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A New ER Trafficking Signal Regulates the Subunit Stoichiometry of Plasma Membrane KATP Channels
Abstract
Proper ion channel function often requires specific combinations of pore-forming alpha and regulatory beta subunits, but little is known about the mechanisms that regulate the surface expression of different channel combinations. Our studies of ATP-sensitive K+ channel (K(ATP)) trafficking reveal an essential quality control function for a trafficking motif present in each of the alpha (Kir6.1/2) and beta (SUR1) subunits of the K(ATP) complex. We show that this novel motif for endoplasmic reticulum (ER) retention/retrieval is required at multiple stages of K(ATP) assembly to restrict surface expression to fully assembled and correctly regulated octameric channels. We conclude that exposure of a three amino acid motif (RKR) can explain how assembly of an ion channel complex is coupled to intracellular trafficking.
... Endoplasmic reticulum (ER) retention sequence is critical in regulating the surface expression of ion channels. It serves as a quality control mechanism allowing ion channels that are correctly assembled, in which the retention signals are sterically masked, to traffic to the cell surface (1)(2)(3)(4)(5). Several ER retention signals in ion channel subunits involved in this process have been identified. ...
... Several ER retention signals in ion channel subunits involved in this process have been identified. The RKR signals in Kir6.1, Kir6.2, and SUR subunits of ATP-sensitive potassium channels (K ATP ) prevent incompletely assembled channels from being exported to cell surface (2). RSRR signal in γ-aminobutyric acid B1 (GABA B1 ) subunit is masked by assembly with GABA B2 subunit, allowing the correctly assembled heterodimeric receptors to traffic to the plasma membrane (1). ...
... Similar theory has been proposed in several ion channels. The RKR signals in Kir6.1, Kir6.2, and SUR subunits of ATP-sensitive potassium channels (K ATP ) and the RSRR signal in γ-aminobutyric acid B1 (GABA B1 ) subunit prevent incompletely assembled channels from being exported to cells surface (1,2). It would be of interest to know if TMEM63B monomers can traffic to cell surface. ...
TMEM63B is a mechanosensitive cation channel activated by hypoosmotic stress and mechanic stimulation. We recently reported a brain-specific alternative splicing of Exon 4 in TMEM63B. The short variant lacking Exon 4, which constitutes the major isoform in the brain, exhibits enhanced responses to hypoosmotic stimulation compared to the long isoform containing Exon 4. However, the mechanisms affecting this differential response are unclear. Here, we showed that the short isoform exhibited stronger cell surface expression compared to the long variant. Using mutagenesis screening of the coding sequence of Exon 4, we identified an RXR-type endoplasmic reticulum (ER) retention signal (RER). We found that this motif was responsible for binding to the COPI retrieval vesicles, such that the longer TMEM63B isoforms were more likely to be retrotranslocated to the ER than the short isoforms. In addition, we demonstrated long TMEM63Bs could form heterodimers with short isoforms and reduce their surface expression. Taken together, our findings revealed an ER retention signal in the alternative splicing domain of TMEM63B that regulates the surface expression of TMEM63B protein and channel function.
... 354-356 (RER) (Figure 2.2A). In addition to conservation of these residues, amino acid sequences containing arginine (R)-any amino acid (X)-arginine (R), or 'RXR', motifs retain ion channels and GPCRs in the endoplasmic reticulum (Zerangue et al., 1999;Margeta-Mitrovic et al., 2000;Ma et al., 2001;Cunningham et al., 2012). ...
... RXR motifs were first shown to mediate retrieval and retention of the ATPsensitive potassium channel α (Kir6.1/2) and β (SUR1) subunits in the ER (Zerangue et al., 1999). Complete assembly of subunits masks the RXR sequence and allows channel export from the ER. ...
... RXR motifs interact with COPI through distinct sites (β-COP and δ-COP) from where canonical dilysine motifs interact (α-COP and β'-COP) (Eugster et al., 2004;Michelsen et al., 2007;Jackson et al., 2012;Ma and Goldberg, 2013). Lysines typically cannot substitute for arginines in other RXR motifs both in terms of COPI interaction and functional retention of proteins (Zerangue et al., 1999;Yuan et al., 2003;Michelsen et al., 2005). However, lysine substitution in DOR RXR motifs preserves signal-regulated DOR retention, though we did not test the effect of lysine substitution on the DOR COPI interaction. ...
G protein-coupled receptors (GPCRs) transduce diverse signals, including light, ions, hormones, and neurotransmitters, into equally diverse cellular responses. These cellular responses underlie complex physiological processes, including sensation, learning and memory, cardiac function, and immune function. Understanding the variables which contribute to GPCR signaling diversity at a cellular level is essential to understanding the role of GPCRs in physiology and disease. The subcellular location from which GPCR signaling occurs is an increasingly recognized variable which contributes to signaling diversity. I have used the delta opioid receptor (DOR) as a prototype GPCR to investigate mechanisms regulating GPCR localization and the effects of subcellular location on GPCR function. DOR is an ideal and therapeutically relevant prototype GPCR to study these questions. In neuronal cells, DOR localizes to multiple membrane compartments, including the plasma membrane and the Golgi apparatus. Relocation of DOR from intracellular sites to the plasma membrane is associated with enhanced pain-relieving effects of DOR agonists, which highlights the therapeutically relevant link between DOR localization and function. I first investigated the mechanisms which regulate DOR localization to the Golgi in a rat neuroendocrine cell line which shares common mechanisms with primary neurons in regulation of DOR trafficking. Through systematic mutagenesis of the DOR C-terminal primary amino acid sequence and high-resolution imaging, we identified conserved dual RXR amino acid motifs which are required for signal-regulated retention of DOR in the Golgi. Using biochemical approaches, we showed that these RXR motifs also mediate interaction with the coatomer protein I (COPI) complex. These data support a model in which DOR retention in the Golgi is mediated by active retrograde trafficking within the biosynthetic pathway. I next explored the effect of subcellular location on DOR activation. GPCR activation and coupling to effectors is driven by conformational changes in the receptor upon agonist binding. We used fluorescently tagged biosensors which recognize these conformational changes and high-resolution imaging to visualize DOR activation in different subcellular locations. We found that DOR in the plasma membrane and the Golgi differentially recruit two active conformation biosensors in response to the same agonist. These results indicate that subcellular location drives distinct engagement of effectors and suggest the exciting possibility that subcellular location may alter GPCR conformational landscapes upon ligand binding. I also determined the effect of subcellular location on DOR signaling using biosensors for second messenger signaling molecules cAMP and calcium. We found that DOR activation in both the plasma membrane and the Golgi inhibits cAMP production, suggesting that DOR couples to inhibitory G proteins regardless of compartment-specific effects on effector engagement or conformational landscapes. In a rat neuroendocrine cell line, DOR activation at the plasma membrane modulates calcium release from intracellular stores in a Gi/o, Gq/11, and phospholipase C- dependent manner. Modulation of calcium is specific to DOR signaling from the plasma membrane and is not observed upon DOR activation in the Golgi. These data suggest that DOR subcellular location influences the signaling profile of active receptors. Together this work adds to our understanding of how GPCR subcellular localization is regulated and how subcellular location can drive distinct GPCR activation and signaling. In the future, this mechanistic understanding could be applied to tune localization of therapeutically relevant GPCRs like DOR or to target GPCRs in specific subcellular compartments for desired therapeutic effects.
... A key-regulatory element within the channel protein is the ER retention signal, RKR, in the C-terminal domain of both Kir6.2 and SUR1 37 . RKR serves as a point of control to ensure that only appropriately assembled channels with the correct stoichiometry and subunit composition traffic to the plasma membrane 37 . ...
... Extracellular localization of the N-terminal 6xHis tag or EGFP was investigated in non-permeabilized cells, whereas total Kir6.2 membrane expression was evaluated in permeabilized cells, which allows access to antigens present on both the extracellular and cytoplasmic side of the membrane. Previous immunohistochemistry experiments have relied on hemagglutinin (HA) tags inserted into the Kir6.2 protein in the extracellular loop of the protein, facilitating detection of surface expression 37 . Importantly, these protocols were not extended to explore the possibility of inverted channels, thus potentially inverted channels were not studied. ...
... In wild-type K ATP channels, the RKR retention signal in both Kir6.2 and SUR1 at the cytoplasmic face provides a quality control check that ensures the channels are properly assembled in the ER before further trafficking to the Golgi apparatus 36,37 . Truncated Kir6.2 mutants that lack the RKR sequence are capable of trafficking to the membrane allowing this quality control checkpoint to be bypassed. ...
ATP-sensitive K ⁺ (K ATP ) channels couple cellular metabolism to electrical activity in many cell types. Wild-type K ATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K ⁺ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K ⁺ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.
... The ER has to distinguish between ER-resident proteins and proteins that are passing through. ERresident proteins have sequences that bind to ER-resident chaperones and retain them in the ER (Nilsson et al., 1989;Bonifacino et al., 1990;Cosson and Letourneur, 1994;Zerangue et al., 1999). ...
... In such cases, protein retention in cytoplasmic condensates would serve as checkpoint for full assembly. This idea is again inspired by principles of protein complex assembly of multimeric surface receptors and channels in the ER (Bonifacino et al., 1990;Zerangue et al., 1999). The subunits of multimeric membrane complexes are translated at the ER. ...
... To ensure that only fully assembled channels or receptors leave the ER to be trafficked to the plasma membrane, the individual subunits contain ER retention sites (Nilsson et al., 1989;Cosson and Letourneur, 1994). Upon full assembly, these sites are buried and are no longer accessible on the protein surface, thus allowing the multiprotein complex to leave the ER (Bonifacino et al., 1990;Zerangue et al., 1999). A similar mechanism was shown to take place in the nucleolus for ribosome assembly (Riback et al., 2020). ...
Most cellular processes are carried out by protein complexes, but it is still largely unknown how the subunits of lowly expressed complexes find each other in the crowded cellular environment. Here, we will describe a working model where RNA-binding proteins in cytoplasmic condensates act as matchmakers between their bound proteins (called protein targets) and newly translated proteins of their RNA targets to promote their assembly into complexes. Different RNA-binding proteins act as scaffolds for various cytoplasmic condensates with several of them supporting translation. mRNAs and proteins are recruited into the cytoplasmic condensates through binding to specific domains in the RNA-binding proteins. Scaffold RNA-binding proteins have a high valency. In our model, they use homotypic interactions to assemble condensates and they use heterotypic interactions to recruit protein targets into the condensates. We propose that unoccupied binding sites in the scaffold RNA-binding proteins transiently retain recruited and newly translated proteins in the condensates, thus promoting their assembly into complexes. Taken together, we propose that lowly expressed subunits of protein complexes combine information in their mRNAs and proteins to colocalize in the cytoplasm. The efficiency of protein complex assembly is increased by transient entrapment accomplished by multivalent RNA-binding proteins within cytoplasmic condensates.
... Biochemical and biophysical studies determined that the K ATP channel is a hetero-octameric complex of four Kir6.x and four SURx subunits [22][23][24]. Unassembled or partially assembled channel subunits are retained in the endoplasmic reticulum (ER) owing to an arginine-based ER retention motif present in both Kir6.x and SURx (-RKR-in SUR1, Kir6.1, and Kir6.2; -RKQ-in SUR2A/B; see Figure 1) [25]. K ATP high resolution structures derived from cryoEM single particle imaging confirmed the 4:4 stoichiometry, showing a Kir6 tetramer core surrounded by four SUR proteins (Figure 1), including the SUR1/Kir6.2 ...
... The discovery that truncating the C-terminus of Kir6.2 (Kir6.2ΔC, which removes the ER retention motif -RKR-) allows Kir6.2 channel expression in the plasma membrane in the absence of SUR1 facilitated dissection of the respective role of Kir6.2 and SUR1 in channel response to nucleotides and PIP 2 [25,41]. Comparison of Kir6.2ΔC channels with or without SUR1 combined with mutation-function correlation studies revealed that ATP inhibition, MgADP stimulation and PIP 2 gating are effected through distinct binding sites. ...
KATP channels are ligand-gated potassium channels that couple cellular energetics with membrane potential to regulate cell activity. Each channel is an eight subunit complex comprising four central pore-forming Kir6 inward rectifier potassium channel subunits surrounded by four regulatory subunits known as the sulfonylurea receptor, SUR, which confer homeostatic metabolic control of KATP gating. SUR is an ATP binding cassette (ABC) protein family homolog that lacks membrane transport activity but is essential for KATP expression and function. For more than four decades, understanding the structure-function relationship of Kir6 and SUR has remained a central objective of clinical significance. Here, we review progress in correlating the wealth of functional data in the literature with recent KATP cryoEM structures.
... The third checkpoint involves the recognition of abnormally exposed arginine-framed tripeptides (AFTs). AFTs are transient retention/ retrieval motifs, with a Arg-X-Arg sequence, that couple ER exit to the assembly and folding of multimeric membrane or secretory proteins (Zerangue et al., 1999). AFTs only act as retrieval signal when exposed in misassembled or misfolded proteins, suggesting that the motifs are buried in assembled proteins. ...
... AFTs only act as retrieval signal when exposed in misassembled or misfolded proteins, suggesting that the motifs are buried in assembled proteins. The masking of AFTs in native proteins is the mechanism that allows these motifs to act as retrieval signals in quality control and not as constitutive retention signals (Zerangue et al., 1999). CFTR has at least four AFTs, including Arg29-Gln30-Arg31 located at the N-terminal, Arg516-Tyr517-Arg518 and Arg553-Ala554-Arg-555 located at the nucleotide binding domain 1 (NBD1) and Arg764-Ala765-Arg766 located at the regulatory domain (RD). ...
Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is a complex process that starts with its biosynthesis and folding in the endoplasmic reticulum. Exit from the endoplasmic reticulum (ER) is coupled with the acquisition of a compact structure that can be processed and traffic through the secretory pathway. Once reaching its final destination—the plasma membrane, CFTR stability is regulated through interaction with multiple protein partners that are involved in its post-translation modification, connecting the channel to several signaling pathways. The complexity of the process is further boosted when analyzed in the context of the airway epithelium. Recent advances have characterized in detail the different cell types that compose the surface epithelium and shifted the paradigm on which cells express CFTR and on their individual and combined contribution to the total expression (and function) of this chloride/bicarbonate channel. Here we review CFTR trafficking and its relationship with the knowledge on the different cell types of the airway epithelia. We explore the crosstalk between these two areas and discuss what is still to be clarified and how this can be used to develop more targeted therapies for CF.
... Surface expression Surface expression of wild-type or mutant Kir6.2 was measured based on their ability to chaperone a site-specific tagged SUR1 to the plasma membrane [27]. Briefly, a haemagglutinin (HA) tag (YAYMEKGITD-LAYPYDVPDYA) was inserted into the last extracellular loop of SUR1 (TM16-TM17). ...
... A mutation may affect any of these processes, as well as the rate at which the channel is removed from the membrane. Neither Kir6.2 nor SUR1 traffic to the plasmalemma in the absence of the partner subunit, as they fail to exit the endoplasmic reticulum [27] and are then degraded [12]. The reduction in total SUR1 protein we observed favours the idea that channel assembly is affected, preventing surface trafficking. ...
