK partly blocked the above effects of Ipt. Therefore, our results reveal that Ipt can alleviate the abnormal mitochondrial dynamics and function depending on MitoK, contributing to improve synaptic plasticity and exert antidepressive effects. These findings provide a candidate compound and a novel target for antidepressive therapy.
... We recently demonstrated that intravenous infusion of the ATP sensitive potassium (K ATP ) channel opener levcromakalim induces migraine attacks in migraine without aura patients. 10,11 Since levcromakalim is able to cross the blood-brain barrier, 12 K ATP channels are expressed in glial cells, cortical neurons, and cerebral vasculature, [13][14][15][16] and since CSD can be triggered by an increase of extracellular K + , 17 levcromakalim is a candidate drug for triggering attacks of migraine with aura, thereby potentially serving as an invaluable tool for further research into this condition. ...
Migraine afflicts more than one billion individuals worldwide and is a leading cause of years lived with disability. In about a third of individuals with migraine aura occur in relation to migraine headache. The common pathophysiological mechanisms underlying migraine headache and migraine aura are yet to be identified. Based on recent data, we hypothesized that levcromakalim, an ATP-sensitive potassium channel opener, would trigger migraine attacks with aura in patients. In a randomized, double-blind, placebo-controlled, crossover study, 17 patients aged 21–59 years and diagnosed with migraine with aura exclusively were randomly allocated to receive an infusion of 0.05 mg/min levcromakalim or placebo (isotonic saline) on two different days (ClinicalTrials.gov, ID: NCT04012047). The primary end points were the difference in incidence of migraine attacks with or without aura, headache and the difference in the area under the curve for headache intensity scores (0–12 h). Seventeen patients completed the study. Fourteen of 17 (82%) patients developed migraine attacks with and without aura after levcromakalim compared with 1 of 17 (6%) after placebo (P < 0.001). Ten patients (59%) developed migraine with aura after levcromakalim compared with none after placebo (P = 0.002). One additional patient reported ‘possible’ aura, only partially fulfilling the criteria. Levcromakalim is likely a novel migraine aura-inducing substance in humans. These findings highlight the ATP-sensitive potassium channel as a shared target in migraine aura and migraine headache. Likely, ATP-sensitive potassium channel opening leads to triggering of aura and headache, respectively, via distinct mechanisms.
... To better understand the special vulnerability of the VLS to neuroleptic drugs, the present study aimed to recognize molecular features that distinguish the VLS from other striatal areas. In this regard, potassium channels display highly characteristic distributions in the brain and, especially, in the striatum [28][29][30], suggesting that they may subserve highly specific biological functions. In this regard, the Kv2.2 potassium channel protein and a channel-related protein (KChIP3) recently have been used as markers for characteristic cells in the brain [31,32]. ...
The striatum is the main input structure of the basal ganglia. Distinct striatal subfields are involved in voluntary movement generation and cognitive and emotional tasks, but little is known about the morphological and molecular differences of striatal subregions. The ventrolateral subfield of the striatum (VLS) is the orofacial projection field of the sensorimotor cortex and is involved in the development of orofacial dyskinesias, involuntary chewing-like movements that often accompany long-term neuroleptic treatment. The biological basis for this particular vulnerability of the VLS is not known. Potassium channels are known to be strategically localized within the striatum. In search of possible molecular correlates of the specific vulnerability of the VLS, we analyzed the expression of voltage-gated potassium channels in rodent and primate brains using qPCR, in situ hybridization, and immunocytochemical single and double staining. Here we describe a novel, giant, non-cholinergic interneuron within the VLS. This neuron coexpresses the vesicular GABA transporter, the calcium-binding protein parvalbumin (PV), and the Kv3.3 potassium channel subunit. This novel neuron is much larger than PV neurons in other striatal regions, displays characteristic electrophysiological properties, and, most importantly, is restricted to the VLS. Consequently, the giant striatal Kv3.3-expressing PV neuron may link compromised Kv3 channel function and VLS-based orofacial dyskinesias.
... Nevertheless, while postnatal undernutrition suppresses the genes coding for the K ATP channel subunits Sur2, Kir6.1 and Kir6.2 at postnatal day 30 [23], our data show that postnatal overnutrition, in turn, led to increased expression of genes coding for Sur1 (Abcc8) and Kir6.2 (Kcnj11) at prepubertal stage. Therefore, Kir6.2 seems to be the predominant subunit requested in the ARH due to metabolic demands as previously suggested [57]. Whether sex differences further contribute to the differential expression of genes coding for K ATP channel subunits due to postnatal overweight gain needs to be further investigated. ...
The adipocyte-derived hormone leptin is a potent neurotrophic factor that contributes to the neural plasticity and development of feeding circuitry, particularly in the arcuate nucleus of the hypothalamus (ARH). Postnatal overnutrition affects leptin secretion and sensitivity, but whether postnatal overnutrition produces changes in the development of the synaptic transmission to ARH neurons is currently unknown. We evaluated the excitatory and inhibitory currents to ARH leptin receptor (LepR)-expressing neurons in prepubertal, pubertal and adult female mice. The effects of postnatal overnutrition in the expression of genes that code ion channels subunits in the ARH were also evaluated. We observed that the transition from prepubertal to pubertal stage is characterized by a rise in both excitatory and inhibitory transmission to ARH LepR-expressing neurons in control mice. Postnatal overnutrition induces a further increase in the excitatory synaptic transmission in pubertal and adult animals, whereas the amplitude of inhibitory currents to ARH LepR-expressing cells was reduced. Postnatal overnutrition also contributes to the modulation of gene expression of N-methyl-D-aspartate, GABAB and ATP-sensitive potassium channel subunits in ARH. In summary, the synaptic transmission to ARH cells is profoundly influenced by postnatal overnutrition. Thus, increased adiposity during early postnatal period induces long-lasting effects on ARH cellular excitability.
... This probably also explains the differences between the trigeminal and spinal pain processing systems in relation to K ATP channels. K ATP channels are widely expressed within the brain, which theoretically could be a target site (48,49). However, there is no evidence that either levcromakalim 50 or glibenclamide cross the blood-brain barrier. ...
Background
Recently, the adenosine triphosphate (ATP) sensitive potassium channel opener levcromakalim was shown to induce migraine attacks with a far higher incidence than any previous provoking agent such as calcitonin gene-related peptide. Here, we show efficacy of ATP sensitive potassium channel inhibitors in two validated rodent models of migraine.
Methods
In female spontaneous trigeminal allodynic rats, the sensitivity of the frontal region of the head was tested by an electronic von Frey filament device. In mice, cutaneous hypersensitivity was induced by repeated glyceryl trinitrate or levcromakalim injections over nine days, as measured with von Frey filaments in the hindpaw. Release of calcitonin gene-related peptide from dura mater and trigeminal ganglion was studied ex vivo.
Results
The ATP sensitive potassium channel inhibitor glibenclamide attenuated the spontaneous cephalic hypersensitivity in spontaneous trigeminal allodynic rats and glyceryl trinitrate-induced hypersensitivity of the hindpaw in mice. It also inhibited CGRP release from dura mater and the trigeminal ganglion isolated from spontaneous trigeminal allodynic rats. The hypersensitivity was also diminished by the structurally different ATP sensitive potassium channel inhibitor gliquidone. Mice injected with the ATP sensitive potassium channel opener levcromakalim developed a progressive hypersensitivity that was completely blocked by glibenclamide, confirming target engagement.
Conclusion
The results suggest that ATP sensitive potassium channel inhibitors could be novel and highly effective drugs in the treatment of migraine.
... ATP-sensitive potassium channels (K-ATP) are widely distributed in the hippocampus, prefrontal cortex, and hypothalamus [15] and represent an important component of homeostasis maintenance during stress exposure [13]. Experiments showed that K-ATP participate in the pathogenesis of depressive-like reactions [4] induced by chronic mild stress in mice, while clinical observations demonstrate low bioenergetic metabolism and abnormal ATP biosynthesis in patients with depression [12]. ...
We studied the influence of intraperitoneal injection of ATP-sensitive potassium channels inhibitor glibenclamide in doses of 0.01, 0.1, 1, and 10 mg/kg on the effects of a new pyrazolo[C]pyridine derivative GIZh-72 (4,6-dimethyl-2-(4-chlorphenyl)-2,3-dihydro-1Hpyrazolo[ 4,3-C]pyridine-3-on, chloral hydrate; 20 mg/kg, intraperitoneally) in the marble burying and open-field tests in mice. It was found that glibenclamide produced an anxiolytic effect in the open-field test (in a dose of 0.01 mg/kg) and anticompulsive effect in the marble burying test (in doses of 1 and 10 mg/kg). The observed behavioral effects of glibenclamide did not depend on blood glucose level. At the same time, glibenclamide in subeffective (0.01 and 0.1 mg/kg) and effective (1 and 10 mg/kg) doses potentiated the psychotropic effects of GIZh-72 in these tests. It can be assumed that the psychotropic effects of GIZh-72 depend on functional activity of ATP-sensitive potassium channels.
... [6,7]. Notably, Kir6.1 and Kir6.2 are abundant in neurons, whereas Kir6.1 is also expressed in glial cells such as astrocytes [8,9]. Recently, both Kir6.1 and Kir6.2 mouse mutants were established, and their brain phenotypes have been reported. ...
ATP-sensitive K⁺ (KATP) channels are predominantly expressed in the brain and consist of four identical inward-rectifier potassium ion channel subunits (Kir6.1 or Kir6.2) and four identical high-affinity sulfonylurea receptor subunits (SUR1, SUR2A, or SUR2B). We previously observed that chronic corticosterone-treated (CORT) mice exhibited enhanced anxiety-like behaviors and cued fear memory. In the present study, the protein and mRNA expression levels of Kir6.1, but not Kir6.2, were decreased in the lateral amygdala (LA) of CORT mice. Heterozygous Kir6.1-null (Kir6.1+/−) mice also showed enhanced tone (cued) fear memory and long-term potentiation (LTP) in the cortico-LA pathway compared to those in wild-type mice. However, LTP was not enhanced in the hippocampal CA1 regions of Kir6.1+/− mice. Consistent with increased cued fear memory, both Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) activities were significantly elevated in the LAs of Kir6.1+/− mice after tone stimulation. Our results indicate that increased CaMKII and ERK activities may induce LTP in the LA in Kir6.1+/− mice, leading to aberrant cued fear memory. The changes in neural plasticity in the LA of Kir6.1+/− mice were associated with anxiety-like behaviors and may be related to the pathogenic mechanisms of anxiety disorders in human patients.
... In rats with chronic cerebral hypoperfusion-induced WMI, the pericytes were damaged and its biomarker Kir6.1/Kir6.2 was expressed in heterogeneity, suggesting that the Kir6.1/K-ATP channel might be involved in the pathological process of chronic WMI ( Table 2; Thomzig et al., 2005). ...
Pericytes are functional components of the neurovascular unit (NVU) that are located around the blood vessels, and their roles in the regulation of cerebral health and diseases has been reported. Currently, the potential properties of pericytes as emerging therapeutic targets for cerebrovascular diseases have attracted considerable attention. Nonetheless, few reviews have comprehensively discussed pericytes and their roles in cerebrovascular diseases. Therefore, in this review, we not only summarized and described the basic characteristics of pericytes but also focused on clarifying the new understanding about the roles of pericytes in the pathogenesis of cerebrovascular diseases, including white matter injury (WMI), hypoxic–ischemic brain damage, depression, neovascular insufficiency disease, and Alzheimer’s disease (AD). Furthermore, we summarized the current therapeutic strategies targeting pericytes for cerebrovascular diseases. Collectively, this review is aimed at providing a comprehensive understanding of pericytes and new insights about the use of pericytes as novel therapeutic targets for cerebrovascular diseases.
... Earlier pharmacological studies have indicated K ATP channel agonists could be used as a therapeutic during morphine tolerance (Seth et al., 2010;Cao et al., 2016). Our pharmacological data are in alignment with a previous study indicating the nonselective SUR1/SUR2 agonist cromakalim and the SUR1 agonist diazoxide could inhibit phenotypic indicators of morphine withdrawal in rodents including the jumping and fore-paw tremors (Robles et al., 1994;Thomzig et al., 2005). In our studies, the paw withdrawal thresholds on Day 6 after morphine tolerance was significantly attenuated by NN414, but not diazoxide. ...
