Kir6.2 mutations causing neonatal diabetes prevent endocytosis of ATP-sensitive potassium channels

ATP-sensitive potassium (KATP) channels couple the metabolic status of a cell to its membrane potential-a property that endows pancreatic beta-cells with the ability to regulate insulin secretion in accordance with changes in blood glucose. The channel comprises four subunits each of Kir6.2 and the sulphonylurea receptor (SUR1). Here, we report that KATP channels undergo rapid internalisation from the plasma membrane by clathrin-mediated endocytosis. We present several lines of evidence to demonstrate that endocytosis is mediated by a tyrosine based signal (330YSKF333) located in the carboxy-terminus of Kir6.2 and that SUR1 has no direct role. We show that genetic mutations, Y330C and F333I, which cause permanent neonatal diabetes mellitus, disrupt this motif and abrogate endocytosis of reconstituted mutant channels. The resultant increase in the surface density of KATP channels would predispose beta-cells to hyperpolarise and may account for reduced insulin secretion in these patients. The data imply that endocytosis of KATP channels plays a crucial role in the (patho)-physiology of insulin secretion.     

Compound heterozygous mutations in the SUR1 (ABCC 8) subunit of pancreatic K(ATP) channels cause neonatal diabetes by perturbing the coupling between Kir6.2 and SUR1 subunits

KATP channels regulate insulin secretion by coupling β-cell metabolism to membrane excitability. These channels are comprised of a pore-forming Kir6.2 tetramer which is enveloped by four regulatory SUR1 subunits. ATP acts on Kir6.2 to stabilize the channel closed state while ADP (coordinated with Mg(2+)) activates channels via the SUR1 domains. Aberrations in nucleotide-binding or in coupling binding to gating can lead to hyperinsulinism or diabetes. Here, we report a case of diabetes in a 7-mo old child with compound heterozygous mutations in ABCC8 (SUR1[A30V] and SUR1[G296R]). In unison, these mutations lead to a gain of KATP channel function, which will attenuate the β-cell response to increased metabolism and will thereby decrease insulin secretion. (86)Rb(+) flux assays on COSm6 cells coexpressing the mutant subunits (to recapitulate the compound heterozygous state) show a 2-fold increase in basal rate of (86)Rb(+) efflux relative to WT channels. Experiments on excised inside-out patches also reveal a slight increase in activity, manifested as an enhancement in stimulation by MgADP in channels expressing the compound heterozygous mutations or homozygous G296R mutation. In addition, the IC 50 for ATP inhibition of homomeric A30V channels was increased ~6-fold, and was increased ~3-fold for both heteromeric A30V+WT channels or compound heterozygous (A30V +G296R) channels. Thus, each mutation makes a mechanistically distinct contribution to the channel gain-of-function that results in neonatal diabetes, and which we predict may contribute to diabetes in related carrier individuals.     

Ankyrin regulates KATP channel membrane trafficking and gating in excitable cells

KATP channels play critical roles in many cellular functions by coupling cell metabolic status to electrical activity. First discovered in cardiomyocytes 1, KATP channels (comprised of Kir6.x and SUR subunits) have since been found in many other tissues, including pancreatic beta cells, skeletal muscle, smooth muscle, brain, pituitary, and kidney. By linking cellular metabolic state with membrane potential, KATP channels are able to regulate a number of cellular functions such as hormone secretion, vascular tone, and excitability. Specifically, a reduction in metabolism causes a decrease in the ATP:ADP ratio, opening of KATP channels and allowing K+ efflux, membrane hyperpolarization, and suppression of electrical activity. Conversely, increased cellular metabolism causes a decrease in the ATP:ADP ratio that leads to closure of the KATP channel, membrane depolarization, and stimulation of cell electrical activity.     

The C terminus of SUR1 is required for trafficking of KATP channels.

In beta cells from the pancreas, ATP-sensitive potassium channels, or KATP channels, are composed of two subunits, SUR1 and KIR6.2, assembled in a (SUR1/KIR6.2)4 stoichiometry. The correct stoichiometry of channels at the cell surface is tightly regulated by the presence of novel endoplasmic reticulum (ER) retention signals in SUR1 and KIR6.2; incompletely assembled KATP channels fail to exit the ER/cis-Golgi compartments. In addition to these retrograde signals, we show that the C terminus of SUR1 has an anterograde signal, composed in part of a dileucine motif and downstream phenylalanine, which is required for KATP channels to exit the ER/cis-Golgi compartments and transit to the cell surface. Deletion of as few as seven amino acids, including the phenylalanine, from SUR1 markedly reduces surface expression of KATP channels. Mutations leading to truncation of the C terminus of SUR1 are one cause of a severe, recessive form of persistent hyperinsulinemic hypoglycemia of infancy. We propose that the complete loss of beta cell KATP channel activity seen in this form of hyperinsulinism is a failure of KATP channels to traffic to the plasma membrane.     