Aims/hypothesis
The ATP-sensitive potassium (KATP) channel couples beta cell electrical activity to glucose-stimulated insulin secretion. Loss-of-function mutations in either the pore-forming (inwardly rectifying potassium channel 6.2 [Kir6.2], encoded by KCNJ11) or regulatory (sulfonylurea receptor 1, encoded by ABCC8) subunits result in congenital hyperinsulinism, whereas gain-of-function mutations cause neonatal diabetes. Here, we report a novel loss-of-function mutation (Ser118Leu) in the pore helix of Kir6.2 paradoxically associated with sulfonylurea-sensitive diabetes that presents in early adult life.
Methods
A 31-year-old woman was diagnosed with mild hyperglycaemia during an employee screen. After three pregnancies, during which she was diagnosed with gestational diabetes, the patient continued to show elevated blood glucose and was treated with glibenclamide (known as glyburide in the USA and Canada) and metformin. Genetic testing identified a heterozygous mutation (S118L) in the KCNJ11 gene. Neither parent was known to have diabetes. We investigated the functional properties and membrane trafficking of mutant and wild-type KATP channels in Xenopus oocytes and in HEK-293T cells, using patch-clamp, two-electrode voltage-clamp and surface expression assays.
Results
Functional analysis showed no changes in the ATP sensitivity or metabolic regulation of the mutant channel. However, the Kir6.2-S118L mutation impaired surface expression of the KATP channel by 40%, categorising this as a loss-of-function mutation.
Conclusions/interpretation
Our data support the increasing evidence that individuals with mild loss-of-function KATP channel mutations may develop insulin deficiency in early adulthood and even frank diabetes in middle age. In this case, the patient may have had hyperinsulinism that escaped detection in early life. Our results support the importance of functional analysis of KATP channel mutations in cases of atypical diabetes.
Graphical Abstract
... Mutations linked to neonatal diabetes (which is caused by gain-of-function mutations) and congenital hyperinsulinism affect Kir6.2 surface expression to varying degrees (Flanagan et al., 2009;Hattersley and Ashcroft, 2005;Marthinet et al., 2005;Lin et al., 2003). Beyond known trafficking signals (e.g., ER and Golgiexport signal sequences; Li et al., 2016;Ma et al., 2001;Ma et al., 2002;Ma et al., 2011;Stockklausner et al., 2001;Zerangue et al., 1999), mutations along the entire Kir primary sequence can disrupt surface expression (Zangerl-Plessl et al., 2019). Several additional factors control surface expression, such as protein stability or interactions with trafficking partners and complexes that stabilize channels in the membrane (Ma et al., 2001;Ma et al., 2002;Leonoudakis et al., 2004). ...
... Our finding that variants with deleterious surface score are underreported in available database supports the emerging theme that many disease-causing variants are linked to trafficking defects (O'Donnell et al., 2017;Peters et al., 2003;Fallen et al., 2009;Zangerl-Plessl et al., 2019;Li et al., 2016;Ma et al., 2001;Ma et al., 2002;Ma et al., 2011;Stockklausner et al., 2001;Zerangue et al., 1999;Lin et al., 2006). Other large-scale mutational analysis, as undertaken for the voltagedependent K + channel Kv11.1 (Anderson et al., 2014;Kozek et al., 2020), has similarly shown that 88% of long QT-linked variants have trafficking-deficient mechanisms. ...
A longstanding goal in protein science and clinical genetics is to develop quantitative models of sequence, structure, and function relationships and delineate the mechanisms by which mutations cause disease. Deep Mutational Scanning (DMS) is a promising strategy to map how amino acids contribute to protein structure and function and to advance clinical variant interpretation. Here, we introduce 7,429 single residue missense mutations into the Inward Rectifier K ⁺ channel Kir2.1 and determine how this affects folding, assembly, and trafficking, as well as regulation by allosteric ligands and ion conduction. Our data provide high-resolution information on a cotranslationally-folded biogenic unit, trafficking and quality control signals, and segregated roles of different structural elements in fold-stability and function. We show that Kir2.1 surface trafficking mutants are underrepresented in variant effect databases, which has implications for clinical practice. By comparing fitness scores with expert-reviewed variant effects, we can predict the pathogenicity of 'variants of unknown significance' and disease mechanisms of known pathogenic mutations. Our study in Kir2.1 provides a blueprint for how multiparametric DMS can help us understand the mechanistic basis of genetic disorders and the structure-function relationships of proteins.
... Based on de novo protein structure prediction, a plausible mechanism leading to the trafficking deficit is the exposure of the RKR (arginine-lysine-arginine) motif in Kir6.2-A28V during protein folding. If the RKR motif is not properly concealed, the protein can be aberrantly retained in the endoplasmic reticulum (ER) (4). ...
... We first determined the cellular distributions of WT and mutant K ATP channels in INS-1 cells transiently transfected with HA-tagged SUR1 and WT Kir6.2 or mutant Kir6.2-A28V by costaining for the protein and organelle markers. A de novo protein structure simulation suggested that Ala to Val mutation at position 28 would rearrange the RKR ER-retention motif (3,4), meaning that the mutant K ATP channels might remain in the ER (4). INS-1 cells expressing HA-tagged SUR1 and WT Kir6.2 or mutant Kir6.2-A28V were treated with vehicle or 10 μM Carb, followed by immunofluorescence staining with HA and KEDL (C-terminal tetrapeptide ER retention signal) antibodies. ...
Pancreatic β-cells express ATP-sensitive potassium (KATP) channels, consisting of octamer complexes containing four SUR1 and four Kir6.2 subunits. Loss of KATP channel function causes persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a rare but debilitating condition if not treated. We previously showed that the sodium-channel blocker carbamazepine (Carb) corrects KATP channel surface expression defects induced by PHHI-causing mutations in SUR1. In this study, we show that Carb treatment can also ameliorate the trafficking deficits associated with a recently discovered PHHI-causing mutation in Kir6.2 (Kir6.2-A28V). In HEK293 or INS-1 cells expressing this mutant KATP channel (SUR1 and Kir6.2-A28V), biotinylation and immunostaining assays revealed that Carb can increase surface expression of the mutant KATP channels. We further examined the subcellular distributions of mutant KATP channels before and after Carb treatment; without Carb treatment, we found that mutant KATP channels were aberrantly accumulated in the Golgi apparatus. However, after Carb treatment, co-immunoprecipitation of mutant KATP channels and Golgi marker GM130 was diminished, and KATP staining was also reduced in lysosomes. Intriguingly, Carb treatment also simultaneously increased autophagic flux and p62 accumulation, suggesting that autophagy-dependent degradation of the mutant channel was stimulated, but also interrupted. In summary, our data suggest that surface expression of Kir6.2-A28V KATP channels is rescued by Carb treatment via promotion of mutant KATP channel exit from the Golgi apparatus and reduction of autophagy-mediated protein degradation.
... SURs are essential to Kir6 channel function because in their absence, Kir6.1 or 6.2 α subunits fail to reach the cell surface and are therefore nonfunctional. This is because of the RKR motif, an endoplasmic reticulum (ER) retention signal that lies within each of the Kir6 and SUR subunits (Zerangue et al. 1999), which ensures ER retention of the homomeric subunits. Co-expression of the two subunit types permits each to reciprocally mask the RKR motifs of the other, permitting maturation and surface trafficking of the entire K ATP channel complex. ...
... Co-expression of the two subunit types permits each to reciprocally mask the RKR motifs of the other, permitting maturation and surface trafficking of the entire K ATP channel complex. The Kir6.2 ER retention signal lies within the last 26 residues of the distal C-terminus; accordingly, deletion of this portion of the α subunit permits surface expression and channel activity (Tucker et al. 1997;Zerangue et al. 1999). Thus, SURs are essential modulators in K ATP channels by virtue of an ER retention motif that has evolved to prevent the α subunits from reaching the cell surface alone. ...
Potassium channels facilitate and regulate physiological processes as diverse as electrical signaling, ion, solute and hormone secretion, fluid homeostasis, hearing, pain sensation, muscular contraction, and the heartbeat. Potassium channels are each formed by either a tetramer or dimer of pore-forming α subunits that co-assemble to create a multimer with a K+-selective pore that in most cases is capable of functioning as a discrete unit to pass K+ ions across the cell membrane. The reality in vivo, however, is that the potassium channel α subunit multimers co-assemble with ancillary subunits to serve specific physiological functions. The ancillary subunits impart specific physiological properties that are often required for a particular activity in vivo; in addition, ancillary subunit interaction often alters the pharmacology of the resultant complex. In this chapter the modes of action of ancillary subunits on K+ channel physiology and pharmacology are described and categorized into various mechanistic classes.
... A study done by Zerangue et al. disclosed that a novel endoplasmic reticulum (ER) retention sequence, RKR is present in both of the heterologous K ATP channels subunits (Zerangue et al. 1999). In Kir6.2, RKR is located in the C-terminus and in SUR1, it is located upstream of the first nucleotide binding fold. ...
... In Kir6.2, RKR is located in the C-terminus and in SUR1, it is located upstream of the first nucleotide binding fold. With this finding, it is now understood why Kir6.2 does not form functional channels when expressed alone but does when co-expressed with SUR1 (Inagaki et al. 1995;Tucker et al. 1997 (Zerangue et al. 1999). ...
KATP channels are ubiquitously expressed and link metabolic state to electrical excitability. In heart, in response to ischaemic stress, they play a protective role and in vascular smooth muscle regulation of vascular tone (vasorelaxation). Functional KATP channels are hetero-octamers composed of two subunits, a pore forming Kir6, which is a member of the inwardly rectifying potassium channels family and a regulatory sulphonylurea receptor (SUR). In response to nucleotides and pharmacological agents, SUR allosterically regulate KATP channel gating. Multidisciplinary techniques (molecular biology, biochemistry, electrophysiology, pharmacology) were used to study the allosteric regulation between these two heterologous subunits in KATP channels.
This project was divided into three major sub-projects: 1) Application of site directed mutagenesis and biochemical techniques to identify the cognate interaction domain on Kir6.2 for SUR2A-NBD2 (nucleotide binding domain 2). 2) Electrophysiological techniques to investigate the allosteric information transfer between heterologous subunits Kir6 and SUR2A. 3) Recombinant fusion protein to express and purify the cytoplasmic domains of Kir6.2 for structural analysis of the interaction between the two subunits.
This study reports on the identification of three cytoplasmic electrostatic interfaces between Kir6 and SUR2A involved in determining the sensitivity of KATP channel agonist, pinacidil, and antagonist, glibenclamide, from SUR2A to the Kir6 channel pore.
For structural study of cytoplasmic domains of Kir6.2, bacterial TM1070 was used as fusion partner with Kir6.2. A TM1070-Kir6.2 NC (CT-His6 tag) fusion construct expressed in Arctic Express competent cells permitted successful expression of folded cytoplasmic domains of Kir6.2 in near native form. Immobilized metal ion affinity chromatography, IMAC (Ni2+), and gel filtration chromatography (GFC) column as second purification step were performed to purify this recombinant protein. The purification was confirmed by CBS and Western blot analysis.
Possibly, this new information on channel structure-function relationships may contribute to the design of novel and more effective drugs.
... Such that, as a result of Kir6.2 mutation, persistent hyperinsulinemic hypoglycemia picture occurs in infants due to uncontrolled beta cell depolarization and insulin secretion [45] (Fig. 3). Not all of the complexes formed by beta subinides reach the cell membrane, and some of them are linked to the smooth endoplasmic reticulum and are regulated by Ca + 2 signaling [47]. Although this bond is more common in the SUR2A subtype, which is located on smooth muscle and cardiac muscle and regulates contraction, it has also been observed in the SUR1 subtype located in pancreatic beta cells [48,49]. ...
Pancreatic beta cells play a crucial role in maintaining glucose homeostasis through the regulation of insulin secretion. The electrophysiological properties of these cells, including ion channel function, electrical activity, and secretory mechanisms, are essential for their proper physiological function. In this comprehensive review, we provide an in-depth analysis of the electrophysiology of pancreatic beta cells. We discuss the various ion channels involved in the generation and modulation of electrical signals, such as voltage-gated ion channels, ATP-sensitive potassium channels, and calcium channels. Additionally, we examine the intricate interplay between intracellular calcium dynamics and insulin release. Furthermore, we explore the physiological and pathological factors that can influence the electrophysiology of pancreatic beta cells. A comprehensive understanding of the electrophysiological mechanisms governing pancreatic beta cell function is crucial for unraveling the pathogenesis of diabetes mellitus and developing novel therapeutic strategies.
... Two SUR1 bands were detected for wild-type channels: an immature core-glycosylated form in the endoplasmic reticulum (ER) that corresponds to 170 kDa and a mature complex glycosylation that has exited the ER and traversed the Golgi apparatus (200 kDa). 7 In both HEK293 and INS-1 cells, the p.His103Tyr variant exhibited distinct 170-kDa and 200-kDa bands. ...
Congenital hyperinsulinism (CHI; OMIM: 256450) is characterized by persistent insulin secretion despite severe hypoglycemia. The most common causes are variants in the ATP‐binding cassette subfamily C member 8(ABCC8) and potassium inwardly‐rectifying channel subfamily J member 11(KCNJ11) genes. These encode ATP‐sensitive potassium (KATP) channel subunit sulfonylurea receptor 1 (SUR1) and inwardly rectifying potassium channel (Kir6.2) proteins. A 7‐day‐old male infant presented with frequent hypoglycemic episodes and was clinically diagnosed with CHI, underwent trio‐whole‐exome sequencing, revealing compound heterozygous ABCC8 variants (c.307C>T, p.His103Tyr; and c.3313_3315del, p.Ile1105del) were identified. In human embryonic kidney 293 (HEK293) and rat insulinoma cells (INS‐1) transfected with wild‐type and variant plasmids, KATP channels formed by p.His103Tyr were delivered to the plasma membrane, whereas p.Ile1105del or double variants (p.His103Tyr coupled with p.Ile1105del) failed to be transported to the plasma membrane. Compared to wild‐type channels, the channels formed by the variants (p.His103Tyr; p.Ile1105del) had elevated basal [Ca²⁺]i, but did not respond to stimulation by glucose. Our results provide evidence that the two ABCC8 variants may be related to CHI owing to defective trafficking and dysfunction of KATP channels.
... SURs have no recognized transporter function, but instead, regulate K ATP channel complexes, conferring Mg-nucleotide activation and pharmacological sensitivity and modulating ATP inhibition. 2 Functional K ATP channel expression at the plasma membrane requires co-assembly of four K ir 6 subunits with four SUR subunits. [3][4][5][6][7][8][9] Extensive study has shown that truncation of SUR proteins impairs or abolishes surface expression of K ATP channels 10,11 and truncations of SUR1 are associated with congenital hyperinsulinism due to loss of pancreatic K ATP function. 12 SUR2-containing K ATP channels are well described in multiple tissues, including smooth, cardiac and skeletal muscle. ...
Loss-of-function mutation of ABCC9, the gene encoding the SUR2 subunit of ATP sensitive-potassium (KATP) channels, was recently associated with autosomal recessive ABCC9-related intellectual disability and myopathy syndrome (AIMS).
Here we identify nine additional subjects, from seven unrelated families, harboring different homozygous LoF variants in ABCC9 and presenting with a conserved range of clinical features. All variants are predicted to result in severe truncations or in-frame deletions within SUR2, leading to the generation of non-functional SUR2-dependent KATP channels.