ATP-sensitive potassium (KATP) channels are found in the nervous system and are downstream targets of opioid receptors. KATP channel activity can effect morphine efficacy and may beneficial for relieving chronic pain in the peripheral and central nervous system. Unfortunately, the KATP channels exists as a heterooctomers, and the exact subtypes responsible for the contribution to chronic pain and opioid signaling in either dorsal root ganglia (DRG) or the spinal cord are yet unknown. Chronic opioid exposure (15 mg/kg morphine, s.c., twice daily) over 5 days produces significant downregulation of Kir6.2 and SUR1 in the spinal cord and DRG of mice. In vitro studies also conclude potassium flux after KATP channel agonist stimulation is decreased in neuroblastoma cells treated with morphine for several days. Mice lacking the KATP channel SUR1 subunit have reduced opioid efficacy in mechanical paw withdrawal behavioral responses compared to wild-type and heterozygous littermates (5 and 15 mg/kg, s.c., morphine). Using either short hairpin RNA (shRNA) or SUR1 cre-lox strategies, downregulation of SUR1 subtype KATP channels in the spinal cord and DRG of mice potentiated the development of morphine tolerance and withdrawal. Opioid tolerance was attenuated with intraplantar injection of SUR1 agonists, such as diazoxide and NN-414 (100 μM, 10 μL) compared to vehicle treated animals. These studies are an important first step in determining the role of KATP channel subunits in antinociception, opioid signaling, and the development of opioid tolerance, and shed light on the potential translational ability of KATP channel targeting pharmaceuticals and their possible future clinical utilization. These data suggest that increasing neuronal KATP channel activity in the peripheral nervous system may be a viable option to alleviate opioid tolerance and withdrawal.
... There are four subpopulations of tanycytes: α1, α2, β1, and β2. β1 tanycytes express the glucose transporter, GLUT2, in the apical region, as well as other proteins involved in the peripheral glucose-sensing mechanism, such as glucokinase (GK) and the potassium channel subunit, kir 6.1 [6][7][8][9][10]. Recently, studies of hypothalamic sections and primary cultures have shown that β1 tanycytes respond to changes in glucose concentrations with increased intracellular calcium [4,5,11]. ...
Our data proposes that glucose is transferred directly to the cerebrospinal fluid (CSF) of the hypothalamic ventricular cavity through a rapid “fast-track-type mechanism” that would efficiently stimulate the glucosensing areas. This mechanism would occur at the level of the median eminence (ME), a periventricular hypothalamic zone with no blood-brain barrier. This “fast-track” mechanism would involve specific glial cells of the ME known as β2 tanycytes that could function as “inverted enterocytes,” expressing low-affinity glucose transporters GLUT2 and GLUT6 in order to rapidly transfer glucose to the CSF. Due to the large size of tanycytes, the presence of a high concentration of mitochondria and the expression of low-affinity glucose transporters, it would be expected that these cells accumulate glucose in the endoplasmic reticulum (ER) by sequestering glucose-6-phosphate (G-6-P), in a similar way to that recently demonstrated in astrocytes. Glucose could diffuse through the cells by micrometric distances to be released in the apical region of β2 tanycytes, towards the CSF. Through this mechanism, levels of glucose would increase inside the hypothalamus, stimulating glucosensing mechanisms quickly and efficiently.
Key messages
• Glucose diffuses through the median eminence cells (β2 tanycytes), towards the hypothalamic CSF.
• Glucose is transferred through a rapid “fast-track-type mechanism” via GLUT2 and GLUT6.
• Through this mechanism, hypothalamic glucose levels increase, stimulating glucosensing.
... Tanycytes (mainly α tanycytes and in a lesser extend β tanycytes) have mainly been shown to be able to detect changes in glucose levels in the CSF and release paracrine factors (e.g., ATP), that activate neighboring tanycytes (13) but could also potentially activate neighboring hypothalamic neurons (59,60). The idea that tanycytes act as glucose-sensors has gained credence with the demonstration that selective glucose puffing onto tanycyte cell bodies induces Ca 2+ waves in brain slice preparations (13) or in primary tanycyte cultures (46), as well as the immunodetection of molecules known to be essential components of glucose metabolism in pancreatic β-cells (61), such as the glucose transporter GLUT2 (62), glucokinase (63,64), and the K ATP channel subunits Kir6.1 (62,65). However, non-metabolizable glucose analogs (e.g., 2-deoxy-D-glucose and methyl-α-D-glucopyranoside) are also capable of evoking these signals in tanycytes (13), suggesting that tanycytes would not completely mimic β-cell sensing and/or that different mechanisms exist according to tanycyte subtypes. ...
Animal survival relies on a constant balance between energy supply and energy expenditure, which is controlled by several neuroendocrine functions that integrate metabolic information and adapt the response of the organism to physiological demands. Polarized ependymoglial cells lining the floor of the third ventricle and sending a single process within metabolic hypothalamic parenchyma, tanycytes are henceforth described as key components of the hypothalamic neural network controlling energy balance. Their strategic position and peculiar properties convey them diverse physiological functions ranging from blood/brain traffic controllers, metabolic modulators, and neural stem/progenitor cells. At the molecular level, these functions rely on an accurate regulation of gene expression. Indeed, tanycytes are characterized by their own molecular signature which is mostly associated to their diverse physiological functions, and the detection of variations in nutrient/hormone levels leads to an adequate modulation of genetic profile in order to ensure energy homeostasis. The aim of this review is to summarize recent knowledge on the nutritional control of tanycyte gene expression.
... PKG-dependent activation of TASK-1 and TREK-1 (Toyoda et al., 2010) as well as inhibition of heteromeric TASK-1/TASK-3 (Gonzalez-Forero et al., 2007) channels have been described. In addition, inward rectifier K + currents have been shown to be inhibited in a cGMP-dependent manner (Dixon and Copenhagen, 1997) and a number of Kir channel subtypes (including members of the Kir2, Kir3 and Kir6 families) are expressed in thalamic cells ( Thomzig et al., 2005). In TC neurons of different species several K 2P (TASK-1, TASK-3, TREK-1, TREK-2) and Kir channels (Kir2, Kir3) are expressed and have important contributions to the RMP, anomalous rectification and firing pattern (Meuth et al., 2003(Meuth et al., , 2006Bista et al., 2015), thereby pointing to a complex scenario when cGMP-dependent effector functions are considered. ...
The hyperpolarization-activated inward current, Ih, plays a key role in the generation of rhythmic activities in thalamocortical (TC) relay neurons. Cyclic nucleotides, like 3′,5′-cyclic adenosine monophosphate (cAMP), facilitate voltage-dependent activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels by shifting the activation curve of Ih to more positive values and thereby terminating the rhythmic burst activity. The role of 3′,5′-cyclic guanosine monophosphate (cGMP) in modulation of Ih is not well understood. To determine the possible role of the nitric oxide (NO)-sensitive cGMP-forming guanylyl cyclase 2 (NO-GC2) in controlling the thalamic Ih, the voltage-dependency and cGMP/cAMP-sensitivity of Ih was analyzed in TC neurons of the dorsal part of the lateral geniculate nucleus (dLGN) in wild type (WT) and NO-GC2-deficit (NO-GC2−/−) mice. Whole cell voltage clamp recordings in brain slices revealed a more hyperpolarized half maximal activation (V1/2) of Ih in NO-GC2−/− TC neurons compared to WT. Different concentrations of 8-Br-cAMP/8-Br-cGMP induced dose-dependent positive shifts of V1/2 in both strains. Treatment of WT slices with lyase enzyme (adenylyl and guanylyl cyclases) inhibitors (SQ22536 and ODQ) resulted in further hyperpolarized V1/2. Under current clamp conditions NO-GC2−/− neurons exhibited a reduction in the Ih-dependent voltage sag and reduced action potential firing with hyperpolarizing and depolarizing current steps, respectively. Intrathalamic rhythmic bursting activity in brain slices and in a simplified mathematical model of the thalamic network was reduced in the absence of NO-GC2. In freely behaving NO-GC2−/− mice, delta and theta band activity was enhanced during active wakefulness (AW) as well as rapid eye movement (REM) sleep in cortical local field potential (LFP) in comparison to WT. These findings indicate that cGMP facilitates Ih activation and contributes to a tonic activity in TC neurons. On the network level basal cGMP production supports fast rhythmic activity in the cortex.
... 7 The differential expression of SUR subunits may also contribute to breadth of pathologies induced by K ATP channelopathy. Since Kir6.2 is expressed in the hypothalamus, 35,36 we expect the expression of the dominant negative A28V hKir6.2 protein in hypothalamic neurons could impact the K ATP channels, disrupt hypothalamic function and thereby effect pituitary function. Previous studies have identified multiple roles for K ATP channels in the hypothalamus, ranging from regulation of food intake and glucose homeostasis to hormonal functions. ...
The ATP-sensitive potassium channel (KATP) functions as a metabo-electric transducer in regulating insulin secretion from pancreatic β-cells. The pancreatic KATP channel is composed of a pore-forming inwardly-rectifying potassium channel, Kir6.2, and a regulatory subunit, sulphonylurea receptor 1 (SUR1). Loss-of-function mutations in either subunit often lead to the development of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). PHHI is a rare genetic disease and most patients present with immediate onset within the first few days after birth. In this study, we report an unusual form of PHHI, in which the index patient developed hyperinsulinemic hypoglycemia after 1 year of age. The patient failed to respond to routine medication for PHHI and underwent a complete pancreatectomy. Genotyping of the index patient and his immediate family members showed that the patient and other family members with hypoglycemic episodes carried a heterozygous novel mutation in KCNJ11 (C83T), which encodes Kir6.2 (A28V). Electrophysiological and cell biological experiments revealed that A28V hKir6.2 is a dominant-negative, loss-of-function mutation and that KATP channels carrying this mutation failed to reach the cell surface. de novo protein structure prediction indicated that this A28V mutation reoriented the ER retention motif located at the C-terminal of the hKir6.2, and this result may explain the trafficking defect caused by this point mutation. Our study is the first report of a novel form of late-onset PHHI that is caused by a dominant mutation in KCNJ11 and exhibits a defect in proper surface expression of Kir6.2.
... Impaired autonomous activity of STN neurons in BACHD mice is due to increased activation of K ATP channels NMDAR receptor-generated mitochondrial oxidant stress in BACHD may lead to the activation of K ATP channels, which act as metabolic sensors and homeostatic regulators of excitability in multiple cell types (Nichols, 2006). STN neurons abundantly express all the molecular components of K ATP channels including the Kir6.2 pore-forming subunit of the K ATP channel (Thomzig et al., 2005) and the SUR1, SUR2A and SUR2B regulatory subunits (Karschin et al., 1997;Zhou et al., 2012). To determine whether K ATP channels are responsible for impaired firing in BACHD mice, the effects of the K ATP channel inhibitor glibenclamide (100 nM) on WT and phenotypic BACHD autonomous firing ex vivo were compared. ...
The subthalamic nucleus (STN) is an element of cortico-basal ganglia-thalamo-cortical circuitry critical for action suppression. In Huntington's disease (HD) action suppression is impaired, resembling the effects of STN lesioning or inactivation. To explore this potential linkage, the STN was studied in BAC transgenic and Q175 knock-in mouse models of HD. At <2 and 6 months of age autonomous STN activity was impaired due to activation of KATP channels. STN neurons exhibited prolonged NMDA receptor-mediated synaptic currents, caused by a deficit in glutamate uptake, and elevated mitochondrial oxidant stress, which was ameliorated by NMDA receptor antagonism. STN activity was rescued by NMDA receptor antagonism or the break down of hydrogen peroxide. At 12 months of age approximately 30% of STN neurons had been lost, as in HD. Together, these data argue that dysfunction within the STN is an early feature of HD that may contribute to its expression and course.
... ATP-sensitive K + (KATP) channels, discovered originally in cardiac muscle (Noma 1983), have been found ubiquitously distributed in various cells and tissues (Inagaki et al., 1995b), such as pancreatic β-cells (Cook et al., 1988;Ashcroft and Kakei, 1989), pituitary GH3 cells (Wu et al., 2000), skeletal muscle (Allard and Lazdunski, 1993), neurons and glial cells of brain (Zhou et al., 2002;Thomzig et al., 2005), kidney (Hurst et al., 1993;Zhou et al., 2007b;Zhou et al., 2008) and testis (Acevedo et al., 2006;Zhou et al., 2011). KATP channels close at high intracellular ATP concentrations and open at lower concentrations during ischemia (Yokoshiki et al., 1998;Yuan et al., 2004). ...
ATP-sensitive K+ (KATP) channel subunits Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B in the rat pituitary gland were first investigated by RT-PCR assay and immunohistochemical staining. The results of RTPCR analysis showed that the rat pituitary gland expressed the five KATP channel subunits mentioned above. Immunohistochemical staining showed that these KATP channel subunits were widely localized in the anterior lobe, intermediate lobe, and posterior lobe at different intensities. Immunofluorescence double and triple staining showed that these KATP channel subunits colocalized with cells containing adrenocorticotropic hormone (ACTH) in the anterior lobe of the pituitary gland. Interestingly, neither Kir6.1 nor Kir6.2 colocalized with cells containing prolactin (PRL), follicular stimulating hormone (FSH), and growth hormone (GH). These results suggest that ACTH cells contain four types of KATP channels: Kir6.1/SUR2A, Kir6.2/SUR2A, Kir6.1/SUR2B, and Kir6.2/SUR2B. KATP channels may play some important roles in ACTH cells in the pituitary gland. The different compositions of KATP channel subunits in corticotrophs but not in the PRL, FSH, and GH cells might be due to the different metabolic situations of these cells. Key words: ATP-sensitive K+ channel; Kir6.x; SUR2; immunohistochemistry; pituitary gland; rat
... K ATP channel subunit expression is initiated in the peri-weaning period, after which leptin only inhibits NAG neurons [30]. Kir6.2 is the predominant inward rectifying subunit expressed in the mediobasal hypothalamus throughout the post-weaning period ( Figure 6D and [56,57]. SUR2-containing K ATP channels appear to develop before SUR1-containing channels ( Figure 6A, B), but electrophysiological recordings provide evidence that both types are active in NAG neurons by P30 ( Figure 7D-F). ...
Objective:
Humans and animals exposed to undernutrition (UN) during development often experience accelerated "catch-up" growth when food supplies are plentiful. Little is known about the mechanisms regulating early growth rates. We previously reported that actions of leptin and presynaptic inputs to orexigenic NPY/AgRP/GABA (NAG) neurons in the arcuate nucleus of the hypothalamus are almost exclusively excitatory during the lactation period, since neuronal and humoral inhibitory systems do not develop until after weaning. Moreover, we identified a critical step that regulates the maturation of electrophysiological responses of NAG neurons at weaning - the onset of genes encoding ATP-dependent potassium (KATP) channel subunits. We explored the possibility that UN promotes subsequent catch-up growth, in part, by delaying the maturation of negative feedback systems to neuronal circuits driving food intake.
Methods:
We used the large litter (LL) size model to study the impacts of postnatal UN followed by catch-up growth. We evaluated the maturation of presynaptic and postsynaptic inhibitory systems in NAG neurons using a combination of electrophysiological and molecular criteria, in conjunction with leptin's ability to suppress fasting-induced hyperphagia.
Results:
The onset of KATP channel subunit expression and function, the switch in leptin's effect on NAG neurons, the ingrowth of inhibitory inputs to NAG neurons, and the development of homeostatic feedback to feeding circuits were delayed in LL offspring relative to controls. The development of functional KATP channels and the establishment of leptin-mediated suppression of food intake in the peri-weaning period were tightly linked and were not initiated until growth and adiposity of LL offspring caught up to controls.
Conclusions:
Our data support the idea that initiation of KATP channel subunit expression in NAG neurons serves as a molecular gatekeeper for the maturation of homeostatic feeding circuits.
ATP-sensitive potassium (KATP) channels enable ATP to control the membrane potential and insulin secretion. Humans affected by severe activating mutations in KATP channels suffer from developmental delay, epilepsy and neonatal diabetes (DEND syndrome). While the diabetes in DEND syndrome is well understood, the pathophysiology of the neurological symptoms remains unclear. We hypothesized that parvalbumin-positive interneurons (PV-INs) are key for the pathophysiology and found, by using electrophysiology, that expressing the DEND mutation Kir6.2-V59M selectively in PV-INs reduced intrinsic gamma frequency preference and short-term depression as well as disturbed cognition-associated gamma oscillations and hippocampal sharp waves. Furthermore, risk of seizures is increased and day-night shift in gamma activity disrupted. Thus, PV-INs play a key role in DEND syndrome and this provides a framework for establishing treatment options.
Aims:
Chronic liver disease (CLD) is a serious medical condition affecting patients globally and pain management poses a unique challenge. ATP-sensitive potassium channels (KATP) are expressed in nociceptive neurons and hepatic cells. We tested the hypothesis whether morphine and nicorandil, KATP channel opener, alone and in combination possess hepatoprotective, antinociceptive effect and alter morphine physical dependence.
Main methods:
Intraperitoneal injection (i.p.) of carbon tetrachloride (CCl4) induced liver fibrosis in male Wistar rats. Nicorandil (15 mg/kg/day) was administered Per os for two weeks. Morphine (3.8, 5, 10 mg/kg, i.p.) was administered prior to antinociception testing in tail flick and formalin tests. Morphine physical dependence following naloxone injection, Fibrotic, oxidative stress markers, and liver histopathology were assessed.
Key findings:
Morphine alone, produced insignificant changes of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), hepatic hydroxyproline (Hyp), malondialdehyde (MDA), and superoxide dismutase (SOD) levels and exerted significant antinociception in the pain models. Nicorandil alone protected against liver damage (decreased serum ALT, AST, HA, hepatic Hyp, MDA, increased SOD levels, improved fibrosis scores). Nicorandil/morphine combination produced remarkable hepatoprotection and persistent analgesia compared to morphine alone as evidenced by reduced (EC50) of morphine. Nicorandil augmented morphine analgesia and markedly decreased withdrawal signs in morphine-dependent rats.
Significance:
The data showed for the first time, the hepatoprotection and augmented antinociception mediated by nicorandil/morphine combination in liver fibrosis via antioxidant and antifibrotic mechanisms. Nicorandil ameliorated withdrawal signs in morphine dependence in CLD. Thus, combining nicorandil/morphine provides a novel treatment strategy to ameliorate hepatic injury, potentiate antinociception and overcome morphine-induced physical dependence in liver fibrosis.
Glucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (K ATP ) channels formed with KCNJ11 and ABCC8 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through K ATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function.
Patients with Alzheimer's disease (AD) demonstrate severely impaired olfactory systems, which occur in the early stages of the disease. Olfactory bulbectomy (OBX) in mice elicits cognitive deficits, and reduces cholinergic activity in the hippocampus. Here, we confirmed that the novel AD drug memantine rescues cognitive deficits via ATP-sensitive potassium (KATP) channel inhibition in OBX mice. Repeated memantine administration at 1–3 mg/kg p.o. for 14 days starting at 10 days after OBX surgery significantly rescued cognitive deficits in OBX mice, as assessed using Y-maze, novel object recognition, and passive avoidance tasks. Consistent with the rescued cognitive deficits in OBX mice, long-term potentiation (LTP) in the hippocampal cornu ammonis (CA) 1 region was markedly restored with memantine administration. As demonstrated by immunoblotting, the reductions of calcium/calmodulin-dependent protein kinase II (CaMKII) α (Thr-286) autophosphorylation and calcium/calmodulin-dependent protein kinase IV (CaMKIV; Thr-196) phosphorylation in the CA1 region of OBX mice were significantly restored with memantine. Conversely, pre-treatment with pinacidil, a KATP channel opener, failed to reinstate hippocampal LTP and CaMKII/CaMKIV activities in the CA1 region. Finally, improvement of cognitive deficits by memantine treatments was observed in OBX-operated Kir6.1 heterozygous (+/−) mice but not in OBX-operated Kir6.2 heterozygous (+/−) mice. Overall, our study demonstrates that memantine rescues OBX-induced cognitive deficits via Kir6.2 channel inhibition in the CA1 region.
ATP‐sensitive potassium (KATP) channels couple intracellular metabolism to the electrical activity by regulating K⁺ flux across the plasma membrane, thus playing an important role in both normal and pathophysiology. To understand the mechanism of ATP regulating biological ion channels, developing an ATP‐responsive artificial nanochannel is an appealing but challenging topic because KATP channel is a heteromultimer of two subunits (potassium channel subunit (Kir6.x) and sulfonylurea receptor (SUR)) and exhibit dynamic functions with adjustability and reversibility. Inspired by the structure of KATP channels, we designed a smart copolymer modified nanochannel that may address the challenge. In the tricomponent poly(N-isopropylacrylamide) (PNIPAAm, PNI)-based copolymer system, phenylthiourea was used to bind the phosphate units of nucleotides and phenylboronic acid was introduced to combine the pentose ring of the nucleoside unit. Besides, a -COOH group with electron-withdrawing property was added into the phenylthiourea units, which may promote the hydrogen-bond-donating ability of thiourea. Specially, the smart copolymer not only provided static binding sites for recognition but also translated the recognition of ATP into their dynamic conformational transitions by changing the hydrogen-bonding environments surrounding PNIPAAm chains, thus achieving the gating function of nanochannel, which resembled the integration and coordination of Kir6.x and SUR units in active KATP. The ATP-regulated ion channel exhibited excellent stability and reversibility. This study is the first example showing how to learn from nature to assemble the ATP‐responsive artificial nanochannel and demonstrate the possible mechanism of ATP gating.
Glucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (K ATP ) channels formed with Kir6.2 and SUR1 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through K ATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function.
HIGHLIGHTS
Most cortical neurons subtypes express pancreatic beta-cell like K ATP channels.
Lactate enhances spiking activity via its uptake and closure of K ATP channels.
Cortical neurons take up and oxidize lactate.
Cortical neurons produce ATP mainly by oxidative phosphorylation.
Aberrant depressive-like behaviors in olfactory bulbectomized (OBX) mice have been documented by previous studies. Here, we show that memantine enhances adult neurogenesis in the subgranular zone of the hippocampal dentate gyrus (DG) and improves depressive-like behaviors via inhibition of the ATP-sensitive potassium (KATP) channel in OBX mice. Treatment with memantine (1–3 mg/kg; per os (p.o.)) for 14 days significantly improved depressive-like behaviors in OBX mice, as assessed using the tail-suspension and forced-swim tests. Treatment with memantine also increased the number of BrdU-positive neurons in the DG of OBX mice. In the immunoblot analysis, memantine significantly increased phosphorylation of CaMKIV (Thr-196) and Akt (Ser-473), but not ERK (Thr-202/Tyr-204), in the DG of OBX mice. Furthermore, phosphorylation of GSK3β (Ser-9) and CREB (Ser-133), and BDNF protein expression levels increased in the DG of OBX mice, possibly accounting for the increased adult neurogenesis owing to Akt activation. In contrast, both the improvement of depressive-like behaviors and increase in BrdU-positive neurons in the DG following treatment with memantine were unapparent in OBX-treated Kir6.1 heterozygous (+/-) mice but not OBX-treated Kir6.2 heterozygous (+/-) mice. Furthermore, the increase in CaMKIV (Thr-196) and Akt (Ser-473) phosphorylation and BDNF protein expression levels was not observed in OBX-treated Kir6.1 +/- mice. Overall, our study shows that memantine improves OBX-induced depressive-like behaviors by increasing adult neurogenesis in the DG via Kir6.1 channel inhibition.