Affected individuals show psychomotor delay and intellectual disability of variable severity, microcephaly, corpus callosum and white matter abnormalities, seizures, spasticity, short stature, muscle fatigability, and weakness. Heterozygous parents do not show any conserved clinical pathology but report multiple incidences of intrauterine fetal death, which were also observed in an eighth family included in this study. In vivo studies of abcc9 LoF in zebrafish revealed an exacerbated motor response to pentylenetetrazole, a pro-convulsive drug, consistent with impaired neurodevelopment associated with an increased seizure susceptibility.
Our findings define an ABCC9 LoF related phenotype, expanding the genotypic and phenotypic spectrum of AIMS and reveal novel human pathologies arising from KATP channel dysfunction.
... This is supported by our overexpression data, in which a significant increase in the proliferative ability of HPV-C33A cells was only observed when both channel subunits were transfected. This also fits with the current assembly hypothesis for K ATP channels, whereby neither subunit can be trafficked beyond the endoplasmic reticulum unless fully assembled into hetero-octameric channels [69][70][71]. ...
Persistent infection with high-risk human papillomaviruses (HPVs) is the causal factor in multiple human malignancies, including >99% of cervical cancers and a growing proportion of oropharyngeal cancers. Prolonged expression of the viral oncoproteins E6 and E7 is necessary for transformation to occur. Although some of the mechanisms by which these oncoproteins contribute to carcinogenesis are well-characterised, a comprehensive understanding of the signalling pathways manipulated by HPV is lacking. Here, we present the first evidence to our knowledge that the targeting of a host ion channel by HPV can contribute to cervical carcinogenesis. Through the use of pharmacological activators and inhibitors of ATP-sensitive potassium ion (KATP) channels, we demonstrate that these channels are active in HPV-positive cells and that this activity is required for HPV oncoprotein expression. Further, expression of SUR1, which forms the regulatory subunit of the multimeric channel complex, was found to be upregulated in both HPV+ cervical cancer cells and in samples from patients with cervical disease, in a manner dependent on the E7 oncoprotein. Importantly, knockdown of SUR1 expression or KATP channel inhibition significantly impeded cell proliferation via induction of a G1 cell cycle phase arrest. This was confirmed both in vitro and in in vivo tumourigenicity assays. Mechanistically, we propose that the pro-proliferative effect of KATP channels is mediated via the activation of a MAPK/AP-1 signalling axis. A complete characterisation of the role of KATP channels in HPV-associated cancer is now warranted in order to determine whether the licensed and clinically available inhibitors of these channels could constitute a potential novel therapy in the treatment of HPV-driven cervical cancer.
... C terminus (Kir6.2Δ36), allows a degree of surface expression (Zerangue et al., 1999). We therefore transfected HEK293 cells with Kir6.2Δ36 in the absence of SURx. ...
Background: K ATP channels have diverse roles, including regulation of insulin secretion and blood flow, and protection against biological stress responses and are excellent therapeutic targets. Different subclasses of K ATP channels exist in various tissue types due to the unique assemblies of specific pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against the various K ATP channel subclasses.
Methods and Results: We used 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures of channels in both the open and closed states to identify a potential agonist binding pocket in a functionally critical area of the channel. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds yielded 15 top-ranked “hits”, which were tested for activity against K ATP channels using patch clamping and thallium (Tl ⁺ ) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl ⁺ fluxes. One of them (CL-705G) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC 50 of 9 µM and 11 μM, respectively). Remarkably, compound CL-705G had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1, or Kir3.1/Kir3.4 channels, or Na ⁺ currents of TE671 medulloblastoma cells. CL-705G activated Kir6.2Δ36 in the presence of SUR2A, but not when expressed by itself. CL-705G activated Kir6.2/SUR2A channels even after PIP 2 depletion. The compound has cardioprotective effects in a cellular model of pharmacological preconditioning. It also partially rescued activity of the gating-defective Kir6.2-R301C mutant that is associated with congenital hyperinsulinism.
Conclusion: CL-705G is a new Kir6.2 opener with little cross-reactivity with other channels tested, including the structurally similar Kir6.1. This, to our knowledge, is the first Kir-specific channel opener.
... Second, there is an MKRR sequence in TASK that seems to be crucial since it makes the difference between an active channel and a non-active channel (TASK-1ΔSSV). This MKRR sequence resembles a retention sequence (RKR) to the endoplasmic reticulum (ER), that was previously described in a Kir potassium channel (Zerangue et al., 1999). The authors hypothesize that this sequence would be masked by the p11 protein when it binds to TASK-1, thus allowing the correct transport of the channel to the cell membrane rather than being retained in the ER. ...
Two-pore domain (K2P) potassium channels belong to a large family of ion channels implicated in determining and maintaining the resting cell membrane potential. K2P channels are proteins extensively conserved throughout evolution, being present in almost all animal cells. In the nematode Caenorhabditis elegans, 47 genes code K2P channels sub-units, but only three of them have been characterized and reported in the literature. By tagging a certain number of them with fluorescent proteins (CRISPR/Cas9), we have found that nine channels are co-expressed in body wall muscle, showing a highly specific sub-cellular distribution. The most fascinating distribution was the one of TWK-28, which exhibits a polarized comet-like pattern that occupy only the anterior tip of each body wall muscle cell. In order to elucidate the cellular mechanisms underlying this particular distribution, we performed a genetic screen on the novel TWK-28 gain-of-function strain. We revealed that genes belonging to Dystrophin-Associated Protein Complex (DAPC) are involved in determining the amount of this channel at the muscle cell surface. DAPC is composed of at least 10 intra and extracellular proteins and plays a key role in physically connecting the extracellular matrix to the actin cytoskeleton. Interestingly, when tagging multiple components of DAPC with fluorescent proteins by CRISPR/Cas9 gene editing, we found that most of the dystrophin-associated proteins, such as syntrophin/STN-1, dystrobrevin/DYB-1 or even sarcoglycans (SGCA-1 and SGCB-1), show a particularly asymmetric distribution in muscle. We also revealed the, to date excluded, presence of dystroglycan/DGN-1 in body wall muscle of C. elegans. Finally, the asymmetric distribution of TWK-28 along the antero-posterior axis on a cellular and tissue scale, suggests that the Planar Cell Polarity pathways might be implicated. By gene candidate approach of the WNT pathway, we showed that proteins such as Disheveled, ROR/CAM-1 or WNT ligand/EGL-20 can modify the localization of TWK-28 by driving it into a new posterior sub-complex in the muscle cells
... K ATP channel is composed of four pore-forming inward rectifier potassium channel (Kir6) subunits and four regulatory sulfonylurea receptor (SUR) subunits. Both Kir6 and SUR subunits have the ER retention motif (RKR), and the formation of K ATP channel octamer has been proposed to shield this motif to facilitate ER exit [3]. Further, SUR subunits are glycosylated in the Golgi apparatus before reaching the cell surface [4,5]. ...
ATP-sensitive K⁺ (KATP) channel couples membrane excitability to intracellular energy metabolism. Maintaining KATP channel surface expression is key to normal insulin secretion, blood pressure and cardioprotection. However, the molecular mechanisms regulating KATP channel internalization and endocytic recycling, which directly affect the surface expression of KATP channels, are poorly understood. Here we used the cardiac KATP channel subtype, Kir6.2/SUR2A, and characterized Rab35 GTPase as a key regulator of KATP channel endocytic recycling. Electrophysiological recordings and surface biotinylation assays showed decreased KATP channel surface density with co-expression of a dominant negative Rab35 mutant (Rab35-DN), but not other recycling-related Rab GTPases, including Rab4, Rab11a and Rab11b. Immunofluorescence images revealed strong colocalization of Rab35-DN with recycling Kir6.2. Rab35-DN minimized the recycling rate of KATP channels. Rab35 also regulated KATP channel current amplitude in isolated adult cardiomyocytes by affecting its surface expression but not channel properties, which validated its physiologic relevance and the potential of pharmacologic target for treating the diseases with KATP channel trafficking defects.
... Neither Kir6.2 nor SUR1 reach the cell membrane when expressed alone because both contain ER retention signals, a three-amino acid motif (RKR), which are masked only in the presence of their partner subunit. When formed, the fully assembled octameric channels are then translocated to the cell surface [29]. K ATP channel surface density is also regulated by internalization, recycling, and/or protein degradation. ...
Objective: Lipotoxicity-induced pancreatic β cells-dysfunction results in decreased insulin secretion in response to multiple stimulus. In this study, we investigated the reversible effects of palmitate (PA) or oleate (OA) on insulin secretion and the relationship with pancreatic β cell ATP-sensitive potassium (KATP) channels.
Methods: MIN6 cells were treated with PA and OA for 48 hr and then washed out for 24 hr to determine the changes in expression and endocytosis of the KATP channels and insulin secretion under glucose- (GSIS) and sulfonylureas-stimulated (SUs-SIS).
Results: MIN6 cells exposed to PA or OA showed both impaired GSIS and SUs-SIS, the former was not restorable, while the latter was reversible with wash out of PA or OA. Decreased expressions of both total and surface Kir6.2 and SUR1 and endocytosis of KATP channels were observed which were also recoverable after washed out. When MIN6 cells exposed to FFAs were cotreated with AICAR or dynasore, we found that endocytosis of KATP channels did not change significantly by AICAR, but was almost completely blocked by dynasore. Meanwhile, the inhibition of endocytosis of KATP channels after washed out could be activated by PIP2. The recovery of SUs-SIS after washed out was significantly weakened by PIP2, but the decrease of SUs-SIS induced by FFAs was not alleviated by dynasore.
Conclusions: FFAs can cause reversible impairment of SUs-SIS on pancreatic β cells. The reversibility of the effects is partial because of the changes of expression and endocytosis of Kir6.2 and SUR1 which was mediated by dynamin.
... Retention mechanisms in the ER, on the other hand, control cargo access to COPII vesicles. Arginine-based (RXR) retention motifs, which supposedly mediate cargo interaction with putative ER resident gatekeepers, were identified in inwardly rectifying potassium channels [7] and in the GB1 subunit of the GABA B receptor [8]. We previously found that the Prenylated Rab Acceptor Family member 2 (PRAF2), an ubiquitous membrane-associated protein [9] particularly abundant in the ER [10], was the gatekeeper that retains the GB1 GABA B receptor subunit in the ER via the recognition of a specific RXR-di-leucine (LL) motif [11]. ...
The endoplasmic reticulum exit of some polytopic plasma membrane proteins (PMPs) is controlled by arginin-based retention motifs. PRAF2, a gatekeeper which recognizes these motifs, was shown to retain the GABAB-receptor GB1 subunit in the ER. We report that PRAF2 can interact on a stoichiometric basis with both wild type and mutant F508del Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR), preventing the access of newly synthesized cargo to ER exit sites. Because of its lower abundance, compared to wild-type CFTR, CFTR-F508del recruitment into COPII vesicles is suppressed by the ER-resident PRAF2. We also demonstrate that some pharmacological chaperones that efficiently rescue CFTR-F508del loss of function in CF patients target CFTR-F508del retention by PRAF2 operating with various mechanisms. Our findings open new therapeutic perspectives for diseases caused by the impaired cell surface trafficking of mutant PMPs, which contain
RXR-based retention motifs that might be recognized by PRAF2.
... The mechanism of cargo sorting involves the interaction among the Golgi membrane, specific motif sequences in the cargo molecules, and different subunits of COPI. For example, after the recruitment of Arf1-GTP and COPI onto the cis-Golgi membrane, the α-solenoid domain of β'-and α-COP form an arch above the βδγζ-COP subcomplex by orienting their N-terminal β-propeller domains, such that the dilysine motif (K(X)KXX, K, Lys; X, any amino acid), or motif like RKR (R, Arg; K, Lys) of cargo binding site locates on the Golgi membrane [38,48], and recruits molecules such as escaped ER-resident protein cargoes and soluble ER protein cargoes, which are then retro-transferred from the Golgi apparatus back to the ER [39]. When the COPI complex further concentrates and binds to cargo molecules, the B-subcomplex and the F-subcomplex intertwine to form a triply folded structure, which are connected by flexibly attached domains. ...
Intracellular vesicle trafficking is the fundamental process to maintain the homeostasis of membrane-enclosed organelles in eukaryotic cells. These organelles transport cargo from the donor membrane to the target membrane through the cargo containing vesicles. Vesicle trafficking pathway includes vesicle formation from the donor membrane, vesicle transport, and vesicle fusion with the target membrane. Coat protein mediated vesicle formation is a delicate membrane budding process for cargo molecules selection and package into vesicle carriers. Vesicle transport is a dynamic and specific process for the cargo containing vesicles translocation from the donor membrane to the target membrane. This process requires a group of conserved proteins such as Rab GTPases, motor adaptors, and motor proteins to ensure vesicle transport along cytoskeletal track. Soluble N-ethyl-maleimide-sensitive factor (NSF) attachment protein receptors (SNARE)-mediated vesicle fusion is the final process for vesicle unloading the cargo molecules at the target membrane. To ensure vesicle fusion occurring at a defined position and time pattern in eukaryotic cell, multiple fusogenic proteins, such as synaptotagmin (Syt), complexin (Cpx), Munc13, Munc18 and other tethering factors, cooperate together to precisely regulate the process of vesicle fusion. Dysfunctions of the fusogenic proteins in SNARE-mediated vesicle fusion are closely related to many diseases. Recent studies have suggested that stimulated membrane fusion can be manipulated pharmacologically via disruption the interface between the SNARE complex and Ca ²⁺ sensor protein. Here, we summarize recent insights into the molecular mechanisms of vesicle trafficking, and implications for the development of new therapeutics based on the manipulation of vesicle fusion.
... To study the neurological features of DEND, we generated pan-neuronal nK ATP -GOF mice (SI Appendix, Fig. S1). Because K ATP channels require both Kir6.2 and SUR1 subunits (26), only those neurons that endogenously express SUR will form functional transgenic K ATP channels (14). As expected, Kir6.2 messenger RNA (mRNA) levels were increased in nK ATP -GOF mice, while SUR1 levels were unaffected (SI Appendix, Fig. S1 H, Left), suggesting no changes in channel density in mutant mice. ...
... It has been established that voltage-gated channel auxiliary subunits may assist in increasing membrane expression by promoting their trafficking from the endoplasmic reticulum (ER). These subunits often mask ER retention motifs and/or assist in the proper folding of the pore-forming subunits, thereby facilitating their transfer from the ER and insertion and stabilization in the plasma membrane [48,49]. Initial studies identifying the DPP6 modulation of Kv4-mediated currents in heterologous expression systems were key in confirming that both DPP6 and DPP10 are critical in Kv4 trafficking and membrane stabilization. ...
The concerted action of voltage-gated ion channels in the brain is fundamental in controlling neuronal physiology and circuit function. Ion channels often associate in multi-protein complexes together with auxiliary subunits, which can strongly influence channel expression and function and, therefore, neuronal computation. One such auxiliary subunit that displays prominent expression in multiple brain regions is the Dipeptidyl aminopeptidase-like protein 6 (DPP6). This protein associates with A-type K+ channels to control their cellular distribution and gating properties. Intriguingly, DPP6 has been found to be multifunctional with an additional, independent role in synapse formation and maintenance. Here, we feature the role of DPP6 in regulating neuronal function in the context of its modulation of A-type K+ channels as well as its independent involvement in synaptic development. The prevalence of DPP6 in these processes underscores its importance in brain function, and recent work has identified that its dysfunction is associated with host of neurological disorders. We provide a brief overview of these and discuss research directions currently underway to advance our understanding of the contribution of DPP6 to their etiology.