Epilepsy is characterized by repeated spontaneous seizures and remains untreatable due to its complicate pathogenesis. Low-dose hydrogen sulfide (H2S) has been shown to exert antiepileptic and protective effects in the central nervous system, but the administration of H2S gas to suppress seizures is difficult. In the present study, we synthesized a safe and efficient carbazole-based H2S donor from aldehydes with a one-pot procedure and characterized this specific H2S donor via proton nuclear magnetic resonance (¹HNMR), scanning electron microscopy (SEM), and absorption spectroscopy. In vitro and in vivo risk assessments demonstrated that the H2S donor (up to a 400-μM concentration) had sufficient biocompatibility and membrane permeability and suppressed epileptic seizures. Importantly, the suppression of seizures was determined in the brain when the H2S donor was injected into the lateral ventricle in a rat model of advanced seizures. Furthermore, the mechanism by which the H2S donor suppressed the expression of seizures may have been related to H2S donor-induced increases in the expressions of the ATP-sensitive potassium channel (KATP) subunits, Kir6.2 and SUR1. Taken together, these assays suggested that our novel H2S donor may represent a potential therapeutic strategy for ameliorating seizures in epilepsy.
Oncotic cell death or oncosis represents a major mechanism of cell death in ischaemic stroke, occurring in many central nervous system (CNS) cell types including neurons, glia and vascular endothelial cells. In stroke, energy depletion causes ionic pump failure and disrupts ionic homeostasis. Imbalance between the influx of Na⁺ and Cl⁻ ions and the efflux of K⁺ ions through various channel proteins and transporters creates a transmembrane osmotic gradient, with ensuing movement of water into the cells, resulting in cell swelling and oncosis. Oncosis is a key mediator of cerebral oedema in ischaemic stroke, contributing directly through cytotoxic oedema, and indirectly through vasogenic oedema by causing vascular endothelial cell death and disruption of the blood-brain barrier (BBB). Hence, inhibition of uncontrolled ionic flux represents a novel and powerful strategy in achieving neuroprotection in stroke. In this review, we provide an overview of oncotic cell death in the pathology of stroke. Importantly, we summarised the therapeutically significant pathways of water, Na⁺, Cl⁻ and K⁺ movement across cell membranes in the CNS and their respective roles in the pathobiology of stroke.
The fertility and survival of an individual rely on the ability of the periphery to promptly, effectively and reproducibly communicate with brain neural networks that control reproduction, food intake and energy homeostasis. Tanycytes, a specialized glial cell type lining the wall of the third ventricle in the median eminence of the hypothalamus, appear to act as the linchpin of these processes by dynamically controlling the secretion of neuropeptides into the portal vasculature by hypothalamic neurons and regulating blood-brain and blood-cerebrospinal fluid exchanges, both processes that depend on the ability of these cells to adapt their morphology to the physiological state of the individual. In addition to their barrier properties, they possess the ability to sense blood glucose levels, and play a fundamental and active role in shuttling circulating metabolic signals to hypothalamic neurons that control food intake. Moreover, accumulating data suggest that, in keeping with their putative descent from radial glial cells, tanycytes are endowed with neural stem cell properties and may respond to dietary or reproductive cues by modulating hypothalamic neurogenesis. Tanycytes could thus constitute the missing link in the loop connecting behavior, hormonal changes, signal transduction, central neuronal activation and, finally, behavior again. In this paper, we will examine these recent advances in the understanding of tanycytic plasticity and function in the hypothalamus, and the underlying molecular mechanisms. We will also discuss the putative involvement and therapeutic potential of hypothalamic tanycytes in metabolic and fertility disorders.
Affective disorders including depression and anxiety are among the most prevalent behavioral abnormalities in patients with Alzheimer's disease (AD), which affect the quality of life and progression of the disease. Dysregulation of the hypothalamic-pituitary-adrenal-(HPA) axis has been reported in affective disorders and AD. Recent studies revealed that current antidepressant drugs are not completely effective for treating anxiety- and depression-related disorders in people with dementia. ATP-sensitive-potassium-(KATP) channels are well-known to be involved in AD pathophysiology, HPA axis function and the pathogenesis of depression and anxiety-related behaviors. Thus, targeting of KATP channel may be a potential therapeutic strategy in AD. Hence, we investigated the effects of intracerebroventricular injection of Aβ25-35 alone or in combination with glibenclamide, KATP channel inhibitor on depression- and anxiety-related behaviors as well as HPA axis response to stress in rats. To do this, non-Aβ25-35- and Aβ25-35-treated rats were orally treated with glibenclamide, then the behavioral consequences were assessed using sucrose preference, forced swim, light-dark box and plus maze tests. Stress-induced corticosterone levels following forced swim and plus maze tests were also evaluated as indicative of abnormal HPA-axis-function. Aβ25-35 induced HPA axis hyperreactivity and increased depression- and anxiety-related symptoms in rats. Our results showed that blockade of KATP channels with glibenclamide decreased depression- and anxiety-related behaviors by normalizing HPA axis activity in Aβ25-35-treated rats. This study provides additional evidence that Aβ administration can induce depression- and anxiety-like symptoms in rodents, and suggests that KATP channel inhibitors may be a plausible therapeutic strategy for treating affective disorders in AD patients.
Glucokinase (GK), the hexokinase involved in glucosensing in pancreatic β-cells, is also expressed in arcuate nucleus (AN) neurons and hypothalamic tanycytes, the cells that surround the basal third ventricle (3V). Several lines of evidence suggest that tanycytes may be involved in the regulation of energy homeostasis. Tanycytes have extended cell processes that contact the feeding-regulating neurons in the AN, particularly, agouti-related protein (AgRP), neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC) neurons. In this study, we developed an adenovirus expressing GK shRNA to inhibit GK expression in vivo. When injected into the 3V of rats, this adenovirus preferentially transduced tanycytes. qRT-PCR and Western blot assays confirmed GK mRNA and protein levels were lower in GK knockdown animals compared to the controls. In response to an intracerebroventricular glucose injection, the mRNA levels of anorexigenic POMC and CART and orexigenic AgRP and NPY neuropeptides were altered in GK knockdown animals. Similarly, food intake, meal duration, frequency of eating events and the cumulative eating time were increased, whereas the intervals between meals were decreased in GK knockdown rats, suggesting a decrease in satiety. Thus, GK expression in the ventricular cells appears to play an important role in feeding behavior.
ATP-sensitive potassium (KATP) channels are known as the potassium-conducting channels coupling cellular metabolic status to membrane electrical activity. Either an increase in ADP or decrease in ATP levels opens KATP channels and hyperpolarizes the membrane potential. Knocking out the inward rectifier K+ channel (Kir6.2) subunit of the KATP channels or pharmacologically blocking KATP channels increases brain injury. Overexpression of the Kir6.2 subunit or pharmacologically opening KATP channel reduces neuronal injury from ischemic insults. Hypoxic preconditioning (HPC) provides neuroprotection against subsequent ischemic brain injury. Similar to its effects in heart, KATP channels contribute to the hypoxic preconditioning-induced neuroprotection. KATP channels may therefore serve as therapeutic targets in ischemic or hypoxic-ischemic brain injury.
Here, we report a novel target of the drug memantine, ATP-sensitive K(+) (KATP) channels, potentially relevant to memory improvement. We confirmed that memantine antagonizes memory impairment in Alzheimer's model APP23 mice. Memantine increased CaMKII activity in the APP23 mouse hippocampus, and memantine-induced enhancement of hippocampal long-term potentiation (LTP) and CaMKII activity was totally abolished by treatment with pinacidil, a specific opener of KATP channels. Memantine also inhibited Kir6.1 and Kir6.2 KATP channels and elevated intracellular Ca(2+) concentrations in neuro2A cells overexpressing Kir6.1 or Kir6.2. Kir6.2 was preferentially expressed at postsynaptic regions of hippocampal neurons, whereas Kir6.1 was predominant in dendrites and cell bodies of pyramidal neurons. Finally, we confirmed that Kir6.2 mutant mice exhibit severe memory deficits and impaired hippocampal LTP, impairments that cannot be rescued by memantine administration. Altogether, our studies show that memantine modulates Kir6.2 activity, and that the Kir6.2 channel is a novel target for therapeutics to improve memory impairment in Alzheimer disease patients.Molecular Psychiatry advance online publication, 25 October 2016; doi:10.1038/mp.2016.187.
Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by beta-amyloid (Aβ) deposition, neurofibrillary tangles and cognitive decline. Recent pharmacologic studies have found that KATP channels may play a role in AD and could be a potential therapeutic target. Interestingly, these channels are found in both neurons and astrocytes. One of the hallmarks associated with AD is reactive gliosis and dysfunction of astrocytes has been identified in several neuropathological conditions including AD. Thus the goal of this study was to examine whether the pore-forming subunits of KATP channels, Kir6.1 and Kir6.2, are altered in the hippocampus of the 3xTg mouse model of AD and in human AD tissue obtained from the Chinese brain bank in a cell type specific manner. Specifically, in the old 3xTg-AD mice, and age-matched controls, we examined glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), Kir6.1 and Kir6.2 in hippocampal region CA1 with a combination of immunoblotting and immunohistochemistry (IHC). A time point was selected when memory impairment and histopathological changes have been reported to occur in 3xTg-AD mice. In human AD and age-matched control tissue IHC experiments were performed using GFAP and Kir6.2. In the hippocampus of 3xTg-AD mice, compared to wild type controls, western blots showed a significant increase in GFAP indicating astrogliosis. Further, there was an increase in Kir6.2, but not Kir6.1 in the plasma membrane fraction. IHC examination of hippocampal region CA1 in 3xTg-AD sections revealed an increase in Kir6.2 immunoreactivity in astrocytes as assessed with GFAP and GS. In human AD tissue similar data were obtained. There was an increase in GFAP-IR in the stratum oriens and alveus of CA1 concomitant with an increase in Kir6.2-IR in cells with an astrocytic-like morphology. Dual immunofluorescence revealed a dramatic increase in co-localization of Kir6.2-IR and GFAP-IR. Taken together, these data demonstrate that increased Kir6.2 is seen in reactive astrocytes in old 3xTg-AD mice and human AD tissue. These changes could dramatically alter astrocytic function and subsequently contribute to AD phenotype in either a compensatory or pathophysiological manner.
The habenular complexes represent phylogenetically constant structures in the diencephalon of all vertebrates. Available evidence suggests that this area is engaged in a variety of important biological functions, such as reproductive behaviors, central pain processing nutrition, sleep-wake cycles, stress responses, and learning. Based on Nissl-stained sections, one medial nucleus and two lateral nuclei (divisions) have been widely accepted in the rat. Cytochemical, hodologic, and functional studies suggest a considerably more complex subnuclear structure. To improve our knowledge of the precise structural composition of the habenular complexes, we have systematically investigated their fine ultrastructure in the rat. Based on the detailed analysis of complete series of large, semithin sections supplemented with electron photomicrographs of selected fields, clear criteria for the delineation of five distinct subnuclei of the medial and ten subnuclei of the lateral habenular complexes were elaborated for the first time. All 15 subnuclei were reconstructed, and their dimensions were determined. A medial and lateral stria medullaris were described. Different roots of the fasciculus retroflexus were differentiated within the medial and lateral habenular complexes. The topographical relationships with respect to the adjacent habenular areas as well as to the neighboring thalamic nuclei were identified and demonstrated. The new understanding of the subnuclear organization of the habenular complexes certainly will facilitate further functional investigations. Whether the newly identified subnuclei finally will be recognized as functionally distinct awaits ongoing immunocytochemical, hodologic, and functional studies. (C) 1999 Wiley-Liss, Inc.
The recognition of the components of the central nervous system is directly related to questions concerning the interconnections of neuronal systems within the brain. Therefore, it is necessary to combine a description of structural details with their exact topographical orientation with respect to other structures. Thus, an analysis of neuronal systems requires a profound knowledge of the localization of the smallest areas. It necessitates a specific preparatory method in order to investigate distinct areas by light and electron microscopic means.
The major physiological stimulus for the secretion of insulin from the
pancreatic β-cell is an increase in the plasma glucose
concentration. It is well established that glucose-stimulated insulin
secretion is associated with the appearance of electrical activity in
the β-cell1,2 glucose concentrations above the threshold
level for insulin release produce a slow membrane depolarization
followed by either oscillatory bursts of action potentials (5-15 mM
glucose) or continuous spiking (>16mM glucose). Tracer flux
studies3 and microelectrode measurements using intact islets
of Langerhans4 have indicated that the initial depolarization
induced by glucose is caused by a decrease in the resting membrane
permeability to potassium. Evidence also suggests that the
electrical5, ionic6 and secretory
responses7,8 to glucose are mediated by the metabolism of the
sugar within the β-cell. By using cell-attached membrane
patches9 from isolated rat pancreatic β-cells, we have
now identified a potassium channel (G-channel) that is active at the
resting potential and is inhibited by glucose. Closure of this channel
requires glucose metabolism. This is the first report of a potassium
channel whose activity is modulated by glucose, and which may couple
metabolic and ionic events involved in the secretion of insulin.
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.
The sulphonylureas, tolbutamide (0.1–10 m m ) and glibenclamide (0.1–100 μ m ) were shown not to inhibit ATP‐K ⁺ channel currents when applied to inside‐out membrane patches excised from rat cultured cerebral cortex or freshly‐dispersed ventromedial hypothalmic nucleus (VMHN) neurones.
Saturable binding sites for [ ³ H]‐glibenclamide, with similar affinity constants are present in rat cerebral cortex and hypothalamic membranes. The density of binding sites was lower in the hypothalamus than cortex.
Intracellular recordings from glucoreceptive VMHN neurones in hypothalamic slices were obtained. In the absence of glucose, tolbutamide (0.1 m m ) depolarized these cells, increased membrane resistance and elicited action potentials.
Tolbutamide (0.1 m m ) inhibited ATP‐K ⁺ channel currents and induced action current activity in cell‐attached recordings from glucoreceptive VMHN neurones.
Glibenclamide (10–500 n m ) had no effect per se on glucoreceptive VMHN neurones but did antagonize the actions of tolbutamide.
It is concluded that the hypothalamic (and perhaps cortical) sulphonylurea receptors are not directly coupled to ATP‐K ⁺ channels.
Intracellular recordings were made from neurones located in the ventromedial hypothalamic nucleus (VMHN) of slices from rat hypothalamus. These neurones were hyperpolarized on removal of extracellular glucose, resulting in an inhibition of firing, actions which were reversed on the re-introduction of glucose. No reversal of the inhibition of firing was observed when 10 mM mannoheptulose, an inhibitor of glucose metabolism, was present in addition to glucose. Increasing the mannoheptulose concentration to 20 mM resulted in further hyperpolarization. Cell-attached recordings from isolated neurones revealed that an increase in extracellular glucose inhibited a K+ channel and increased action current activity. ATP induced closure of this K+ channel when applied to inside-out membrane patches. Closure was also induced by Mg-free ATP or the non-hydrolysable ATP-analogue, adenylylimidodiphosphate indicating no requirement for ATP metabolism. We suggest that the closure of ATP-sensitive potassium channels underlies increased hypothalamic firing following an increase in extracellular glucose.
The major physiological stimulus for the secretion of insulin from the pancreatic beta-cell is an increase in the plasma glucose concentration. It is well established that glucose-stimulated insulin secretion is associated with the appearance of electrical activity in the beta-cell; glucose concentrations above the threshold level for insulin release produce a slow membrane depolarization followed by either oscillatory bursts of action potentials (5-15 mM glucose) or continuous spiking (greater than 16 mM glucose). Tracer flux studies and microelectrode measurements using intact islets of Langerhans have indicated that the initial depolarization induced by glucose is caused by a decrease in the resting membrane permeability to potassium. Evidence also suggests that the electrical, ionic and secretory responses to glucose are mediated by the metabolism of the sugar within the beta-cell. By using cell-attached membrane patches from isolated rat pancreatic beta-cells, we have now identified a potassium channel (G-channel) that is active at the resting potential and is inhibited by glucose. Closure of this channel requires glucose metabolism. This is the first report of a potassium channel whose activity is modulated by glucose, and which may couple metabolic and ionic events involved in the secretion of insulin.
Preconditioning with sublethal ischemia protects against neuronal damage after subsequent lethal ischemic insults in hippocampal neurons. A pharmacological approach using agonists and antagonists at the adenosine A1 receptor as well as openers and blockers of ATP-sensitive K+ channels has been combined with an analysis of neuronal death and gene expression of subunits of glutamate and gamma-aminobutyric acid receptors, HSP70, c-fos, c-jun, and growth factors. It indicates that the mechanism of ischemic tolerance involves a cascade of events including liberation of adenosine, stimulation of adenosine A1 receptors, and, via these receptors, opening of sulfonylurea-sensitive ATP-sensitive K+ channels.
The presence of adenosine triphosphate-regulated potassium channels (K-ATPs) in midbrain dopamine neurons is currently in dispute. This was investigated using whole-cell patch-clamp recordings from dopamine neurons in slices of midbrain from 9-12-d-old rats. Intracellular dialysis with Mg2+ ATP-free solutions resulted in a membrane hyperpolarization (14 +/- 6 mV), or outward current (102 +/- 27 pA) in voltage clamp, which developed over 14 +/- 1.6 min. These hyperpolarizations and outward currents were reversed by the K-ATP-blocking sulfonylureas tolbutamide (100 microM) and glibenclamide (3 microM). This sulfonylurea-sensitive outward current was associated with an increase in a nonrectifying (between -50 and -130 mV) conductance of approximately 2 nS, with a reversal potential of -100 mV (in 2.5 mM extracellular potassium), consistent with a potassium conductance increase. When the dialyzate contained Mg2+ATP (2 mM), no slowly developing hyperpolarization or outward current occurred, and tolbutamide (200 microM) and glibenclamide (10 microM) did not affect membrane potential or current. Additionally, the "potassium channel activators" (KCAs) lemakalim (200 microM) and pinacidil (50 microM) were also without effect on the membrane potential or holding current in these cells. The hyperpolarizations and outward currents caused by baclofen and quinpirole, agonists at GABAB and D2 receptors, respectively, were neither blocked by sulfonylureas nor occluded by the current resulting from depletion of intracellular ATP. Thus, these K-ATPs appear independent of the potassium channels coupled to GABAB and D2 receptors in these cells. This ATP-regulated potassium conductance may constitute a protective mechanism during anoxia or hypoglycemia, by restricting membrane depolarization of dopamine neurons when intracellular ATP levels fall.
Depending on its severity and duration, O2 deprivation activates mechanisms that can lead to profound deleterious changes in neuronal structure and function. Hypoxia also evokes inherent adaptive mechanisms that can possibly delay injury and increase neuronal survival. One of these neuronal adaptive mechanisms is believed to be the activation of K+ channels, but direct evidence for their activation is lacking. We performed experiments to test the hypothesis that hypoxia induces activation of K+ channels via changes in cytosolic and membrane factors such as ATP, Ca2+, and membrane potential. The effect of hypoxia on single-channel currents was studied in rat substantia nigra neurons, since these have a high density of glibenclamide binding sites. In cell-attached patches, hypoxia or cyanide reversibly activated an outward current. This hypoxia-activated current in excised inside-out patches was K+ selective and voltage dependent, and had a high sensitivity to internal ATP, ADP, and AMP-PNP, a nonhydrolyzable ATP analog. Activation of this channel required the presence of free Ca2+ on the cytosolic side, but charybdotoxin or apamin did not have any effect on this channel. The effect of ATP on channel activity was not a result of Ca2+ chelation because Mg.ATP in high Mg2+ background and K2.ATP in high Ca2+ environment inhibited the channel. These results suggest that although this hypoxia-activated K+ channel shares properties with ATP-sensitive K+ (KATP) channels in other tissues, substantia nigra neurons seem to have a different subtype or isoform of KATP channels. Gating this channel by multiple factors simultaneously would allow this channel to be particularly suitable for activation during metabolic stress.
The adenohypophysis contains high-affinity binding sites for antidiabetic sulfonylureas that are specific blockers of ATP-sensitive K+ channels. The binding protein has a M(r) of 145,000 +/- 5000. The presence of ATP-sensitive K+ channels (26 pS) has been demonstrated by electrophysiological techniques. Intracellular perfusion of adenohypophysis cells with an ATP-free medium to activate ATP-sensitive K+ channels induces a large hyperpolarization (approximately 30 mV) that is antagonized by antidiabetic sulfonylureas. Diazoxide opens ATP-sensitive K+ channels in adenohypophysis cells as it does in pancreatic beta cells and also induces a hyperpolarization (approximately 30 mV) that is also suppressed by antidiabetic sulfonylureas. As in pancreatic beta cells, glucose and antidiabetic sulfonylureas depolarize the adenohypophysis cells and thereby indirectly increase Ca2+ influx through L-type Ca2+ channels. The K+ channel opener diazoxide has an opposite effect. Opening ATP-sensitive K+ channels inhibits growth hormone secretion and this inhibition is eliminated by antidiabetic sulfonylureas.
We have isolated a cDNA encoding a novel isoform of the sulfonylurea receptor from a mouse heart cDNA library. Coexpression of this isoform and BIR (Kir6.2) in a mammalian cell line elicited ATP-sensitive K+ (KATP) channel currents. The channel was effectively activated by both diazoxide and pinacidil, which is the feature of smooth muscle KATP channels. Sequence analysis indicated that this clone is a variant of cardiac type sulfonylurea receptor (SUR2). The 42 amino acid residues located in the carboxyl-terminal end of this novel sulfonylurea receptor is, however, divergent from that of SUR2 but highly homologous to that of the pancreatic one (SUR1). Therefore, this short part of the carboxyl terminus may be important for diazoxide activation of KATP channels. The reverse transcription-polymerase chain reaction analysis showed that mRNA of this clone was ubiquitously expressed in diverse tissues, including brain, heart, liver, urinary bladder, and skeletal muscle. These results suggest that this novel isoform of sulfonylurea receptor is a subunit reconstituting the smooth muscle KATP channel.
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.
Whole‐cell patch‐clamp recordings were made from rat striatal cholinergic interneurones in slices of brain tissue in vitro. In the absence of ATP in the electrode solution, these neurones were found to gradually hyperpolarize through the induction of an outward current at −60 mV. This outward current and the resultant hyperpolarization were blocked by the sulphonylureas tolbutamide and glibenclamide and by the photorelease of caged ATP within neurones.
This ATP‐sensitive outward current was not observed when 2 mM ATP was present in the electrode solution. Under these conditions, 500 μM diazoxide was found to induce an outward current that was blocked by tolbutamide.
Using permeabilized patch recordings, neurones were shown to hyperpolarize in response to glucose deprivation or metabolic poisoning with sodium azide (NaN 3 ). The resultant hyperpolarization was blocked by tolbutamide.
In cell‐attached recordings, metabolic inhibition with 1 mM NaN 3 revealed the presence of a tolbutamide‐sensitive channel exhibiting a unitary conductance of 44.1 pS.
Reverse transcription followed by the polymerase chain reaction using cytoplasm from single cholinergic interneurones demonstrated the expression of the ATP‐sensitive potassium (K ATP ) channel subunits Kir6.1 and SUR1 but not Kir6.2 or SUR2.
It is concluded that cholinergic interneurones within the rat striatum exhibit a K ATP channel current and that this channel is formed from Kir6.1 and SUR1 subunits.
ATP-sensitive potassium (K-ATP) channels couple the metabolic state to cellular excitability in various tissues. Several isoforms of the K-ATP channel subunits, the sulfonylurea receptor (SUR) and inwardly rectifying K channel (Kir6.X), have been cloned, but the molecular composition and functional diversity of native neuronal K-ATP channels remain unresolved. We combined functional analysis of K-ATP channels with expression profiling of K-ATP subunits at the level of single substantia nigra (SN) neurons in mouse brain slices using an RT-multiplex PCR protocol. In contrast to GABAergic neurons, single dopaminergic SN neurons displayed alternative co-expression of either SUR1, SUR2B or both SUR isoforms with Kir6.2. Dopaminergic SN neurons expressed alternative K-ATP channel species distinguished by significant differences in sulfonylurea affinity and metabolic sensitivity. In single dopaminergic SN neurons, co-expression of SUR1 + Kir6.2, but not of SUR2B + Kir6.2, correlated with functional K-ATP channels highly sensitive to metabolic inhibition. In contrast to wild-type, surviving dopaminergic SN neurons of homozygous weaver mouse exclusively expressed SUR1 + Kir6.2 during the active period of dopaminergic neurodegeneration. Therefore, alternative expression of K-ATP channel subunits defines the differential response to metabolic stress and constitutes a novel candidate mechanism for the differential vulnerability of dopaminergic neurons in response to respiratory chain dysfunction in Parkinson's disease.