... The rise in cAMP induces the closure of K ATP þ channels evoking b-cell membrane depolarization, Ca 2þ influx, and insulin release. Sulfonylureas, which are benzoic acid derivatives, bind to the SUR1 subunits of the K ATP þ channel, inhibiting the K þ influx, triggering the same process train as elevated cAMP resulting in hormone secretion [41]. It should be noted, however, that this sulfonylurea-triggered production of insulin in b-cells is essentially independent of extracellular glucose [57] Review levels, given that the binding of sulfonylureas diminishes the ability of K ATP þ channels to sense intracellular ATP and ADP fluctuations in response to the rise and fall in extracellular glucose. ...
Background:
Optogenetic modalities as well as optochemical and photopharmacological strategies, collectively termed optical methods, have revolutionized the control of cellular functions via light with great spatiotemporal precision. In comparison to the major advances in the photomodulation of signaling activities noted in neuroscience, similar applications to endocrine cells of the pancreas, particularly insulin-producing β-cells, have been limited. The availability of tools allowing light-mediated changes in the trafficking of ions such as K+ and Ca2+ and signaling intermediates such as cyclic adenosine monophosphate (cAMP), renders β-cells and their glucose-stimulated insulin secretion (GSIS) amenable to optoengineering for drug-free control of blood sugar.
Scope of review:
The molecular circuit of the GSIS in β-cells is described with emphasis on intermediates which are targetable for optical intervention. Various pharmacological agents modifying the release of insulin are reviewed along with their documented side effects. These are contrasted with optical approaches, which have already been employed for engineering β-cell function or are considered for future such applications. Principal obstacles are also discussed as the implementation of optogenetics is pondered for tissue engineering and biology applications of the pancreas.
Conclusions:
Notable advances in optogenetic, optochemical and photopharmacological tools are rendering feasible the smart engineering of pancreatic cells and tissues with light-regulated function paving the way for novel solutions for addressing pancreatic pathologies including diabetes.
... Therefore, we speculate that there are cytoplasmic retention signals on cytosolic regions of NALCN, and the binding of UNIM-A and UNIM-B onto the UNC79-UNC80 heterodimer masks such signals to facilitate the surface localization of the NALCN channel. This is akin to the K ATP channel, in which the SUR subunits mask the ER retention signal of Kir6 subunits to promote the surface localization of the fully assembled K ATP channel 27 . ...
NALCN channel mediates sodium leak currents and is important for maintaining proper resting membrane potential. NALCN and FAM155A form the core complex of the channel, the activity of which essentially depends on the presence of both UNC79 and UNC80, two auxiliary proteins. NALCN, FAM155A, UNC79, and UNC80 co-assemble into a large hetero-tetrameric channel complex. Genetic mutations of NALCN channel components lead to neurodevelopmental diseases. However, the structure and mechanism of the intact channel complex remain elusive. Here, we present the cryo-EM structure of the mammalian NALCN-FAM155A-UNC79-UNC80 quaternary complex. The structure shows that UNC79-UNC80 form a large piler-shaped heterodimer which was tethered to the intracellular side of the NALCN channel through tripartite interactions with the cytoplasmic loops of NALCN. Two interactions are essential for proper cell surface localization of NALCN. The other interaction relieves the self-inhibition of NALCN by pulling the auto-inhibitory CTD Interacting Helix (CIH) out of its binding site. Our work defines the structural mechanism of NALCN modulation by UNC79 and UNC80. NALCN channel mediates sodium leak currents and is essential for neuronal activity. Here authors uncover the mechanism of how UNC79 and UNC80 interact and promote the function of NALCN channel.
... and AtADS2 contain a putative di-arginine (-RXR-) ER retrieval and retention motif [139] close to the C terminus, and when transiently expressed as yellow fluorescent protein-fusions in Nicotiana benthamiana leaves, they colocalize with ER membrane markers [137]. In the other five ADS proteins predicted to be ER-localized, the di-arginine motif is atypical, with arginine replaced with lysine residues (-KXK-) [140]. ...
Monounsaturated fatty acids are straight-chain aliphatic monocarboxylic acids comprising a unique carbon‑carbon double bond, also termed unsaturation. More than 50 distinct molecular structures have been described in the plant kingdom, and more remain to be discovered. The evolution of land plants has apparently resulted in the convergent evolution of non-homologous enzymes catalyzing the dehydrogenation of saturated acyl chain substrates in a chemo-, regio- and stereoselective manner. Contrasted enzymatic characteristics and different subcellular localizations of these desaturases account for the diversity of existing fatty acid structures. Interestingly, the location and geometrical configuration of the unsaturation confer specific characteristics to these molecules found in a variety of membrane, storage, and surface lipids. An ongoing research effort aimed at exploring the links existing between fatty acid structures and their biological functions has already unraveled the importance of several monounsaturated fatty acids in various physiological and developmental contexts. What is more, the monounsaturated acyl chains found in the oils of seeds and fruits are widely and increasingly used in the food and chemical industries due to the physicochemical properties inherent in their structures. Breeders and plant biotechnologists therefore develop new crops with high monounsaturated contents for various agro-industrial purposes.
... To study the neurological features of DEND, we generated pan-neuronal nK ATP -GOF mice (SI Appendix, Fig. S1). Because K ATP channels require both Kir6.2 and SUR1 subunits (26), only those neurons that endogenously express SUR will form functional transgenic K ATP channels (14). As expected, Kir6.2 messenger RNA (mRNA) levels were increased in nK ATP -GOF mice, while SUR1 levels were unaffected (SI Appendix, Fig. S1 H, Left), suggesting no changes in channel density in mutant mice. ...
Significance
Gain-of-function (GOF) mutations in the ATP-sensitive potassium (K ATP ) channel cause neonatal diabetes, with some individuals exhibiting developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome. In this study, we uncover the direct effects of neuronal expression of K ATP -GOF mutations, and not diabetes per se, on the neurological features of DEND. Our results show a close link between neuronal K ATP -GOF expression and cognitive dysfunction in DEND and reveal that antidiabetic sulfonylureas, which successfully treat diabetes, mitigate some sensorimotor problems but not cognitive deficits. These results have critical implications for humans, revealing the need for novel drugs to treat learning and memory deficits not only for K ATP -induced DEND but also for other pathologies arising from altered ion channels in the brain.
... TRPM4 is Ca 2+ activated, and is one of only two known ion channels in the mammalian genome that exclusively and non-selectively conducts monovalent cations [5,58]. TRPM4 is an independently functional ion channel, whereas SUR1 requires binding to a pore-forming subunit for functional capability [59,60]. SUR1 co-associates with TRPM4 (previously SUR1-regulated NCCa-ATP channels/SUR1-NCCa-ATP) and forms a hetero-octameric structure comprised of four SUR1 subunits and four TRPM4 subunits (the pore-forming complex), as schematized in Figure 1 [5,11]. ...
Sulfonylurea receptor 1 (SUR1) is a member of the adenosine triphosphate (ATP)-binding cassette (ABC) protein superfamily, encoded by Abcc8, and is recognized as a key mediator of central nervous system (CNS) cellular swelling via the transient receptor potential melastatin 4 (TRPM4) channel. Discovered approximately 20 years ago, this channel is normally absent in the CNS but is transcriptionally upregulated after CNS injury. A comprehensive review on the pathophysiology and role of SUR1 in the CNS was published in 2012. Since then, the breadth and depth of understanding of the involvement of this channel in secondary injury has undergone exponential growth: SUR1-TRPM4 inhibition has been shown to decrease cerebral edema and hemorrhage progression in multiple preclinical models as well as in early clinical studies across a range of CNS diseases including ischemic stroke, traumatic brain injury, cardiac arrest, subarachnoid hemorrhage, spinal cord injury, intracerebral hemorrhage, multiple sclerosis, encephalitis, neuromalignancies, pain, liver failure, status epilepticus, retinopathies and HIV-associated neurocognitive disorder. Given these substantial developments, combined with the timeliness of ongoing clinical trials of SUR1 inhibition, now, another decade later, we review advances pertaining to SUR1-TRPM4 pathobiology in this spectrum of CNS disease—providing an overview of the journey from patch-clamp experiments to phase III trials.
... hydrophobic amino acid) 81 on cytosolic domains 107,108 to assist a quality control mediated by COPI vesicles, so as to prevent the transport of dysfunctional complexes to cell membrane 81 . ...
Coat protein complex I (COPI) vesicles coated with the heptameric complex coatomer mediate retrograde cargo trafficking from Golgi to endoplasmic reticulum as well as intra-Golgi transport. Whether paralogous subunits of coatomer have different functions is currently unclear. In this thesis, we reveal distinct roles of paralogous coatomer subunits γ1-COP and γ2-COP during the neuronal differentiation of mouse pluripotent cells. Following genome editing experiments, our work shows that γ1-COP specifically facilitates neurite extension and underlines a paralogue-specific function of the COPI pathway and offers evidence for the role of COPI coated vesicles in neuronal polarization. Furthermore, to explore the mechanism of γ1-COP specific functions during pluripotent cells differentiation into neurons, the combination proximity-dependent biotinylation with affinity purification and mass spectrometry (AP-MS) was applied to analyze whether the interactome of γ-COPs reveals paralogue-specific cargos or regulators. In the light of label free quantification (LFQ) analysis, various neurogenesis-related proteins were significantly enriched in the γ1-COP interactome indicating that γ1-COP may preferentially traffic such proteins during the process of pluripotent cells neuronal differentiation. Zusammenfassung Mit dem heptameren Komplex-Coatomer beschichtete Coat Protein Complex I (COPI) -Vesikel vermitteln den retrograden Frachthandel von Golgi zum endoplasmatischen Retikulum sowie den Intra-Golgi-Transport. Ob paraloge Untereinheiten des Coatomers unterschiedliche Funktionen haben, ist derzeit unklar. In dieser Arbeit zeigen wir unterschiedliche Rollen der paralogen Coatomer-Untereinheiten γ1-COP und γ2-COP während der neuronalen Differenzierung pluripotenter Mauszellen. Nach Experimenten zur Bearbeitung des Genoms zeigen unsere Arbeiten, dass γ1-COP die Neuritenverlängerung spezifisch erleichtert, eine paralogspezifische Funktion des COPI-Signalwegs unterstreicht und Hinweise auf die Rolle von COPI-beschichteten Vesikeln bei der neuronalen Polarisation liefert. Um den Mechanismus der γ1-COP-spezifischen Funktionen während der Differenzierung pluripotenter Zellen in Neuronen zu untersuchen, wurde die kombinationsnäherungsabhängige Biotinylierung mit Affinitätsreinigung und Massenspektrometrie (AP-MS) angewendet, um zu analysieren, ob das Interaktom von γ-COPs Paralog zeigt. spezifische Ladungen oder Regulierungsbehörden. Im Lichte der Analyse der markierungsfreien Quantifizierung (LFQ) wurden verschiedene Neurogenese-verwandte Proteine im γ1-COP-Interaktom signifikant angereichert, was darauf hinweist, dass γ1-COP solche Proteine während des Prozesses der neuronalen Differenzierung pluripotenter Zellen bevorzugt transportieren kann.
... The assembly status is thus coupled to forward transport in a quality control process resembling "hide and run" (Michelsen et al., 2005). This process referred originally to Arg-based ER motifs in polytopic membrane proteins that are part of functional heteromultimeric complexes, including, for instance, the ATPsensitive K + channel (Zerangue et al., 1999), γ-aminobutyric acid B receptor (Margeta-Mitrovic et al., 2000), and glutamate receptors (Scott et al., 2003;Xia et al., 2001). Given the broad range of substrates and associated pathways controlled by γ-secretase, tuning and regulating its assembly after ER exit but before entrance into the Golgi provides a spatiotemporal mechanism to regulate overall intramembrane proteolysis by γ-secretase. ...
γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer's disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in post-Golgi compartments.
... The predicted subcellular localisation results showed that all three Elovls are located in the ER, the cellular site where LC-PUFA biosynthesis takes place [1, 35,41]. Retention in the ER is crucial for Elovl to exert its function and, consistently, the E. marinus Elovl4 and Elovl1/7like, but not Elovl6, have the RXR motif that is also associated with ER retention [50] and predicted in the P. trituberculatus Elovl4 [39]. Collectively, the molecular characterisation results showed that the E. marinus Elovl4 and Elovl1/7-like have distinctive features with respect to Elovl6, which can partly account for the substrate specificities revealed in our functional assays in yeast. ...
Long-chain (C20–24) polyunsaturated fatty acids (LC-PUFAs) are essential nutrients that are mostly produced in marine ecosystems. Previous studies suggested that gammarids have some capacity to endogenously produce LC-PUFAs. This study aimed to investigate the repertoire and functions of elongation of very long-chain fatty acid (Elovl) proteins in gammarids. Our results show that gammarids have, at least, three distinct elovl genes with putative roles in LC-PUFA biosynthesis. Phylogenetics allowed us to classify two elongases as Elovl4 and Elovl6, as they were bona fide orthologues of vertebrate Elovl4 and Elovl6. Moreover, a third elongase was named as “Elovl1/7-like” since it grouped closely to the Elovl1 and Elovl7 found in vertebrates. Molecular analysis of the deduced protein sequences indicated that the gammarid Elovl4 and Elovl1/7-like were indeed polyunsaturated fatty acid (PUFA) elongases, whereas Elovl6 had molecular features typically found in non-PUFA elongases. This was partly confirmed in the functional assays performed on the marine gammarid Echinogammarus marinus Elovl, which showed that both Elovl4 and Elovl1/7-like elongated PUFA substrates ranging from C18 to C22. E. marinus Elovl6 was only able to elongate C18 PUFA substrates, suggesting that this enzyme does not play major roles in the LC-PUFA biosynthesis of gammarids.
Sarcolemmal ATP-sensitive potassium (K ATP ) channels play a vital role in cardioprotection. Cardiac K ATP channels are enriched in caveolae and physically interact with the caveolae structural protein caveolin-3 (Cav3). Disrupting caveolae impairs the regulation of K ATP channels through several signaling pathways. However, the direct functional effect of Cav3 on K ATP channels is still poorly understood. Here we used the cardiac K ATP channel subtype, Kir6.2/SUR2A, and showed that Cav3 greatly reduced K ATP channel surface density and current amplitude in a caveolae-independent manner. A screen of Cav3 functional domains revealed that a 25 amino acid region in the membrane attachment domain of Cav3 is the minimal effective segment (MAD1). The peptide corresponding to the MAD1 segment decreased K ATP channel current in a concentration-dependent manner with an IC50 of ~5 μM. The MAD1 segment prevented K ATP channel recycling, thus decreased K ATP channel surface density and abolished the cardioprotective effect of ischemic preconditioning. Our research identified the Cav3 MAD1 segment as a novel negative regulator of K ATP channel recycling, providing pharmacological potentials in the treatment of diseases with K ATP channel trafficking defects.