ATP-sensitive K+ channels (KATP channels) play important roles in many cellular functions by coupling cell metabolism to electrical activity. The KATP channels in pancreatic beta-cells are thought to be critical in the regulation of glucose-induced and sulfonylurea-induced insulin secretion. Until recently, however, the molecular structure of the KATP channel was not known. Cloning members of the novel inwardly rectifying K+ channel subfamily Kir6.0 (Kir6.1 and Kir6.2) and the sulfonylurea receptors (SUR1 and SUR2) has clarified the molecular structure of KATP channels. The pancreatic beta-cell KATP channel comprises two subunits: a Kir6.2 subunit and an SUR1 subunit. Molecular biological and molecular genetic studies have provided insights into the physiological and pathophysiological roles of the pancreatic beta-cell KATP channel in insulin secretion.
ATP-sensitive K+ channels (KATP channels) play important roles in many cellular functions by coupling cell metabolism to electrical activity. The KATP channels in pancreatic beta-cells are thought to be critical in the regulation of glucose-induced and sulfonylurea-induced insulin secretion. Until recently, however, the molecular structure of the KATP channel was not known. Cloning members of the novel inwardly rectifying K+ channel subfamily Kir6.0 (Kir6.1 and Kir6.2) and the sulfonylurea receptors (SUR1 and SUR2) has clarified the molecular structure of KATP channels. The pancreatic beta-cell KATP channel comprises two subunits: a Kir6.2 subunit and an SUR1 subunit. Molecular biological and molecular genetic studies have provided insights into the physiological and pathophysiological roles of the pancreatic beta-cell KATP channel in insulin secretion.
Ischemic brain injury produced by stroke or cardiac arrest is a major cause of human neurological disability. Steady advances in the neurosciences have elucidated the pathophysiological mechanisms of brain ischemia and have suggested many therapeutic approaches to achieve neuroprotection of the acutely ischemic brain that are directed at specific injury mechanisms. In the second portion of this two-part review, the following potential therapeutic approaches to acute ischemic injury are considered: 1) modulation of nonglutamatergic neurotransmission, including monoaminergic systems (dopamine, norepinephrine, serotonin), γ-aminobutyric acid, and adenosine; 2) mild-to-moderate therapeutic hypothermia; 3) calcium channel antagonism; 4) an tagonism of oxygen free radicals; 5) modulation of the nitric oxide system; 6) antagonism of cytoskeletal proteolysis; 7) growth factor administration; 8) therapy directed at cellular mediators of injury; and 9) the rationale for combination pharmacotherapy. The Neuroscientist 1:164-175, 1995
ATP-sensitive K+(KATP) channels are abundantly expressed in the heart and may be involved in the pathogenesis of myocardial ischemia. These channels are heteromultimeric, consisting of four pore-forming subunits (Kir6.1, Kir6.2) and four sulfonylurea receptor (SUR) subunits in an octameric assembly. Conventionally, the molecular composition of KATPchannels in cardiomyocytes and pancreatic β -cells is thought to include the Kir6.2 subunit and either the SUR2A or SUR1 subunits, respectively. However, Kir6.1 mRNA is abundantly expressed in the heart, suggesting that Kir6.1 and Kir6.2 subunits may co-assemble to form functional heteromeric channel complexes. Here we provide two independent lines of evidence that heteromultimerization between Kir6.1 and Kir6.2 subunits is possible in the presence of SUR2A. We generated dominant negative Kir6 subunits by mutating the GFG residues in the channel pore to a series of alanine residues. The Kir6.1-AAA pore mutant subunit suppressed both wt-Kir6.1/SUR2A and wt-Kir6.2/SUR2A currents in transfected HEK293 cells. Similarly, the dominant negative action of Kir6.2-AAA does not discriminate between either of the wild-type subunits, suggesting an interaction between Kir6.1 and Kir6.2 subunits within the same channel complex. Biochemical data support this concept: immunoprecipitation with Kir6.1 antibodies also co-precipitates Kir6.2 subunits and conversely, immunoprecipitation with Kir6.2 antibodies co-precipitates Kir6.1 subunits. Collectively, our data provide direct electrophysiological and biochemical evidence for heteromultimeric assembly between Kir6.1 and Kir6.2. This paradigm has profound implications for understanding the properties of native KATPchannels in the heart and other tissues.
Select groups of neurons within the brain alter their firing rate when ambient glucose levels change. These glucose-responsive neurons are integrated into systems which control energy balance in the body. They contain an ATP-sensitive K+ channel (KATP) which mediates this response. KATP channels are composed of an inwardly rectifying pore-forming unit (Kir6.1 or Kir6.2) and a sulfonylurea binding site. Here, we examined the anatomical distribution and phenotype of cells containing Kir6.2 mRNA within the rat brain by combinations of in situ hybridization and immunocytochemistry. Cells containing Kir6.2 mRNA were widely distributed throughout the brain without apparent concentration in areas known to contain specific glucose-responsive neurons. Kir6.2 mRNA was present in neurons expressing neuron-specific enolase, tyrosine hydroxylase, neuropeptide Y (NPY) and the glutamic acid decarboxylase isoform, GAD65. No astrocytes expressing glial fibrillary acidic protein or oligodendrocytes expressing carbonic anhydrase II were found to co-express Kir6.2 mRNA. Virtually all of the NPY neurons in the hypothalamic arcuate n. and catecholamine neurons in the substantia nigra, pars compacta and locus coeruleus contained Kir6.2 mRNA. Epinephrine neurons in the C2 area also expressed high levels of Kir6.2, while noradrenergic neurons in A5 and A2 areas expressed lower levels. The widespread distribution of Kir6.2 mRNA suggests that the KATP channel may serve a neuroprotective role in neurons which are not directly involved in integrating signals related to the body's energy homeostasis.
The Kir6.1/uKATP-1, subunit of ATP-sensitive K+ channels (KATP), was localized in adult rat brain by in situ hybridization and immunohistochemistry. The mRNA of this subunit was ubiquitously expressed in various neurons and nuclei of the adult rat brain. Interestingly, Kir6.1/uKATP-1 mRNA was also expressed in glial cells. Kir6.1/uKATP-1 protein staining gave a dispersed array of fine dots throughout all neurons and glial cells examined. Under electron microscope, the immunoreactive products were specifically restricted to the mitochondria. The present study indicates that this KATP subunit is localized in the mitochondria and may play a fundamental role in vital brain function.
Functional and molecular properties of ATP‐sensitive K ⁺ (K ATP ) channels were studied in dorsal vagal neurons (DVNs) of rat brainstem slices using patch‐clamp and single‐cell antisense RNA amplification‐polymerase chain reaction (PCR) techniques.
In the cell‐attached configuration, 1 mM cyanide resulted in block of spontaneous firing and concomitant opening of single channels with a mean single open time of 2‐3 ms and a burst duration of up to several hundred milliseconds. Inhibition of such single‐channel activity with 200 μM tolbutamide led to the reappearance of spontaneous discharge.
Whole‐cell recordings during anoxia revealed a hyperpolarization of the DVNs. Harvesting of cytoplasm, antisense RNA amplification and subsequent PCR showed coexpression for single DVNs of mRNA for the sulphonylurea receptor SUR1 isoform and for the inwardly rectifying K ⁺ channel subunit Kir6.2, but not for the SUR2 or Kir6.1 isoforms of these channel/receptor subclasses.
Upon anoxia, a stable depolarization by less than 10 mV was observed in non‐excitable cells in the dorsal vagal nucleus. These cells, which expressed glial fibrillary acidic protein (GFAP), showed a high level of mRNA for Kir6.2, a weak signal for SUR1, whereas SUR2 or Kir6.1 were not detected.
The results suggest that functional K ATP channels in DVNs are constituted by the formation of Kir6.2 subunits with SUR1 receptors.
Previous studies in our laboratory have shown that cromakalim activates a tetraethylammonium-sensitive K+ current in cultured embryonic rat hippocampal neurons. This phenomenon was further characterized using whole-cell voltage-clamp and single-channel recording techniques. Glyburide (1-25 microM), an antagonist of ATP-sensitive K+ channels, produced a concentration-dependent depression of the cromakalim-activated current. In contrast, charybdotoxin (100 nM), an antagonist of some Ca(2+)-dependent and other K+ channels, not only failed to block the effect of cromakalim but actually produced a moderate enhancement of the cromakalim-activated K+ current. Neither glyburide nor charybdotoxin affected resting or voltage-activated K+ currents in the absence of cromakalim. Exposure of the cells to energy-depleting conditions (0.24 micrograms/ml oligomycin and 10 mM 2-deoxy-D-glucose) also activated an outward current. Single-channel recordings in the cell-attached configuration showed that cromakalim (100 microM) stimulated the opening of flickery single channels having a unitary conductance of approximately 26 pS and a prolonged burst duration (mean open time, approximately 131 msec); similar channel openings were observed in patches from cells exposed to energy-depleting conditions. In patches containing a single K+ channel, the open probability in the presence of cromakalim was approximately 0.6 and in the presence of energy-depleting conditions was approximately 0.8; in the absence of either of these treatments, channel openings were not observed. Glyburide produced a reversible inhibition of the channels activated by cromakalim and energy-depleting conditions. These data provide additional support for the existence of ATP-sensitive K+ channels in central neurons and indicate that the K+ channels whose opening is stimulated by cromakalim are likely to be of the ATP-sensitive type.
Sulfonylurea-sensitive adenosine triphosphate (ATP)-regulated potassium (KATP) channels are present in brain cells and play a role in neurosecretion at nerve terminals. KATP channels in substantia nigra, a brain region that shows high sulfonylurea binding, are inactivated by high glucose concentrations and by antidiabetic sulfonylureas and are activated by ATP depletion and anoxia. KATP channel inhibition leads to activation of gamma-aminobutyric acid (GABA) release, whereas KATP channel activation leads to inhibition of GABA release. These channels may be involved in the response of the brain to hyper- and hypoglycemia (in diabetes) and ischemia or anoxia.
It has been known for some years that skeletal muscle develops a high potassium permeability in conditions that produce rigor, where ATP concentrations are low and intracellular Ca2+ is high. It has seemed natural to attribute this high permeability to K channels that are opened by internal Ca2+, especially as the presence of such channels has been demonstrated in myotubes and in the transverse tubular membrane system of adult skeletal muscle. However, as we show here, the surface membrane of frog muscle contains potassium channels that open at low internal concentrations of ATP (less than 2 mM). ATP induces closing of these channels without being split, apparently holding the channels in one of a number of closed states. The channels have at least two open states whose dwell times are voltage-dependent. Surprisingly, we find that these may be the most common K channels of the surface membrane of skeletal muscle.
ATP-sensitive channels were observed in isolated inside-out membrane patches from rat cultured central neurones. Two types of ATP-sensitive K+ channels were present in cortical neurones, one which had its open-state probability increased, the other its open-state probability decreased by application of ATP to the cytoplasmic membrane surface. Another, ATP-sensitive channel differing in ion conductance from all previously reported ATP-sensitive channels was also seen in patches from cortical neurones. This channel was nonselective with respect to Na+, K+ and Cl- ions and ATP produced a "flickery" type of block. The non-hydrolysable analogue, AMPPNP, did not mimic ATP and prevented ATP action. Preliminary experiments indicate that similar, but not, identical ATP-sensitive channels exist in cerebellar neurones.