ATP-sensitive potassium ion channels (KATP) are transmembrane proteins that modulate insulin release and muscle contraction. KATP channels are composed of two types of subunit, Kir6 and SUR, which exist in two and three isoforms respectively with different tissue distribution. In this work, we identify a previously undescribed ancestral vertebrate gene encoding a Kir6-related protein that we have named Kir6.3, which may not have a SUR binding partner, unlike the other two Kir6 proteins. Whereas Kir6.3 was lost in amniotes including mammals, it is still present in several early-diverging vertebrate lineages such as frogs, coelacanth, and rayfinned fishes. Molecular dynamics (MD) simulations using homology models of Kir6.1, Kir6.2, and Kir6.3 from the coelacanth Latimeria chalumnae showed that the three proteins exhibit subtle differences in their dynamics. Steered MD simulations of Kir6-SUR pairs suggest that Kir6.3 has a lower binding affinity for the SUR proteins than either Kir6.1 or Kir6.2. As we found no additional SUR gene in the genome of the species that have Kir6.3, it most likely forms a lone tetramer. These findings invite studies of the tissue distribution of Kir6.3 in relation to the other Kir6 as well as SUR proteins to determine the functional roles of Kir6.3.
Metabotropic glutamate receptor 6 (mGluR6) predominantly localizes to the postsynaptic sites of retinal ON-bipolar cells, at which it recognizes glutamate released from photoreceptors. The C-terminal domain (CTD) of mGluR6 contains a cluster of basic amino acids resembling motifs for endoplasmic reticulum (ER) retention. We herein investigated whether these basic residues are involved in regulating the subcellular localization of mGluR6 in 293 T cells expressing mGluR6 CTD mutants using immunocytochemistry, immunoprecipitation, and flow cytometry. We showed that full-length mGluR6 localized to the ER and cell surface, whereas mGluR6 mutants with 15- and 20-amino acid deletions from the C terminus localized to the ER, but were deficient at the cell surface. We also demonstrated that the cell surface deficiency of mGluR6 mutants was rescued by introducing an alanine substitution at basic residues within the CTD. The surface-deficient mGluR6 mutant still did not localize to the cell surface and was retained in the ER when co-expressed with surface-expressible constructs, including full-length mGluR6, even though surface-deficient and surface-expressible constructs formed heteromeric complexes. The co-expression of the surface-deficient mGluR6 mutant reduced the surface levels of surface-expressible constructs. These results indicate that basic residues in the mGluR6 CTD served as ER retention signals. We suggest that exposed ER retention motifs in the aberrant assembly containing truncated or misfolded mGluR6 prevent these protein complexes from being transported to the cell surface.
The endoplasmic reticulum (ER) is the most widespread organelle within eukaryotic cells, performing various essential functions such as protein synthesis, post-translational modifications, and lipid metabolism. Abnormal fluctuations of biologically active species and microenvironments in the ER can disrupt homeostasis and eventually lead to ER stress, which is closely linked to the occurrence and progression of many human diseases. Therefore, the ER has been regarded as an important analytical object as well as a promising therapeutic target in both bio sensing and biomedicine. Recently, there has been a growing interest in developing photon-excited molecular tools to uncover the physio pathological roles of ER and treat ER-related disorders. This review presents a comprehensive summary of recent advances in ER-targeted small-molecule probes and their applications for fluorescent sensing and phototherapy, mainly focusing on targeting strategies and probe design principles. Last, we discuss the challenges involved with ER-targeted probes and highlight potential prospects in this field.
The 2TM domain family of K channels are also known as the inward-rectifier K channel family. This family includes the strong inward-rectifier K channels (Kir2.x) that are constitutively active, the G-protein-activated inward-rectifier K channels (Kir3.x) and the ATP-sensitive K channels (Kir6.x, which combine with sulphonylurea receptors (SUR1-3)). The pore-forming α subunits form tetramers, and heteromeric channels may be formed within subfamilies (e.g. Kir3.2 with Kir3.3).
TRESK (K2P18.1) possesses unique structural proportions within the K2P background potassium channel family. The previously described TRESK regulatory mechanisms are based on the long intracellular loop between the second and third transmembrane segments (TMS). However, the functional significance of the exceptionally short intracellular C-terminal region (iCtr) following the fourth TMS has not yet been examined. In the present study, we investigated TRESK constructs modified at the iCtr by two-electrode voltage clamp and the newly developed epithelial sodium current ratio (ENaR) method in Xenopus oocytes. The ENaR method allowed the evaluation of channel activity by exclusively using electrophysiology, and provided data that are otherwise not readily available under whole-cell conditions. TRESK homodimer was connected with two ENaC (epithelial Na+ channel) heterotrimers and the Na+ current was measured as an internal reference, proportional to the number of channels in the plasma membrane. Modifications of TRESK iCtr resulted in diverse functional effects, indicating a complex contribution of this region to K+ channel activity. Mutations of positive residues in proximal iCtr locked TRESK in a low activity, calcineurin-insensitive state, although this phosphatase binds to distant motifs in the loop region. Accordingly, mutations in proximal iCtr may prevent the transmission of modulation to the gating machinery. Replacing distal iCtr with a sequence designed to interact with the inner surface of the plasma membrane increased the activity of the channel to unprecedented levels, as indicated by ENaR and single channel measurements. In conclusion, the distal iCtr is a major positive determinant of TRESK function.
Congenital hyperinsulinism (CHI) is the most common cause of persistent hypoglycemia in infancy/childhood and is a serious condition associated with severe recurrent attacks of hypoglycemia due to dysregulated insulin secretion. Timely diagnosis and effective treatment are crucial to prevent severe hypoglycemia that may lead to life-long neurological complications. In pancreatic β-cells, adenosine triphosphate (ATP)-sensitive K+ (KATP) channels are a central regulator of insulin secretion vital for glucose homeostasis. Genetic defects that lead to loss of expression or function of KATP channels are the most common cause of HI (KATP-HI). Much progress has been made in our understanding of the molecular genetics and pathophysiology of KATP-HI in the past decades; however, treatment remains challenging, in particular for patients with diffuse disease who do not respond to the KATP channel activator diazoxide. In this review, we discuss current approaches and limitations on the diagnosis and treatment of KATP-HI, and offer perspectives on alternative therapeutic strategies.
Commonly used techniques, such as CryoEM or X-ray, are not able to capture the structural reorganizations of disordered regions of proteins (IDR); therefore, it is difficult to assess their functions in proteins based exclusively on experiments. To fill this gap, we used computational molecular dynamics (MD) simulation methods to capture IDR dynamics and trace biological function-related interactions in the Kir6.2/SUR1 potassium channel. This ATP-sensitive octameric complex, one of the critical elements in the insulin secretion process in human pancreatic β-cells, has four to five large, disordered fragments. Using unique MD simulations of the full Kir6.2/SUR1 channel complex, we present an in-depth analysis of the dynamics of the disordered regions and discuss the possible functions they could have in this system. Our MD results confirmed the crucial role of the N-terminus of the Kir6.2 fragment and the L0-loop of the SUR1 protein in the transfer of mechanical signals between domains that trigger insulin release. Moreover, we show that the presence of IDRs affects natural ligand binding. Our research takes us one step further toward understanding the action of this vital complex.
Potassium channels are multi-subunit transmembrane proteins that permit the selective passage of potassium and play fundamental roles in physiological processes, such as action potentials in the nervous system and organismal salt and water homeostasis, which is mediated by the kidney. Like all ion channels, newly translated potassium channels enter the endoplasmic reticulum (ER) and undergo the error-prone process of acquiring post-translational modifications, folding into their native conformations, assembling with other subunits, and trafficking through the secretory pathway to reach their final destinations, most commonly the plasma membrane. Disruptions in these processes can result in detrimental consequences, including various human diseases. Thus, multiple quality control checkpoints evolved to guide potassium channels through the secretory pathway and clear potentially toxic, aggregation-prone misfolded species. We will summarize current knowledge on the mechanisms underlying potassium channel quality control in the secretory pathway, highlight diseases associated with channel misfolding, and suggest potential therapeutic routes.
Stimulator of interferon genes (STING) plays important roles in innate immunology. In this study, we isolated the STING gene in Nile tilapia, termed OnSTING. Using quantitative RT–PCR, we explored the expression patterns of the OnSTING gene. Using dual-luciferase reporter assays, we revealed the effect of STING overexpression on nuclear factor κB (NF-κB), IFN and AP activation in HEK 293 cells. Using coimmunoprecipitation, the interaction of STING and TRIF was studied. The effect of OnSTING overexpression on the antibacterial activity in tilapia was investigated. The results showed that upon stimulation with Streptococcus agalactiae, the OnSTING transcript was upregulated in all the tested tissues. OnSTING mRNA levels were very stable from 2.5 to 8.5 dpf. Moreover, OnSTING, OnIFN and IRF3 expression was induced by LPS, Poly (I:C), S. agalactiae WC1535 and DCPS in Nile tilapia macrophages. Overexpression of OnSTING and OnDDX41 increased NF-κB activation in HEK293T cells and slightly increased IFN-β activation but had no effect on AP-1 activation. OnSTING interacted with OnDDX41 and OnTBK1. However, OnSTING did not interact with TRIF. OnSTING overexpression in vivo decreased the sensitivity of tilapia to S. agalactiae infection. These results are helpful for clarifying the innate immune response against bacterial infection in Nile tilapia.
Persistent infection with high-risk human papillomaviruses (HPVs) is the causal factor in multiple human malignancies, including >99% of cervical cancers and a growing proportion of oropharyngeal cancers. Prolonged expression of the viral oncoproteins E6 and E7 is necessary for transformation to occur. Although some of the mechanisms by which these oncoproteins contribute to carcinogenesis are well-characterised, a comprehensive understanding of the signalling pathways manipulated by HPV is lacking. Here, we present the first evidence to our knowledge that the targeting of a host ion channel by HPV can contribute to cervical carcinogenesis. Through the use of pharmacological activators and inhibitors of ATP-sensitive potassium ion (K ATP ) channels, we demonstrate that these channels are active in HPV-positive cells and that this activity is required for HPV oncoprotein expression. Further, expression of SUR1, which forms the regulatory subunit of the multimeric channel complex, was found to be upregulated in both HPV+ cervical cancer cells and in samples from patients with cervical disease, in a manner dependent on the E7 oncoprotein. Importantly, knockdown of SUR1 expression or K ATP channel inhibition significantly impeded cell proliferation via induction of a G1 cell cycle phase arrest. This was confirmed both in vitro and in in vivo tumourigenicity assays. Mechanistically, we propose that the pro-proliferative effect of K ATP channels is mediated via the activation of a MAPK/AP-1 signalling axis. A complete characterisation of the role of K ATP channels in HPV-associated cancer is now warranted in order to determine whether the licensed and clinically available inhibitors of these channels could constitute a potential novel therapy in the treatment of HPV-driven cervical cancer.
Sarcolemmal/plasmalemmal ATP-sensitive K ⁺ (K ATP ) channels have key roles in many cell types and tissues. Hundreds of studies have described how the K ATP channel activity and ATP sensitivity can be regulated by changes in the cellular metabolic state, by receptor signaling pathways and by pharmacological interventions. These alterations in channel activity directly translate to alterations in cell or tissue function, that can range from modulating secretory responses, such as insulin release from pancreatic β-cells or neurotransmitters from neurons, to modulating contractile behavior of smooth muscle or cardiac cells to elicit alterations in blood flow or cardiac contractility. It is increasingly becoming apparent, however, that K ATP channels are regulated beyond changes in their activity. Recent studies have highlighted that K ATP channel surface expression is a tightly regulated process with similar implications in health and disease. The surface expression of K ATP channels is finely balanced by several trafficking steps including synthesis, assembly, anterograde trafficking, membrane anchoring, endocytosis, endocytic recycling and degradation. This review aims to summarize the physiological and pathophysiological implications of K ATP channel trafficking and mechanisms that regulate K ATP channel trafficking. A better understanding of this topic has potential to identify new approaches to develop therapeutically useful drugs to treat K ATP channel-related diseases.
Sialylation, the final stage of post-translational modification of proteins, is achieved in the Golgi apparatus and is related to the malignant phenotype of cancer. Disialylation of ganglioside (GD3) by St8sia1 and polysialylation by St8sia2 and 4 have been shown to be related to malignant phenotypes; however, di/oligosialylation by St8sia6 is still unknown. In this study, we analyzed the malignant phenotype of St8sia6 and found that upregulation of St8sia6 in melanoma B16 cells increased anchorage-independent cell growth, which was not due to sialic acid cleavage by a sialidase. Moreover, unlike other sialyltransferases, St8sia6 localized to the endoplasmic reticulum (ER). We found that the localization to the Golgi apparatus could be regulated by swapping experiments using St8sia2; however, the malignant phenotype did not change. These data demonstrate that the enhancement of anchorage-independent cell growth by St8sia6 is not due to its localization of ER, but is due to the expression of the protein itself.
The 2TM domain family of K channels are also known as the inward-rectifier K channel family. This family includes the strong inward-rectifier K channels (Kir2.x) that are constitutively active, the G-protein-activated inward-rectifier K channels (Kir3.x) and the ATP-sensitive K channels (Kir6.x, which combine with sulphonylurea receptors (SUR1-3)). The pore-forming α subunits form tetramers, and heteromeric channels may be formed within subfamilies (e.g. Kir3.2 with Kir3.3).
For the past two decades several scholarly reviews have appeared on the inwardly rectifying potassium (Kir) channels. We would like to highlight two efforts in particular, which have provided comprehensive reviews of the literature up to 2010 (Hibino et al., Physiol Rev 90(1):291-366, 2010; Stanfield et al., Rev Physiol Biochem Pharmacol 145:47-179, 2002). In the past decade, great insights into the 3-D atomic resolution structures of Kir channels have begun to provide the molecular basis for their functional properties. More recently, computational studies are beginning to close the time domain gap between in silico dynamic and patch-clamp functional studies. The pharmacology of these channels has also been expanding and the dynamic structural studies provide hope that we are heading toward successful structure-based drug design for this family of K+ channels. In the present review we focus on placing the physiology and pharmacology of this K+ channel family in the context of atomic resolution structures and in providing a glimpse of the promising future of therapeutic opportunities.
Developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome, the most severe end of neonatal diabetes mellitus, is caused by mutation in the ATP-sensitive potassium (KATP) channel. In addition to diabetes, DEND patients present muscle weakness as one of the symptoms, and although the muscle weakness is considered to originate in the brain, the pathological effects of mutated KATP channels in skeletal muscle remain elusive. Here, we describe the local effects of the KATP channel on muscle by expressing the mutation present in the KATP channels of the DEND syndrome in the murine skeletal muscle cell line C2C12 in combination with computer simulation. The present study revealed that the DEND mutation can lead to a hyperpolarized state of the muscle cell membrane, and molecular dynamics simulations based on a recently reported high-resolution structure provide an explanation as to why the mutation reduces ATP sensitivity and reveal the changes in the local interactions between ATP molecules and the channel.
ATP-sensitive potassium (KATP) channels are multimeric protein complexes made of four inward rectifying potassium channel (Kir6.x) subunits and four ABC protein sulfonylurea receptor (SURx) subunits. Kir6.x subunits form the potassium ion conducting pore of the channel, and SURx functions to regulate Kir6.x. Kir6.x and SURx are uniquely dependent on each other for expression and function. In pancreatic β-cells, channels comprising SUR1 and Kir6.2 mediate glucose-stimulated insulin secretion and are the targets of antidiabetic sulfonylureas. Mutations in genes encoding SUR1 or Kir6.2 are linked to insulin secretion disorders, with loss- or gain-of-function mutations causing congenital hyperinsulinism or neonatal diabetes mellitus, respectively. Defects in the KATP channel in other tissues underlie human diseases of the cardiovascular and nervous systems. Key to understanding how channels are regulated by physiological and pharmacological ligands and how mutations disrupt channel assembly or gating to cause disease is the ability to observe structural changes associated with subunit interactions and ligand binding. While recent advances in the structural method of single-particle cryo-electron microscopy (cryoEM) offers direct visualization of channel structures, success of obtaining high-resolution structures is dependent on highly concentrated, homogeneous KATP channel particles. In this chapter, we describe a method for expressing KATP channels in mammalian cell culture, solubilizing the channel in detergent micelles and purifying KATP channels using an affinity tag to the SURx subunit for cryoEM structural studies.
We have used fibroblast clones expressing muscle nicotinic acetylcholine receptor alpha and gamma, and alpha and delta subunits to measure the kinetics of subunit assembly, and to study the properties of the partially assembled products that are formed. We demonstrate by coimmunoprecipitation that assembly intermediates in fibroblasts coexpressing alpha and delta subunits are formed in a time-dependent manner. The alpha and gamma- and the alpha and delta-producing transfected cells form complexes that, when labeled with 125I-alpha-bungarotoxin, migrate in sucrose gradients at 6.3S, a value consistent with a hetero-dimer structure. An additional peak at 8.5S is formed from the alpha and gamma subunits expressed in fibroblasts suggesting that gamma may have more than one binding site for alpha subunit. The stability and specificity of formation of these partially assembled complexes suggests that they are normal intermediates in the assembly of acetylcholine receptor. Comparison of the binding of 125I-alpha-bungarotoxin to intact and detergent-extracted fibroblasts indicate that essentially all of the binding sites are retained in an intracellular pool. The fibroblast delta subunit has the electrophoretic mobility in SDS-PAGE of a precursor that does not contain complex carbohydrates. In addition, alpha gamma and alpha delta complexes had lectin binding properties expected of subunits lacking complex oligosaccharides. Therefore, fibroblasts coexpressing alpha and gamma or alpha and delta subunits produce discrete assembly intermediates that are retained in an intracellular compartment and are not processed by Golgi enzymes.
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.
We have used fibroblast clones expressing muscle nicotinic acetylcholine receptor alpha and gamma, and alpha and delta subunits to measure the kinetics of subunit assembly, and to study the properties of the partially assembled products that are formed. We demonstrate by coimmunoprecipitation that assembly intermediates in fibroblasts coexpressing alpha and delta subunits are formed in a time-dependent manner. The alpha and gamma- and the alpha and delta-producing transfected cells form complexes that, when labeled with 125I-alpha-bungarotoxin, migrate in sucrose gradients at 6.3S, a value consistent with a hetero-dimer structure. An additional peak at 8.5S is formed from the alpha and gamma subunits expressed in fibroblasts suggesting that gamma may have more than one binding site for alpha subunit. The stability and specificity of formation of these partially assembled complexes suggests that they are normal intermediates in the assembly of acetylcholine receptor. Comparison of the binding of 125I-alpha-bungarotoxin to intact and detergent-extracted fibroblasts indicate that essentially all of the binding sites are retained in an intracellular pool. The fibroblast delta subunit has the electrophoretic mobility in SDS-PAGE of a precursor that does not contain complex carbohydrates. In addition, alpha gamma and alpha delta complexes had lectin binding properties expected of subunits lacking complex oligosaccharides. Therefore, fibroblasts coexpressing alpha and gamma or alpha and delta subunits produce discrete assembly intermediates that are retained in an intracellular compartment and are not processed by Golgi enzymes.
Evoked synaptic responses were studied in hippocampal pyramidal neurons, using the in vitro slice preparation. Using both current- and voltage-clamp techniques, the amplitude and time course of the synaptic responses were measured as a function of the membrane potential of the postsynaptic cell. The results provide the first description of the postsynaptic conductance mechanisms responsible for the generation of synaptic signals in a mammalian cortical neuron. Intracellular recordings were made with low-resistance (10-30 MΩ glass micropitettes, which were usually filled with 2 M Cs2SO4 or 4 M K acetate. Under direct visual control, the recording micropipette tips were positioned into the stratum pyramidale (the pyramidal cell body layer) of the CA3 region, and bipolar stimulation electrodes were placed into the stratum granulosum (granule cell body layer) of the dentate gyrus. Low-intensity current was used to stimulate the granule cells, whose mossy fiber axons are known to make monosynaptic en passant synapses onto the proximal apical dendrites of the pyramidal neurons. A 3-kHz time-share single-electrode clamp (SEC) was used for both current- and voltage-clamp experiments. In many of the experiments, cesium ions were injected into the postsynaptic pyramidal neurons to increase their input resistance and reduce outward rectification. Strict criteria were established for accepting voltage-clamp results, criteria that were satisfied in only 12 of the 298 cells we studied. Under current-clamp conditions, when the membrane potential of the postsynaptic pyramidal cell was -60 to -70 mV or more negative, the postsynaptic potential (PSP) typically appeared to consist of a simple monophasic depolarization. However, when the pyramidal cell was depolarized by passing outward current, the PSP became biphasic, consisting of a depolarizing early phase followed by a hyperpolarizing late phase. The early phase is thought to represent the monosynaptic response to the granule cell mossy fiber input, while the late phase is believed to arise from recurrent or feedforward synaptic inhibition. When the pyramidal cell was further depolarized, the amplitude of the hyperpolarizing phase of the PSP greatly increased and the amplitude of the depolarizing phase decreased. When such cells were depolarized more positive than about -30 mV, the entire PSP waveform typically appeared to become hyperpolarizing. However, when the late phase was blocked pharmacologically by adding picrotoxin (5-10 μM) or penicillin (3.3 mM) to the bath, the reversal potential of the remaining PSP shifted in a positive direction to a mean (±SE) value of -0.3 ± 2.9 mV. Using voltage-clamp techniques, it was possible to achieve partial temporal separation of the currents responsible for the early and late phases of the PSP waveform. The early phase was associated with a mean conductance increase of 19.5 ± 3.6 nS, and the currents had a mean reversal potential of -5.7 ± 3.2 mV. The late phase resulted from a much larger conductance increase, with an average value of 93.2 ± 18.9 nS. The reversal potential of the late currents was extremely variable; for technical reasons its value was not determined accurately in the present study. However, the reversal potential of the late current was always negative to the reversal potential for the early currents. When the late current was blocked pharmacologically by adding 5-10 μM picrotoxin to the bath, the reversal potential of the remaining early current was -7.8 ± 5.7 mV. In about 1% of the cells we examined there was no detectable late component even in the absence of the pharmacological blocking agents. In one such cell in which we obtained suitable voltage-clamp measurements, the decay of the synaptic currents could be described by a single exponential. In this cell we were able to examine the decay time constant of the currents as a function of the membrane potential. The decay time constant was shorter at more positive potentials. At a holding potential near the resting potential, the overall current waveform could be reasonably well approximated by an α function. The results indicate that synaptic signal generation produced by granule cell stimulation results from a conductance increase mechanism and the currents display a clear reversal potential, consistent with a chemical mode of synaptic transmission. This characterization of the conductance waveforms and current reversal potential is important for understanding the genesis of synaptic signals and their propagation throughout the dendritic arborization. The ability to separate and quantify the conductances responsible for the excitatory and inhibitory currents will permit tests of several of the possible meachanism that may underlie the interesting forms of use-dependent synaptic plasticity in the hippocampus.
Network oscillations are postulated to be instrumental for synchronizing the activity of anatomically distributed populations of neurons. Results from recent studies on the physiology of cortical interneurons suggest that through their interconnectivity, they can maintain large-scale oscillations at various frequencies (4-12 Hz, 40-100 Hz and 200 Hz). We suggest that networks of inhibitory interneurons within the forebrain impose co-ordinated oscillatory 'contexts' for the 'content' carried by networks of principal cells. These oscillating inhibitory networks may provide the precise temporal structure necessary for ensembles of neurons to perform specific functions, including sensory binding and memory formation.
The Arabidopsis thaliana cDNA, KAT1, encodes a hyperpolarization-activated K+ channel. In the present study, we utilized a combination of random site-directed mutagenesis, genetic screening in a potassium uptake-deficient yeast strain, and electrophysiological analysis in Xenopus oocytes to identify strong modifications in cation selectivity of the inward rectifying K+ channel KAT1. Threonine at position 256 was replaced by 11 other amino acid residues. Six of these mutated KAT1 cDNAs complemented a K+ uptake-deficient yeast strain at low concentrations of potassium. Among these, two mutants (T256D and T256G) showed a sensitivity of yeast growth toward high ammonium concentrations and a dramatic increase in current amplitudes of rubidium and ammonium ions relative to K+ by 39-72-fold. These single site mutations gave rise to Rb+- and NH4(+)-selective channels with Rb+ and NH4+ currents that were approximately 10-13-fold greater in amplitude than K+ currents, whereas the NH4+ to K+ current amplitude ratio of wild type KAT1 was 0.28. This strong conversion in cation specificity without loss of general selectivity exceeds those reported for other mutations in the pore domain of voltage-dependent K+ channels. Yeast growth was greatly impaired by sodium in two other mutants at this site (T256E and T256Q), which were blocked by millimolar sodium (K1/2 = 1.1 mM for T256E), although the wild type channel was not blocked by 110 mM sodium. Interestingly, the ability of yeast to grow in the presence of toxic cations correlated to biophysical properties of KAT1 mutants, illustrating the potential for qualitative K+ channel mutant selection in yeast. These data suggest that the size of the side chain of the amino acid at position 256 in KAT1 is important for enabling cation permeation and that this site plays a crucial role in determining the cation selectivity of hyperpolarization-activated potassium channels.
Signals that can cause retention in the ER have been found in the cytoplasmic domain of individual subunits of multimeric receptors destined to the cell surface. To study how ER retention motifs are masked during assembly of oligomeric receptors, we analyzed the assembly and intracellular transport of the human high-affinity receptor for immunoglobulin E expressed in COS cells. The cytoplasmic domain of the alpha chain contains a dilysine ER retention signal, which becomes nonfunctional after assembly with the gamma chain, allowing transport out of the ER of the fully assembled receptor. Juxtaposition of the cytoplasmic domains of the alpha and gamma subunits during assembly is responsible for this loss of ER retention. Substitution of the gamma chain cytoplasmic domain with cytoplasmic domains of irrelevant proteins resulted in efficient transport out of the ER of the alpha chain, demonstrating that nonspecific steric hindrance by the cytoplasmic domain of the gamma chain accounts for the masking of the ER retention signal present in the cytoplasmic domain of the alpha chain. Such a mechanism allows the ER retention machinery to discriminate between assembled and nonassembled receptors, and thus participates in quality control at the level of the ER.
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Psychophysical measurements indicate that human subjects can store approximately seven short-term memories. Physiological studies suggest that short-term memories are stored by patterns of neuronal activity. Here it is shown that activity patterns associated with multiple memories can be stored in a single neural network that exhibits nested oscillations similar to those recorded from the brain. Each memory is stored in a different high-frequency ("40 hertz") subcycle of a low-frequency oscillation. Memory patterns repeat on each low-frequency (5 to 12 hertz) oscillation, a repetition that relies on activity-dependent changes in membrane excitability rather than reverberatory circuits. This work suggests that brain oscillations are a timing mechanism for controlling the serial processing of short-term memories.
Single-unit activity was recorded from inferior temporal (IT) cortex and the hippocampus in 2 macaques trained on auditory-visual and visual-visual delayed matching-to-sample tasks. The main purpose of the study was to compare the response properties of delay neurons between the 2 areas. The authors noted that (a) IT cortex delay activity was usually selective to a particular stimulus, whereas hippocampal delay activity was usually nonselective; (b) the level of delay activity was generally larger in the hippocampus than in IT cortex; and (c) unlike IT cortex delay activity, hippocampal delay activity tended to increase in magnitude as the delay progressed. The authors also examined the functional significance of delay activity and noted a higher probability of encountering a delay neuron when the monkeys were performing 75%-100% correct as compared with 50%-75% correct. The significance of these findings for visual recognition memory is discussed.
Multiple voltage-gated potassium (K) channel gene products are likely to be involved in regulating neuronal excitability of any single neuron in the mammalian brain. Here we show that two closely related voltage-gated K channel proteins, mKv1.1 and mKv1.2, are present in multiple subcellular locations including cell somata, juxta-paranodal regions of myelinated axons, synaptic terminals, unmyelinated axons, specialized junctions among axons, and proximal dendrites. Staining patterns of the two channel polypeptides overlap in some areas of the brain, yet each has a unique pattern of expression. For example, in the hippocampus, both mKv1.1 and mKv1.2 proteins are present in axons, often near or at synaptic terminals in the middle molecular layer of the dentate gyrus, while only mKv1.1 is detected in axons and synaptic terminals in the hilar/CA3 region. In the cerebellum, both channel proteins are localized to axon terminals and specialized junctions among axons in the plexus region of basket cells. Strong differential staining is observed in the olfactory bulb, where mKv1.2 is localized to cell somata and axons, as well as to proximal dendrites of the mitral cells. This overlapping yet differential pattern of expression and specific subcellular localization may contribute to the unique profile of excitability displayed by a particular neuron.
In MDCK cells, basolateral sorting of most membrane proteins has been shown to depend on distinct cytoplasmic domain determinants. These signals can be divided into those which are related to signals for localization at clathrin-coated pits and those which are unrelated. The LDL receptor bears two tyrosine-containing signals, one of each class, that can independently target receptors from the Golgi complex and from endosomes to the basolateral plasma membrane. We have now investigated the other structural features required for the activity of both determinants. We find that both depend, at least in part, on clusters of 1-3 acidic amino acids located on the COOH-terminal side of each tyrosine. While single residues adjacent to each tyrosine were also found to be critical, the two signals differed in that only the coated pit-unrelated signal could tolerate a phenylalanine in place of its tyrosine residue. We also found that the structural requirements for basolateral targeting of the "coated pit-related" signal were distinct from those required for rapid endocytosis. Apart from sharing a common tyrosine residue, no feature of the NPXY motif for coated pit localization was required for basolateral targeting. We also investigated basolateral targeting of the mouse macrophage Fc receptor (FcRII-B2) which contains a tyrosine-independent coated pit localization signal. Basolateral transport and endocytosis were found to depend on a common dileucine-type motif. Thus, basolateral targeting determinants, like coated pit domains, can contain either tyrosine- or di-leucine-containing signals. The amino acids in the vicinity of these motifs determine whether they function as determinants for endocytosis, basolateral targeting, or both.