Vasodilators are used clinically for the treatment of hypertension and heart failure. The effects of some vasodilators seem
to be mediated by membrane hyperpolarization. The molecular basis of this hyperpolarization has been investigated by examining
the properties of single K+ channels in arterial smooth muscle cells. The presence of adenosine triphosphate (ATP)-sensitive
K+ channels in these cells was demonstrated at the single channel level. These channels were opened by the hyperpolarizing
vasodilator cromakalim and inhibited by the ATP-sensitive K+ channel blocker glibenclamide. Furthermore, in arterial rings
the vasorelaxing actions of the drugs diazoxide, cromakalim, and pinacidil and the hyperpolarizing actions of vasoactive intestinal
polypeptide and acetylcholine were blocked by inhibitors of the ATP-sensitive K+ channels, suggesting that all these agents
may act through a common pathway in smooth muscle by opening ATP-sensitive K+ channels.
The membrane properties of neurones in the guinea-pig ventromedial hypothalamic nucleus (v.m.h.) were studied in in vitro brain slice preparations. The average resting potential was -62.9 +/- 5.4 mV (mean +/- S.D.), input resistance was 155 +/- 58 M omega, and action potential amplitude was 69.9 +/- 6.3 mV. Three types of neurone were identified. The type A neurones were characterized by a short membrane time constant (7.3 +/- 2.0 ms) and a small after-hyperpolarization (a.h.p.) (2.0 +/- 1.2 mV) with a short half decay time of 67 +/- 55 ms after stimulation with a long outward current pulse. Type B had a long time constant (18.8 +/- 5.7 ms) and a large a.h.p. (6.9 +/- 2.4 mV) with a medium half decay time of 203 +/- 90 ms. Type C was characterized by a long time constant (14.3 +/- 2.3 ms) and a large a.h.p. (6.5 +/- 1.5 mV) with a long half decay time of 478 +/- 230 ms. The slopes of the frequency-current (f-I) plots of the three types were different, particularly for the first spike interval. The slopes for the type A (414 +/- 102 impulses s-1 nA-1) and type B neurones (480 +/- 120 impulses s-1 nA-1) were steeper than that for the type C neurones (178 +/- 41 impulses s-1 nA-1). This difference is probably related to the relatively long first interval observed in the type C neurones. In all type B and a few type C neurones, when the membrane potential was hyperpolarized beyond--65 mV the application of orthodromic or direct stimulation generated a burst of spikes, consisting of a low-threshold response (l.t.r.) of low amplitude and superimposed high-frequency spikes. At the original resting potential, outward current pulses produced a train of low-frequency spikes. In type C neurones maintained in a depolarized state (about -50 mV), inward current pulses produced a specific delay of the return to the original membrane potential. This delayed return was thought to be generated by activation of a transient K+ (IA) conductance. Stimulation at the lateral edge of the v.m.h. produced excitatory post-synaptic potentials (e.p.s.p.s) in type A neurones, e.p.s.p.s with l.t.r. in type B neurones and e.p.s.p.-inhibitory post-synaptic potential sequences in type C neurones. About 20% of v.m.h. neurones, particularly the type C cells, were depolarized by glucose application with an associated increase in the input membrane resistance.(ABSTRACT TRUNCATED AT 400 WORDS)
This study quantitatively addresses the hypothesis that there is a systematic relationship between the morphologic characteristics of locus neurons and the particular target regions they innervate. Following horseradish peroxidase injections into selected terminal fields, locus coeruleus cell bodies are heavily labeled by retrograde transport so that somata size and shape, and in many cases primary dendritic pattern can be observed. This allows the classification of neurons as one of six cell types: large multipolar cells within ventral locus coeruleus, large multipolar cells in the anterior pole of locus coeruleus, fusiform cells in dorsal LC, posterior pole cells, medium-sized multipolar cells (termed core cells in this report), and small round cells. It was found that while core cells contribute to the innervation of all terminal fields examined, other cell types project to more restricted sets of targets. The contributions of each type to selected efferents are presented in detail. In particular, fusiform cells project to hippocampus and cortex, large multipolar cells in ventral locus coeruleus project to spinal cord and cerebellum, and small round cells in central and anterior locus coeruleus, as well as large multipolar cells in anterior locus coeruleus, project to hypothalamus. These results, in conjunction with those described in the preceding report, indicate that locus coeruleus is intrinsically organized with respect to efferent projections with much more specificity than has previously been evident. This high degree of organization is consistent with other recent demonstrations of functional specificity exhibited by locus coeruleus neurons.
THE lateral hypothalamic region (LH) is generally referred to as the feeding centre of the brain in the regulation of food intake, and many authors consider the ventromedial hypothalamic nucleus (VMH) to be the satiety centre1. Various hypotheses have been put forward to explain how the cells of these centres are activated, and one of these is the glucostat theory1. The existence of hypothalamic chemoreceptors, such as those sensitive to the concentration of blood glucose, can be inferred from studies of single unit discharges induced by intravenous or intracarotid administration of various solutions2-4 and from work on selective gold thioglucose lesions5. It has, however, been impossible to determine which centre is activated or inhibited first or whether both centres are modulated directly by a change in the concentration of blood glucose, because of the reciprocal relations which exist between the activities of the VMH and the LH2,6. We report here the direct effects of glucose on individual cells of the VMH and LH, which we studied by means of electro-osmotic applications of glucose from micropipettes-the method used by Krnjevic and Whittaker7 in other regions in the brain.
An outward current of unknown nature increases significantly when cardiac cells are treated with cyanide or subjected to hypoxia, and decreases on intracellular injection of ATP. We report here that application of the patch-clamp technique to CN-treated mammalian heart cells reveals specific K+ channels which are depressed by intracellular ATP (ATPi) at levels greater than 1 mM. For these channels, conductance in the outward direction is much larger than the inward rectifier K+ channel which is insensitive to ATP. AMP had no effect on the ATP-sensitive K+ channel, and ADP was less effective than ATP. Thus, the ATP-sensitive K+ channel seems to be important for regulation of cellular energy metabolism in the control of membrane excitability.
Extracellular action potentials were recorded from rat ventromedial hypothalamic nucleus (VMH) tissue slices in vitro. The identified glucoresponsive and non-glucoresponsive neurons were then visually located and observed by intracellular horseradish peroxidase staining. Glucoresponsive neurons, which were predominantly multipolar, were found near the center of the VMH, while non-glucoresponsive neurons, half of which were bipolar and half multipolar, were found randomly throughout the VMH.
1. Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). Locus coeruleus neurons responded to brief hypoxic stimuli (replacement of 95% O2-5% CO2 with 95% N2-5% CO2) with hyperpolarization and a cessation of spontaneous action potentials. When the cells were continuously hyperpolarized by about 15 mV in order to abolish spontaneous firing, hypoxia induced an early depolarization (HD), followed by a hypoxic hyperpolarization (HH) and after reoxygenation, a posthypoxic hyperpolarization (PHH). These responses were accompanied by a decrease in input resistance, which was larger during HH than during HD but, thereafter, became smaller during PHH. 2. The hypoxia-induced currents associated with the changes in membrane potential, at a holding potential of -70 mV, were an early inward current (HIC), a subsequent outward current (HOC) and after reoxygenation, another outward current (PHOC). The HIC did not change with an increasing holding potential. In contrast, the HOC reversed its amplitude at about -95 mV. Finally, the PHOC decreased, but did not reverse its polarity at more negative holding potentials. When the external K+ was elevated from 2.5 to 10.5 mM, the current-voltage (I-V) relation of the HOC and its reversal potential were shifted to the right. 3. In the presence of tetrodotoxin, the HH decreased. A low Ca(2+)-high Mg2+ medium depressed both the HH and PHH. Rauwolscine did not alter either response to hypoxia, while 8-cyclopentyl-1,3-dipropylxanthine decreased the PHH only. S-(p-Nitrobenzyl)-6-thioguanosine potentiated both HH and PHH. 4. Whereas tolbutamide markedly lowered the HH and PHH, glibenclamide was ineffective. Tetraethylammonium also failed to alter the hypoxic responses. Furthermore, ouabain or the removal of K+ from the superfusion medium, depressed PHH. 5. Pressure application of adenosine inhibited the spontaneous firing of LC neurons. DPCPX did not alter the firing, but antagonized the effect of adenosine. Tolbutamide also counteracted the inhibitory effect of adenosine and, additionally, facilitated the firing rate in some neurons. Moreover, tolbutamide abolished the adenosine-induced outward current. 6. Early hypoxic depolarization and PHH are mostly due to the blockade and subsequent reactivation of the K(+)-Na+ pump, respectively. The HH is caused by the opening of ATP-sensitive K+ (KATP) channels in response to the hypoxia-induced decline of intracellular ATP. Adenosine released by hypoxic stimuli may lead to an adenosine A1-receptor-mediated opening of (KATP) channels during the HH and more markedly during the PHH.
ATP-sensitive K+ (KATP) channels are present at high density in membranes of cardiac cells where they regulate cardiac function during cellular metabolic impairment. KATP channels have been implicated in the shortening of the action potential duration and the cellular loss of K+ that occurs during metabolic inhibition. KATP channels have been associated with the cardioprotective mechanism of ischemia-related preconditioning. Intracellular ATP (ATPi) is the main regulator of KATP channels. ATPi has two functions: 1) to close the channel (ligand function) and 2) in the presence of Mg2+, to maintain the activity of KATP channels (presumably through an enzymatic reaction). KATP channel activity is modulated by intracellular nucleoside diphosphates that antagonize the ATPi-induced inhibition of channel opening or induce KATP channels to open. How nucleotides will affect KATP channels depends on the state of the channel. K+ channel-opening drugs are pharmacological agents that enhance KATP channel activity through different mechanisms and have great potential in the management of cardiovascular conditions. KATP channel activity is also modulated by neurohormones. Adenosine, through the activation of a GTP-binding protein, antagonizes the ATPi-induced channel closure. Understanding the molecular mechanisms that underlie KATP channel regulation should prove essential to further define the function of KATP channels and to elucidate the pharmacological regulation of this channel protein. Since the molecular structure of the KATP channel has now become available, it is anticipated that major progress in the KATP channel field will be achieved.
Intracellular recordings were obtained from a pontine slice preparation of the rat brain containing the locus coeruleus (LC). Two openers of ATP‐sensitive potassium (K ATP ) channels, RO 31–6930 (10 μ m ) and cromakalim (100 μ m ) decreased the spontaneous discharge of action potentials without altering their amplitude or duration. Neither compound changed the resting membrane potential.
Of two K ATP channel blockers, tolbutamide (300 μ m ) increased the firing rate, while glibenclamide (3 μ m ) only tended to do so. In addition, both compounds antagonized the effect of RO 31–6930 (10 μ m ). Neither glibenclamide (3 μ m ) nor tolbutamide (300 μ m ) altered the resting membrane potential.
Tetrodotoxin (0.5 μ m ) depressed the firing, but did not influence the inhibitory action of RO 31–6930 (10 μ m ). The excitatory amino acid antagonist, kynurenic acid (500 μ m ), did not change the spontaneous discharge of action potentials.
Small shifts (2–4 mV) of the membrane potential by hyper‐ or depolarizing current injections markedly decreased and increased the firing rate, respectively.
Noradrenaline (100 μ m ) hyperpolarized the cells and decreased their input resistance. This effect was not antagonized by glibenclamide (3 μ m ) or tolbutamide (300 μ m ). Ba ²⁺ (2 m m ), a blocker of both ATP‐sensitive and inwardly rectifying potassium channels, abolished the effects of RO 31–6930 (10 μ m ) and noradrenaline (100 μ m ).
These data suggest that K ATP channels are present on the noradrenergic LC neurones, but are not coupled to α 2 ‐adrenoceptors.
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.
ATP-sensitive K+ channels comprise a complex of at least two proteins: a member of the inwardly rectifying Kir6 family (e.g. Kir6.2) and a sulphonylurea receptor (e.g. SUR1) which belongs to the ATP-binding cassette (ABC) superfamily. Using specific radiolabeled antisense oligonucleotides, the cellular localization of both mRNAs was investigated in the rodent brain by in situ hybridization. The distribution of both transcripts was widespread throughout the brain and showed a high degree of overlap with peak expression levels in the hippocampus, neocortex, olfactory bulb, cerebellum, and several distinct nuclei of the midbrain and brainstem, indicating their important role in vital brain function.