In the nervous system, a wide diversity of K+ channels are formed by the oligomeric assembly of subunits encoded by a large number of K+ channel genes. The physiological functions of a specific K+ channel subunit in vivo will be dictated in part by its subcellular location within neurons. We have used a combined in situ hybridization and immunocytochemical approach to determine the subcellular distribution of Kv1.2, a member of the Shaker subfamily of K+ channel genes. In contrast to other characterized K+ channel subunits, Kv1.2 protein shows a complex differential subcellular distribution in neurons of rat brain. In some of these neurons (e.g., hippocampal and cortical pyramidal cells, and Purkinje cells), Kv1.2 is concentrated in dendrites, while in others (e.g., cerebellar basket cells), Kv 1.2 is predominantly, if not exclusively, localized to nerve terminals. Furthermore, Kv1.2 immunoreactivity was also detected in certain axon tracts. We hypothesize that the differential sorting of Kv1.2 could result from association of Kv1.2 with varying heterologous K+ channel subunits in different cell types, with the implication that Kv1.2 may participate in distinct heteromultimeric K+ channels in different subcellular domains. The findings suggest that Kv1.2-containing K+ channels may play diverse functional roles in several neuronal compartments, regulating presynaptic or postsynaptic membrane excitability, depending on the neuronal cell type.
ACh may set the dynamics of cortical function to those appropriate for learning new information. In models of the putative associative memory function of piriform cortex, selective suppression of intrinsic but not afferent fiber synaptic transmission by ACh prevents recall of previous input from interfering with the learning of new input (Hasselmo, 1993). Selective cholinergic suppression may play a similar role in the hippocampal formation, where Schaffer collateral synapses in stratum radiatum (s. rad) may store associations between activity in region CA3 and the entorhinal cortex input to region CA1 terminating in stratum lacunosum-moleculare (s. I-m). A computational model of region CA1 predicts that for effective associative memory function of the Schaffer collaterals, cholinergic suppression of synaptic transmission should be stronger in s. rad than in s. I-m. In the hippocampal slice preparation, we tested the effect of the cholinergic agonist carbachol (0.01-500 μM) on synaptic transmission in s. rad and s. I- m. Stimulating and recording electrodes were simultaneously placed in both layers, allowing analysis of the effect of carbachol on synaptic potentials in both layers during the same perfusion in each slice. Carbachol produced a significantly stronger suppression of stimulus-evoked EPSPs in s. rad than in s. I-m at all concentrations greater than 1 μM. At 100 μM, EPSP initial slopes were suppressed by 89.1 ± 3.0% in s. rad, but only by 40.1 ± 4.1% in s. I-m. The muscarinic antagonist atropine (1 μM) blocked cholinergic suppression in both layers. These data support the hypothesis that synaptic modification of the Schaffer collaterals may store associations between activity in region CA3 and the afferent input to region CA1 from the entorhinal cortex. In simulations, feedback regulation of cholinergic modulation based on activity in region CA1 sets the appropriate dynamics of learning for novel associations, and recall for familiar associations.
We describe the expression of gpIRK1, an inwardly rectifying K+ channel obtained from guinea pig cardiac cDNA. gpIRK1 is a homologue of the mouse IRK1 channel identified in macrophage cells. Expression of gpIRK1 in Xenopus oocytes produces inwardly rectifying K+ current, similar to the cardiac inward rectifier current IK1. This current is blocked by external Ba2+ and Cs+. Plasmids containing the gpIRK1 coding region under the transcriptional control of constitutive (PGK) or inducible (GAL) promoters were constructed for expression in Saccharomyces cerevisiae. Several observations suggest that gpIRK1 forms functional ion channels when expressed in yeast. gpIRK1 complements a trk1 delta trk2 delta strain, which is defective in potassium uptake. Expression of gpIRK1 in this mutant restores growth on low potassium media. Growth dependent on gpIRK1 is inhibited by external Cs+. The strain expressing gpIRK1 provides a versatile genetic system for studying the assembly and composition of inwardly rectifying K+ channels.
Inwardly rectifying K channel subunits may form homomeric or heteromeric channels with distinct functional properties. Hyperpolarizing commands
delivered to Xenopus oocytes expressing homomeric K 4.1 channels evoke inwardly rectifying K currents which activate rapidly and undergo a pronounced decay at more hyperpolarized potentials. In addition, K 4.1 subunits form heteromeric channels when coexpressed with several other inward rectifier subunits. However, coexpression
of K 4.1 with K 3.4 causes an inhibition of the K 4.1 current. We have investigated this inhibitory effect and show that it is mediated by interactions between the predicted
transmembrane domains of the two subunit classes. Other subunits within the K 3.0 family also exhibit this inhibitory effect which can be used to define subgroups of the inward rectifier family. Further,
the mechanism of inhibition is likely due to the formation of an “inviable complex” which becomes degraded, rather than by
formation of stable nonconductive heteromeric channels. These results provide insight into the assembly and regulation of
inwardly rectifying K channels and the domains which define their interactions.
Cloned K+ channel beta subunits are hydrophilic proteins which associate to pore-forming alpha subunits of the Shaker subfamily. The resulting alphabeta heteromultimers K+ channels have inactivation kinetics significantly more rapid than those of the corresponding alpha homomultimers. This paper reports the cloning and the brain localization of mKvbeta4 (m for mouse), a new beta subunit. This new beta subunit is highly expressed in the nervous system but is also present in other tissues such as kidney. In contrast with other beta subunits, coexpression of the mKvbeta4 subunit with alpha subunits of Shaker-type K+ channel does not modify the kinetic properties or voltage-dependence of these channels in Xenopus oocytes. Instead, mKvbeta4 associates to Kv2.2 (CDRK), a Shab K+ channel, to specifically enhance (a factor of up to 6) its expression level without changing its elementary conductance or kinetics. It is without effect on another closely related Shab K+ channel Kv2.1 (DRK1). Chimeras between Kv2.1 and Kv2. 2 indicate that the COOH-terminal end of the Kv2.2 protein is essential for its mKvbeta4 sensitivity. The functional results associated with the observation of the co-localization of mKvbeta4 and Kv2.2 transcripts in most brain areas strongly suggest that both subunits interact in vivo to form a slowly-inactivating K+ channel. A chaperone-like effect of mKvbeta4 seems to permit the integration of a larger number of Kv2.2 channels at the plasma membrane.
Each organelle of the secretory pathway is required to selectively allow transit of newly synthesized secretory and plasma membrane proteins and also to maintain a unique set of resident proteins that define its structural and functional properties. In the case of the endoplasmic reticulum (ER), residency is achieved in two ways: (a) prevention of residents from entering newly forming transport vesicles and (b) retrieval of those residents that escape. The latter mechanism is directed by discrete retrieval motifs: Soluble proteins have a H/KDEL sequence at their carboxy-terminus; membrane proteins have a dibasic motif, either di-lysine or di-arginine, located close to the terminus of their cytoplasmic domain. Recently it was found that di-lysine motifs bind the complex of cytosolic coat proteins, COP I, and that this interaction functions in the retrieval of proteins from the Golgi to the ER. Also discussed are the potential roles this interaction may have in vesicular trafficking.
ATP-sensitive potassium channels (K-ATP channels) couple cell metabolism to electrical activity and are important in the physiology and pathophysiology of many tissues. In pancreatic beta-cells, K-ATP channels link changes in blood glucose concentration to insulin secretion. They are also the target for clinically important drugs such as sulphonylureas, which stimulate secretion, and the K+ channel opener diazoxide, which inhibits insulin release. Metabolic regulation of K-ATP channels is mediated by changes in intracellular ATP and Mg-ADP levels, which inhibit and activate the channel, respectively. The beta-cell K-ATP channel is a complex of two proteins: an inward-rectifier K+ channel subunit, Kir6.2, and the sulphonylurea receptor, SUR1. We show here that the primary site at which ATP acts to mediate K-ATP channel inhibition is located on Kir6.2, and that SUR1 is required for sensitivity to sulphonylureas and diazoxide and for activation by Mg-ADP.
Structural models of inward rectifier K+ channels incorporate four identical or homologous subunits, each of which has two hydrophobic segments (M1 and M2) which are predicted to span the membrane as alpha helices. Since hydrophobic interactions between proteins and membrane lipids are thought to be generally of a nonspecific nature, we attempted to identify lipid-contacting residues in Kir2.1 as those which tolerate mutation to tryptophan, which has a large hydrophobic side chain. Tolerated mutations were defined as those which produced measurable inwardly rectifying currents in Xenopus oocytes. To distinguish between water-accessible positions and positions adjacent to membrane lipids or within the protein interior we also mutated residues in M1 and M2 individually to aspartate, since an amino acid with a charged side chain should not be tolerated at lipid-facing or interior positions, due to the energy cost of burying a charge in a hydrophobic environment. Surprisingly, 17 out of 20 and 17 out of 22 non-tryptophan residues in M1 and M2, respectively, tolerated being mutated to tryptophan. Moreover, aspartate was tolerated at 15 out of 22 and 15 out of 21 non-aspartate M1 and M2 positions respectively. Periodicity in the pattern of tolerated vs. nontolerated mutations consistent with alpha helices or beta strands did not emerge convincingly from these data. We consider the possibility that parts of M1 and M2 may be in contact with water.
Signals that can cause retention in the ER have been found in the cytoplasmic domain of individual subunits of multimeric receptors destined to the cell surface. To study how ER retention motifs are masked during assembly of oligomeric receptors, we analyzed the assembly and intracellular transport of the human high-affinity receptor for immunoglobulin E expressed in COS cells. The cytoplasmic domain of the alpha chain contains a dilysine ER retention signal, which becomes nonfunctional after assembly with the gamma chain, allowing transport out of the ER of the fully assembled receptor. Juxtaposition of the cytoplasmic domains of the alpha and gamma subunits during assembly is responsible for this loss of ER retention. Substitution of the gamma chain cytoplasmic domain with cytoplasmic domains of irrelevant proteins resulted in efficient transport out of the ER of the alpha chain, demonstrating that nonspecific steric hindrance by the cytoplasmic domain of the gamma chain accounts for the masking of the ER retention signal present in the cytoplasmic domain of the alpha chain. Such a mechanism allows the ER retention machinery to discriminate between assembled and nonassembled receptors, and thus participates in quality control at the level of the ER.
The fact that medial temporal lobe structures, including the hippocampus, are critical for declarative memory is firmly established by now. The understanding of the role that these structures play in declarative memory, however, despite great efforts spent in the quest, has eluded investigators so far. Given the existing scenario, novel ideas that hold the promise of clarifying matters should be eagerly sought. One such idea was recently proposed by Vargha-Khadem and her colleagues on the basis of their study of three young people suffering from anterograde amnesia caused by early-onset hippocampal pathology. The idea is that the hippocampus is necessary for remembering ongoing life's experiences (episodic memory), but not necessary for the acquisition of factual knowledge (semantic memory). We discuss the reasons why this novel proposal makes good sense and why it and its ramifications should be vigorously pursued. We review and compare declarative and episodic theories of amnesia, and argue that the findings reported by Vargha-Khadem and her colleagues fit well into an episodic theory that retains components already publicized, and adds new ones suggested by the Vargha-Khadem et al. study. Existing components of this theory include the idea that acquisition of factual knowledge can occur independently of episodic memory, and the idea that in anterograde amnesia it is quite possible for episodic memory to be more severely impaired than semantic memory. We suggest a realignment of organization of memory such that declarative memory is defined in terms of features and properties that are common to both episodic and semantic memory. The organization of memory thus modified gives greater precision to the Vargha-Khadem et al. neuroanatomical model in which declarative memory depends on perihippocampal cortical regions but not on the hippocampus, whereas episodic memory, which is separate from declarative memory, depends on the hippocampus.
The resting conductance of cultured-cells from murine pancreases was investigated using the whole-cell, cell-attached and isolated patch modes of the patch-clamp technique. Whole-cell experiments revealed a high input resistance of the cells (>20 G per cell or>100 kcm2), if the medium dialysing the cell interior contained 3 mM ATP. The absence of ATP evoked a large additional K+ conductance. In cell-attached patches single K+-channels were observed in the absence of glucose. Adition of glucose (20 mM) to the bath suppressed the channel activity and initiated action potentials. Similar single-channel currents were recorded from isolated patches. In this case the channels were reversibly blocked by adding ATP (3 mM) to the solution at the intracellular side of the membrane. The conductances (51 pS and 56 pS for [K+]0=145 mM, T=21 C) and kinetics (at –70 mV: open=2.2 ms and closed=0.38 ms and 0.33 ms) of the glucose- and ATP-dependent channels were found to be very similar. It is concluded that both channels are identical. The result suggests that glucose could depolarize the-cell by increasing the cytoplasmic concentration of ATP.
the effect of ADP on ATP-sensitive K+ channels in the insulin-secreting RINm5F cell line has been investigated with the help of single-channel current recording from saponin-permeabilized cells. ADP (100–500 μM) markedly activates K+ channels when added to the bath solution in contact with the membrane inside. ADP-β-S cannot mimick this effect. During sustained ATP (500 μM)-evoked inhibition of K+ channel opening, 500 μM ADP markedly and reversibly activates the channels. Conversely ATP markedly reduces the opening probability of ADP-activated channels. It is suggested that the physiological control of K+ channel opening in the insulin-secreting cells is mediated by changes in ATP/ADP ratio rather than being solely determined by the ATP concentration.
The pathways involved in targeting membrane proteins to lysosomes are extraordinarily complex. Newly synthesized proteins in the ER are transported to the Golgi complex, and upon arrival at the trans Golgi network (TGN) are targeted either directly to endosomes, or first to the cell surface from where they can be rapidly internalized into the endocytic pathway for delivery to lysosomes. The routes to endosomes are specified by sorting motifs in the cytoplasmic tails of the proteins that are recognized at the TGN or plasma membrane. The molecular details of these processes are just emerging.
A fundamental and essential property of nearly all salt-transporting epithelia is the tight parallel coupling between the magnitude of the K-conductive pathway at the basolateral membrane and the activity of the Na,K-dependent ATPase (Na,K-ATPase). In the present study, we demonstrate that the coupling response in the renal proximal tubule is governed, at least in part, through the interaction between ATP-sensitive K channels and Na,K-ATPase-mediated changes in intracellular ATP levels. First, we identified a K-selective channel at the basolateral membrane, which is inhibited by the cytosolic addition of ATP. Second, conventional microelectrode analysis in the isolated perfused proximal straight tubule revealed that these channels are the major determinant of the macroscopic K conductance so that ATP-mediated changes in the open probability of the K channel could alter the extent of K recycling. Indeed, the increase in the macroscopic K conductance upon stimulation of transcellular Na transport and pump activity was found to be paralleled by a decrease in intracellular ATP. Finally, a causal link between parallel Na,K-ATPase-K-channel activity and ATP was established by the finding that intracellular ATP loading uncoupled the response. With our recent observations that similar ATP-sensitive K channels are expressed abundantly in other epithelia, we postulate that ATP may act as a universal coupling modulator of parallel Na,K-ATPase-K-channel activity.
The subunit stoichiometry of the mammalian K+ channel KV1.1 (RCK1) was examined by linking together the coding sequences of 2-5 K+ channel subunits in a single open reading frame and tagging the expression of individual subunits with a mutation (Y379K or Y379R) that altered the sensitivity of the channel to block by external tetraethylammonium ion. Two lines of evidence argue that these constructs lead to K+ channel expression only through the formation of functional tetramers. First, currents expressed by tetrameric constructs containing a single mutant subunit have a sensitivity to tetraethylammonium that is well fitted by a single site binding isotherm. Second, a mutant subunit (Y379K) that expresses only as part of a heteromultimer contributes to the expression of functional channels when coexpressed with a trimeric construct but not a tetrameric construct.
Partial complexes of the T cell antigen receptor lacking zeta chains are delivered to lysosomes. Chimeric proteins composed of the Tac antigen fused to the cytoplasmic domains of each CD3 chain has allowed the identification of lysosomal targeting sequences. Tac-gamma and Tac-delta chimeras are retained in the endoplasmic reticulum because of the presence of basic residues reminiscent of sequences responsible for the localization of endoplasmic reticulum resident proteins. Truncation of these retention motifs revealed lysosomal targeting of both Tac-gamma and delta chimeras. A di-leucine- and a tyrosine-based motif are individually sufficient to induce both endocytosis and delivery to lysosomes of Tac. In contrast with chimeras containing only one of these motifs, the chimera containing both was predominantly delivered directly to lysosomes without going through the cell surface. These two sequences may represent two families of targeting motifs that determine the fate of proteins within the peripheral membrane system.
ATP is normally available in cells at millimolar concentrations and is "buffered" by intracellular pools of other high-energy phosphates, such as creatine phosphate. Thus intracellular [ATP] [( ATP]i) may seem an unlikely candidate for a regulatory signal inside cells. Recent evidence suggests, however, that [ATP]i regulates the behavior of a class of potassium (KATP) channels that are found throughout the cardiovascular system. KATP channels are present in cardiac, skeletal, and vascular smooth muscle. The channels are inhibited by micromolar [ATP]i, and this inhibition is relieved by micromolar [ADP]i. We present evidence in support of the idea that variations of [ATP]i and [ADP]i, even within normal concentration ranges, may influence cellular function in the heart and vascular system via a direct action on the KATP channel. Furthermore, very specific modulators of KATP channel activity are available. We discuss the mechanism of action of these agents and their interaction with endogenous modulators and consider their potential roles in cardiovascular therapy.
The intracellular fate of T cell antigen receptor (TCR) subunits (alpha beta gamma delta epsilon zeta 2) is determined by their assembly in the endoplasmic reticulum (ER). To study the structural bases for this tight correlation between assembly and intracellular fate, we sought to define the nature of determinants for both ER degradation and subunit assembly within the TCR-alpha chain. We found that a 9 amino acid transmembrane sequence of the TCR-alpha chain, containing 2 critical charged residues, was sufficient to cause ER degradation when placed in the context of the Tac antigen, used here as a reporter protein. CD3-delta assembled with chimeric proteins containing this short transmembrane sequence, and this assembly resulted in abrogation of targeting for ER degradation. Thus, the colocalization of determinants for ER degradation and sites of subunit interactions explains how the fate of some newly synthesized TCR chains can be decided on the basis of their assembly status.
The resting conductance of cultured beta-cells from murine pancreases was investigated using the whole-cell, cell-attached and isolated patch modes of the patch-clamp technique. Whole-cell experiments revealed a high input resistance of the cells (greater than 20 G omega per cell or greater than 100 k omega X cm2), if the medium dialysing the cell interior contained 3 mM ATP. The absence of ATP evoked a large additional K+ conductance. In cell-attached patches single K+-channels were observed in the absence of glucose. Addition of glucose (20 mM) to the bath suppressed the channel activity and initiated action potentials. Similar single-channel currents were recorded from isolated patches. In this case the channels were reversibly blocked by adding ATP (3 mM) to the solution at the intracellular side of the membrane. The conductances (51 pS and 56 pS for [K+]0 = 145 mM, T = 21 degrees C) and kinetics (at -70 mV: tau open = 2.2 ms and 1.8 ms, tau closed = 0.38 ms and 0.33 ms) of the glucose- and ATP-dependent channels were found to be very similar. It is concluded that both channels are identical. The result suggests that glucose could depolarize the beta-cell by increasing the cytoplasmic concentration of ATP.
The adenoviral transmembrane E3/19K glycoprotein is a resident of the endoplasmic reticulum. Here we show that the last six amino acid residues of the 15-membered cytoplasmic tail are necessary and sufficient for the ER retention. These residues can be transplanted onto the cytoplasmic tail of other membrane-bound proteins such that ER residency is conferred. Deletion analysis demonstrated that no single amino acid residue is responsible for the retention. The identified structural motif must occupy the extreme COOH-terminal position to be functional. An endogenous transmembrane ER protein, UDP-glucuronosyltransferase, also contains a retention signal in its cytoplasmic tail. We suggest that short linear sequences occupying the extreme COOH-terminal position of transmembrane ER proteins serve as retention signals.
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.
1. Paired intracellular recordings were made in rat hippocampal slice cultures, with the use of either sharp microelectrodes or the whole cell configuration of the patch-clamp technique. Unitary synaptic connections were studied between pyramidal and nonpyramidal cells within and between areas CA1 and CA3. 2. Monosynaptic excitatory synaptic responses between CA3 pyramidal neurons were found in 56% of cell pairs (n = 91, 28 postsynaptic cells). Monosynaptic connections from a CA3 cell to a CA1 cell were observed in 76% of cell pairs (n = 125, 26 postsynaptic cells), but from CA1 to CA3 neurons in only 8% of cell pairs (n = 13, 13 postsynaptic cells). Monosynaptic excitatory connections were found in only 16% of CA1/CA1 cell pairs (n = 25, 10 postsynaptic cells). 3. Disynaptic inhibition was commonly observed between CA3 cell pairs (43%), but rarely found between CA3-CA1 pyramidal cell pairs (2%). In 50% of CA3 pyramidal cell pairs, synchronous inhibitory postsynaptic potentials (IPSPs) in both cells could be triggered by an action potential in one pyramidal cell. Reciprocal monosynaptic connections were found between 75% of interneuron and pyramidal cell pairs within area CA3. 4. The latency of monosynaptic CA3- to CA1-cell responses was significantly longer than for responses between two CA3 cells. Within area CA3 the latencies for inhibitory synaptic responses between interneurons and pyramidal cells were significantly shorter than those for excitatory responses between pyramidal cells. Monosynaptic excitatory postsynaptic potentials (EPSPs) in interneurons had a significantly shorter time-to-peak than those recorded in pyramidal neurons. 5. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX)- and D-2-amino-5-phosphonovalerate (AP5)-sensitive components were identified in unitary monosynaptic EPSPs in CA3-CA3 and CA3-CA1 pyramidal cell pairs. The CNQX-sensitive component had a mean time-to-peak and duration of 6.2 +/- 0.3 (SE) ms and 61.2 +/- 2.0 ms, respectively, and an amplitude of approximately 1 mV (n = 93). The AP5-sensitive component of EPSPs was only detected when the cell was depolarized with respect to the resting potential, had a mean time-to-peak of 41 +/- 5 ms and duration of 121 +/- 11 ms (n = 6), and increased in amplitude with postsynaptic depolarization. 6. Unitary monosynaptic IPSPs between an interneuron and a pyramidal cell had a mean amplitude of approximately 1 mV and were fully blocked by gamma-aminobutyric acid-A (GABAA) receptor antagonists (n = 3). 7. Unitary inhibitory responses were found only within, but not between, areas CA3 or CA1.(ABSTRACT TRUNCATED AT 400 WORDS)
The authentic subunit compositions of neuronal K+ channels purified from bovine brain were analyzed using a monoclonal antibody (mAb 5), reactive exclusively with the Kv1.2 subunit of the latter and polyclonal antibodies specific for fusion proteins containing C-terminal regions of four mammalian Kv proteins. Western blotting of the K+ channels isolated from several brain regions, employing the selective blocker alpha-dendrotoxin (alpha-DTX), revealed the presence in each of four different Kvs. Variable amounts of Kv1.1 and 1.4 subunits were observed in the K+ channels purified from cerebellum, corpus striatum, hippocampus, cerebral cortex, and brain stem; on the other hand, contents of Kv1.6 and 1.2 subunits appeared uniform throughout. Each Kv-specific antibody precipitated a different proportion (anti-Kv1.2 > 1.1 > 1.6 > 1.4) of the channels detectable with radioiodinated alpha-DTX in every brain region, consistent with a widespread distribution of these oligomeric subtypes. Such heterooligomeric combinations were further documented by the lack of additivity upon their precipitation with a mixture of antibodies to Kv1.1 and Kv1.2; moreover, cross-blotting of the multimers precipitated by mAb 5 showed that they contain all four Kv proteins. Collectively, these findings demonstrate that subtypes of alpha-DTX-susceptible K+ channels are prevalent throughout mammalian brain which are composed of different Kv proteins assembled in complexes, shown previously to also contain auxiliary beta-subunits [Parcej, D. N., Scott, V. E. S., & Dolly, J.O. (1992) Biochemistry 31, 11084-11088].
Use of alternative initiator methionines in human invariant (Ii) chain mRNA results in the synthesis of two polypeptides, Iip33 and Iip31. After synthesis both isoforms are inserted into the endoplasmic reticulum (ER) as type II membrane proteins. Subsequently, Iip31 is transported out of the ER, guiding MHC class II to the endocytic pathway, whereas Iip33, which differs by only a 16 residue extension at the N-terminus, becomes an ER resident. Mutagenesis of this extension showed that multiple arginines close to the N-terminus were responsible for ER targeting. The minimal requirements of this targeting motif were found to be two arginines (RR) located at positions 2 and 3, 3 and 4 or 4 and 5 or split by a residue at positions 2 and 4 or 3 and 5. Transplanting an RR motif onto transferrin receptor demonstrated that this motif can target other type II membrane proteins to the ER. The characteristics of this RR motif are similar to the KK ER targeting motif for type I membrane proteins. Indeed, RR-tagged transferrin receptor partially localized to the intermediate compartment, suggesting that like the KK motif, the RR motif directs the retrieval of membrane proteins to the ER via a retrograde transport pathway.
An overview of lesion experiments concerned with the involvement of the hippocampus in learning and memory in the rat is presented. Multiple injections of small amounts of ibotenic acid were used to selectively remove the hippocampus (dentate gyrus, hilar cells, CA1-CA3 pyramidal cells). Similar selective, axon-sparing ibotenate lesions of hippocampus were used in a series of learning and memory experiments employing tasks that are thought to be important in hippocampal function. The performance of rats with the hippocampus removed was compared with that of control animals in the acquisition and retention of spatial versus nonspatial information, forgetting of spatial and nonspatial information, contextual learning, recognition memory and concurrent discrimination learning, and complex representational learning (conditional discrimination and negative patterning learning). The general finding that rats without a hippocampus were impaired on those tasks that required the utilization of spatial and contextual information stands in contrast with the spared performance that was found in learning about and handling (even complex) nonspatial information. Rather than support for views that emphasize a role for the hippocampus in specific memory processes (working memory, declarative memory, temporary memory buffer, configural learning), the present results are more compatible with the idea that the hippocampus plays an especially important role in processing and remembering spatial and contextual information. The limited data that are available using more selective lesions of related hippocampal formation structures (entorhinal cortex, subiculum) suggest that these structures also make important contributions to learning and memory, and that some of these contributions may be different from those made by the hippocampus.
A cDNA clone encoding an inwardly-rectifying potassium channel subunit (Kir6.2) was isolated from an insulinoma cDNA library. The mRNA is strongly expressed in brain, skeletal muscle, cardiac muscle and in insulinoma cells, weakly expressed in lung and kidney and not detectable in spleen, liver or testis. Heterologous expression of Kir6.2 in HEK293 cells was only observed when the cDNA was cotransfected with that of the sulphonylurea receptor (SUR). Whole-cell Kir6.2/SUR currents were K(+)-selective, time-independent and showed weak inward rectification. They were blocked by external barium (5 mM), tolbutamide (Kd = 4.5 microM) or quinine (20 microM) and by 5 mM intracellular ATP. The single-channel conductance was 73 pS. Single-channel activity was voltage-independent and was blocked by 1 mM intracellular ATP or 0.5 mM tolbutamide. We conclude that the Kir6.2/SUR channel complex comprises the ATP-sensitive K-channel.
Voltage-gated K+ channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic beta subunits. Here, we show that, in transfected mammalian cells, the predominant beta subunit isoform in brain, Kv beta 2, associates with the Kv1.2 alpha subunit early in channel biosynthesis and that Kv beta 2 exerts multiple chaperone-like effects on associated Kv1.2 including promotion of cotranslational N-linked glycosylation of the nascent Kv1.2 polypeptide, increased stability of Kv beta 2/Kv1.2 complexes, and increased efficiency of cell surface expression of Kv1.2. Taken together, these results indicate that while some cytoplasmic K+ channel beta subunits affect the inactivation kinetics of alpha subunits, a more general, and perhaps more fundamental, role is to mediate the biosynthetic maturation and surface expression of voltage-gated K+ channel complexes. These findings provide a molecular basis for recent genetic studies indicating that beta subunits are key determinants of neuronal excitability.
A group of ATP-binding cassette (ABC) transporters, including the yeast cadmium transporter (YCF1), the mammalian multidrug resistance-associated protein (MRP), the multispecific organic anion transporter and its congener (MOAT and EBCR), as well as the sulfonylurea receptor (SUR), group into a subfamily by sequence comparison. We suggest that these MRP-related proteins are also characterized by a special, common membrane topology pattern. The most studied ABC transporters, the cystic fibrosis transmembrane conductance regulator (CFTR) and the multidrug resistance (MDR) proteins, were shown to contain a tandem repeat of six transmembrane helices, each set followed by an ATP-binding domain. According to the present study, in contrast to various membrane topology predictions proposed for the different MRP-related proteins, they all seem to have a CFTR/MDR-like core structure, and an additional, large, N-terminal hydrophobic region. This latter domain is predicted to contain 4-6 (most probably 5) transmembrane helices, and is occasionally glycosylated on the cell surface. Since all the MRP-related transporters were shown to interact with anionic compounds, the N-terminal membrane-bound domain may have a key role in these interactions.
1. We have studied the electrophysiological properties of cloned ATP-sensitive K+ channels (KATP channels) heterologously expressed in Xenopus oocytes. This channel comprises a sulphonylurea receptor subunit (SUR) and an inwardly rectifying K+ channel subunit (Kir). 2. Oocytes injected with SUR1 and either Kir6.2 or Kir6.1 exhibited large inwardly rectifying K+ currents when cytosolic ATP levels were lowered by the metabolic inhibitors azide or FCCP. No currents were observed in response to azide in oocytes injected with Kir6.2, Kir6.1 or SUR1 alone, indicating that both the sulphonylurea receptor (SUR1) and an inward rectifier (Kir6.1 or Kir6.2) are needed for functional channel activity. 3. The pharmacological properties of Kir6.2-SUR1 currents resembled those of native beta-cell ATP-sensitive K+ channel currents (KATP currents): the currents were > 90% blocked by tolbutamide (500 microM), meglitinide (10 microM) or glibenclamide (100 nM), and activated 1.8-fold by diazoxide (340 microM), 1.4-fold by pinacidil (1 mM) and unaffected by cromakalim (0.5 mM). 4. Macroscopic Kir6.2-SUR1 currents in inside-out patches were inhibited by ATP with a Ki of 28 microM. Kir6.1-SUR1 currents ran down within seconds of patch excision preventing analysis of ATP sensitivity. 5. No sensitivity to tolbutamide or metabolic inhibition was observed when SUR1 was coexpressed with either Kir1.1a or Kir2.1, suggesting that these proteins do not couple in Xenopus ocytes. 6. Our data demonstrate that the Xenopus oocyte constitutes a good expression system for cloned KATP channels and that expression may be assayed by azide-induced metabolic inhibition.