Perfusion of the forebrain with glucose at concentrations which alter neither plasma insulin nor glucose levels leads to sympathetic activation in some rats. We used the expression of Fos-like immunoreactivity (FLI) as an index of neuronal activation to examine the anatomic substrate underlying this phenomenon. Male Sprague-Dawley rats were infused via the right internal carotid artery with glucose (4 mg/kg/min) or equiosmolar mannitol for 60 min. They were killed 3 h after infusion onset and their brains reacted for FLI. As compared to mannitol-infused controls, 105% and 117% more neurons in hypothalamic ventromedial nucleus (VMN) and parvocellular portion of the paraventricular nuclei (PVN) of glucose-infused rats showed FLI, respectively. Importantly, only about half the glucose-infused rats showed increased FLI cells in these areas when compared to controls. In these same animals, glucose also significantly activated cells in the dorsomedial n. There was little FLI expressed in the magnocellular neurons of the PVN. This selective glucose response was bilateral in keeping with the bilateral distribution of India ink to midline hypothalamic structures following unilateral carotid infusions. Retrograde transport of cholera toxin B from medullary and thoracic spinal cord sympathetic outflow areas showed labeling of about 10% of PVN neurons with FLI activated by intracarotid glucose. There was no double labeling of VMN neurons. This supports the presence of anatomic pathways by which a subpopulation of glucose responsive PVN neurons might activate the sympathetic outflow areas in the medulla and spinal cord. The apparent bimodal distribution of glucose-induced activation of VMN and PVN neurons is in keeping with a similar bimodal pattern of sympathetic activation which obesity-prone but not obesity-resistant rats show following glucose infusions. Taken together, these data support a role for glucose-sensitive VMN and parvocellular PVN neurons in the weight gain phenotype specific sympathetic activation to glucose.
The obese (ob) gene encodes a fat cell-derived circulating satiety factor (leptin) that is involved in the regulation of energy homeostasis. In the present study, we examined effects of i.c.v. injection of recombinant human leptin on food intake and body weight gain in rats. We also studied effects of direct microinjections of leptin into the arcuate nucleus (Arc), ventromedial hypothalamus (VMH), and lateral hypothalamus (LH). A single i.c.v. injection of recombinant human leptin (0.25-2.0 micrograms/rat) reduced significantly and dose-dependently food intake and body weight gain in rats. Microinjections (0.125-0.5 microgram/site) into the bilateral Arc, VMH, and LH caused dose-related decreases in food intake and body weight gain as compared with vehicle-treated groups with a rank order of potency; Arc > VMH = LH. The present study provides the first direct evidence that the Arc is a primary site of satiety effect of leptin.
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.
Leptin, the protein encoded by the obese (ob) gene, is secreted from adipose tissue and is thought to act in the central nervous system to regulate food intake and body weight. It has been proposed that leptin acts in the hypothalamus, the main control centre for satiety and energy expenditure. Mutations in leptin or the receptor isoform (Ob-R[L]) present in hypothalamic neurons result in profound obesity and symptoms of non-insulin-dependent diabetes. Here we show that leptin hyperpolarizes glucose-receptive hypothalamic neurons of lean Sprague-Dawley and Zucker rats, but is ineffective on neurons of obese Zucker (fa/fa) rats. This hyperpolarization is due to the activation of a potassium current, and is not easily recovered on removal of leptin, but is reversed by applying the sulphonylurea, tolbutamide. Single-channel recordings demonstrate that leptin activates an ATP-sensitive potassium (K[ATP]) channel. Our data indicate that the K(ATP) channel may function as the molecular end-point of the pathway following leptin activation of the Ob-R(L) receptor in hypothalamic neurons.
ATP-sensitive potassium channels, termed KATP channels, link the electrical activity of cell membranes to cellular metabolism. These channels are heteromultimers of sulfonylurea receptor (SUR) and KIR6.X subunits associated with a 1:1 stoichiometry as a tetramer (SUR/KIR6.X forms the pores, whereas SUR regulates their activity. Changes in [ATP]i and [ADP]i gate the channel. The diversity of KATP channels results from the assembly of SUR and KIR6.X subtypes KIR6.1-based channels differ from KIR6.2 channels mainly by their smaller unitary conductance. SUR1- and SUR2-based channels are distinguished by their differential sensitivity to sulfonylureas, whereas SUR2A-based channels are distinguished from SUR2B channels by their differential sensitivity to diazoxide. Mutations that result in the loss of KATP channels in pancreatic beta-cells have been identified in SUR1 and KIR6.2. These mutations lead to familial hyperinsulinism. Understanding the mutations in SUR and KIR6.X is allowing insight into how these channels respond to nucleotides, sulfonylureas, and potassium channel openers, KCOs.
Select groups of neurons within the brain alter their firing rate when ambient glucose levels change. These glucose-responsive neurons are integrated into systems which control energy balance in the body. They contain an ATP-sensitive K+ channel (KATP) which mediates this response. KATP channels are composed of an inwardly rectifying pore-forming unit (Kir6.1 or Kir6.2) and a sulfonylurea binding site. Here, we examined the anatomical distribution and phenotype of cells containing Kir6.2 mRNA within the rat brain by combinations of in situ hybridization and immunocytochemistry. Cells containing Kir6. 2 mRNA were widely distributed throughout the brain without apparent concentration in areas known to contain specific glucose-responsive neurons. Kir6.2 mRNA was present in neurons expressing neuron-specific enolase, tyrosine hydroxylase, neuropeptide Y (NPY) and the glutamic acid decarboxylase isoform, GAD65. No astrocytes expressing glial fibrillary acidic protein or oligodendrocytes expressing carbonic anhydrase II were found to co-express Kir6.2 mRNA. Virtually all of the NPY neurons in the hypothalamic arcuate n. and catecholamine neurons in the substantia nigra, pars compacta and locus coeruleus contained Kir6.2 mRNA. Epinephrine neurons in the C2 area also expressed high levels of Kir6.2, while noradrenergic neurons in A5 and A2 areas expressed lower levels. The widespread distribution of Kir6.2 mRNA suggests that the KATP channel may serve a neuroprotective role in neurons which are not directly involved in integrating signals related to the body's energy homeostasis.
1. The distribution of ATP-sensitive K+ channels (KATP channels) was investigated in four cell types in hippocampal slices prepared from 10- to 13-day-old rats: CA1 pyramidal cells, interneurones of stratum radiatum in CA1, complex glial cells of the same area and granule cells of the dentate gyrus. The neuronal cell types were identified visually and characterized by the shapes and patterns of their action potentials and by neurobiotin labelling. 2. The patch-clamp technique was used to study the sensitivity of whole-cell currents to diazoxide (0.3 mM), a KATP channel opener, and to tolbutamide (0.5 mM) or glibenclamide (20 microM), two KATP channel inhibitors. The fraction of cells in which whole-cell currents were activated by diazoxide and inhibited by tolbutamide was 26% of pyramidal cells, 89 % of interneurones, 100% of glial cells and 89% of granule cells. The reversal potential of the diazoxide-induced current was at the K+ equilibrium potential and a similar current activated spontaneously when cells were dialysed with an ATP-free pipette solution. 3. Using the single-cell RT-PCR method, the presence of mRNA encoding KATP channel subunits (Kir6.1, Kir6.2, SUR1 and SUR2) was examined in CA1 pyramidal cells and interneurones. Subunit mRNA combinations that can result in functional KATP channels (Kir6.1 together with SUR1, Kir6.2 together with SUR1 or SUR2) were detected in only 17% of the pyramidal cells. On the other hand, KATP channels may be formed in 75% of the interneurones, mainly by the combination of Kir6.2 with SUR1 (58% of all interneurones). 4. The results of these combined analyses indicate that functional KATP channels are present in principal neurones, interneurones and glial cells of the rat hippocampus, but at highly different densities in the four cell types studied.
1. Patch-clamp recordings were made from rat ventromedial hypothalamic neurones in slices of brain tissue in vitro. In cell-attached recordings, removal of extracellular glucose or metabolic inhibition with sodium azide reduced the firing rate of a subpopulation of cells through the activation of a 65 pS channel that was blocked by the sulphonylureas tolbutamide and glibenclamide. 2. In whole-cell patch-clamp recordings, in the absence of ATP in the electrode solution, glucose-receptive neurones gradually hyperpolarized due to the induction of an outward current at -60 mV. This outward current and the resultant hyperpolarization were blocked by the sulphonylureas tolbutamide and glibenclamide. 3. In recordings where the electrode solution contained 4 mM ATP, this outward current was not observed. Under these conditions, 500 microM diazoxide was found to induce an outward current that was blocked by tolbutamide. 4. In cell-attached recordings diazoxide and the active fragment of leptin (leptin 22-56) reduced the firing rate of glucose-receptive neurones by the activation of a channel with similar properties to that induced by removal of extracellular glucose. 5. Reverse transcription followed by the polymerase chain reaction using cytoplasm from single glucose-receptive neurones demonstrated the expression of the ATP-sensitive potassium (KATP) channel subunits Kir6.1 and SUR1 but not Kir6.2 or SUR2. 6. It is concluded that glucose-receptive neurones within the rat ventromedial hypothalamus exhibit a KATP channel current with pharmacological and molecular properties similar to those reported in other tissues.
ATP-sensitive K+ channels (KATP channels) play important roles in many cellular functions by coupling cell metabolism to electrical activity. By cloning members of the novel inwardly rectifying K+ channel subfamily Kir6.0 (Kir6.1 and Kir6.2) and the receptors for sulfonylureas (SUR1 and SUR2), researchers have clarified the molecular structure of KATP channels. KATP channels comprise two subunits: a Kir6.0 subfamily subunit, which is a member of the inwardly rectifying K+ channel family; and a SUR subunit, which is a member of the ATP-binding cassette (ABC) protein superfamily. KATP channels are the first example of a heteromultimeric complex assembled with a K+ channel and a receptor that are structurally unrelated to each other. Since 1995, molecular biological and molecular genetic studies of KATP channels have provided insights into the structure-function relationships, molecular regulation, and pathophysiological roles of KATP channels.
The habenular complexes represent phylogenetically constant structures in the diencephalon of all vertebrates. Available evidence suggests that this area is engaged in a variety of important biological functions, such as reproductive behaviors, central pain processing, nutrition, sleep-wake cycles, stress responses, and learning. Based on Nissl-stained sections, one medial nucleus and two lateral nuclei (divisions) have been widely accepted in the rat. Cytochemical, hodologic, and functional studies suggest a considerably more complex subnuclear structure. To improve our knowledge of the precise structural composition of the habenular complexes, we have systematically investigated their fine ultrastructure in the rat. Based on the detailed analysis of complete series of large, semithin sections supplemented with electron photomicrographs of selected fields, clear criteria for the delineation of five distinct subnuclei of the medial and ten subnuclei of the lateral habenular complexes were elaborated for the first time. All 15 subnuclei were reconstructed, and their dimensions were determined. A medial and lateral stria medullaris were described. Different roots of the fasciculus retroflexus were differentiated within the medial and lateral habenular complexes. The topographical relationships with respect to the adjacent habenular areas as well as to the neighboring thalamic nuclei were identified and demonstrated. The new understanding of the subnuclear organization of the habenular complexes certainly will facilitate further functional investigations. Whether the newly identified subnuclei finally will be recognized as functionally distinct awaits ongoing immunocytochemical, hodologic, and functional studies.
Locus coeruleus (LC) is the significant nucleus for consciousness and it is sensitive to metabolic inhibition. We investigated the effects of a metabolic inhibitor sodium cyanide (NaCN) on the rat dissociated LC neurons using nystatin-perforated patch recordings. Under voltage-clamp (VH=-40 mV), application of NaCN evoked outward currents composed of ATP-sensitive and Ca2+-dependent K+ channel currents (IKATP and IKCa2+). Onset of IKATP was faster than that of IKCa2+. Prolonged application of NaCN brought IKATP rundown but not IKCa2+ rundown. Okadaic acid prevented IKATP rundown, indicating that KATP channels are deactivated by dephosphorylation with protein phosphatase.