Regulation of KATP Channel Expression and Activity by the SUR1 Nucleotide Binding Fold 1

ATP-sensitive K+ (KATP) channels are oligomeric complexes of pore-forming Kir6 subunits and regulatory Sulfonylurea Receptor (SUR) subunits. SUR, an ATP-Binding Cassette (ABC) transporter, confers Mg-nucleotide stimulation to the channel via nucleotide interactions with its two cytoplasmic domains (Nucleotide Binding Folds 1 and 2; NBF1 and NBF2). Regulation of KATP channel expression is a complex process involving subunit assembly in the ER, SUR glycosylation in the Golgi, and trafficking to the plasma membrane. Dysregulation can occur at different steps of the pathway, as revealed by disease-causing mutations. Here, we have addressed the role of SUR1 NBF1 in gating and expression of reconstituted channels. Deletion of NBF1 severely impairs channel expression and abolishes MgADP stimulation. Total SUR1 protein levels are decreased, suggestive of increased protein degradation, but they are not rescued by treatment with sulfonylureas or the proteasomal inhibitor MG-132. Similar effects of NBF1 deletion are observed in recombinant KATP channels obtained by "splitting" SUR1 into two separate polypeptides (a N-terminal "half" and a C-terminal "half"). Interestingly, the location of the "splitting point" in the vicinity of NBF1 has marked effects on the MgADP stimulation of resulting channels. Finally, ablation of the ER retention motif upstream of NBF1 (in either "split" or full-length SUR1) does not rescue expression of channels lacking NBF1. These results indicate that, in addition to NBF1 being required for MgADP stimulation of the channel, it plays an important role in the regulation of channel expression that is independent of the ER retention checkpoint and the proteasomal degradation pathway.     

Activation of mitochondrial ATP-sensitive potassium channels increases cell viability against rotenone-induced cell death

We recently showed that activation of ATP-sensitive potassium (KATP) channels in PC12 cells induces protection against the neurotoxic effect of rotenone, a mitochondrial complex I inhibitor. In this study, we sought to determine the locus of the KATP channels that mediate this protection in PC12 cells. We found that pretreatment of PC12 cells with diazoxide, a mitochondrial KATP channel selective opener, dose-dependently increases cell viability against rotenone-induced cell death as indicated in trypan blue exclusion assays. The protective effect of this preconditioning is attenuated by 5-hydroxydecanoic acid (5-HD), a selective mitochondrial KATP channel antagonist but not in the presence of HMR-1098, a selective plasma membrane KATP potassium channel antagonist. In contrast, P-1075, a selective plasma membrane KATP channel opener, does not induce protection. Using specific antibodies against SUR1 and Kir6.1, we detected immunoreactive proteins of apparent molecular masses 155 and 50 kDa, corresponding to those previously reported for SUR1 and Kir6.1, respectively, in the mitochondria-enriched fraction of PC12 cells. In addition, whole cell patch-clamp studies revealed that inward currents in PC12 cells are insensitive to P-1075, HMR-1098, glibenclamide and diazoxide, indicating that functional plasma membrane KATP channels are negligible. Taken together, our results demonstrate for the first time that activation of mitochondrial KATP channels elicits protection against rotenone-induced cell death.     

Syntaxin-1A inhibits cardiac KATP channels by its actions on nucleotide binding folds 1 and 2 of sulfonylurea receptor 2A

ATP-sensitive potassium (KATP) channels couple the metabolic status of the cell to its membrane potential to regulate a number of cell actions, including secretion (neurons and neuroendocrine cells) and muscle contractility (skeletal, cardiac, and vascular smooth muscle). KATP channels consist of regulatory sulfonylurea receptors (SUR) and pore-forming (Kir6.X) subunits. We recently reported (Pasyk, E. A., Kang, Y., Huang, X., Cui, N., Sheu, L., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 4234-4240) that syntaxin-1A (Syn-1A), known to mediate exocytotic fusion, was capable of binding the nucleotide binding folds (NBF1 and C-terminal NBF2) of SUR1 to inhibit the KATP channels in insulin-secreting pancreatic islet beta cells. This prompted us to examine whether Syn-1A might modulate cardiac SUR2A/KATP channels. Here, we show that Syn-1A is present in the plasma membrane of rat cardiac myocytes and binds the SUR2A protein (of rat brain, heart, and human embryonic kidney 293 cells expressing SUR2A/Kir6. 2) at its NBF1 and NBF2 domains to decrease KATP channel activation. Unlike islet beta cells, in which Syn-1A inhibition of the channel activity was apparently mediated only via NBF1 and not NBF2 of SUR1, both exogenous recombinant NBF1 and NBF2 of SUR2A were found to abolish the inhibitory actions of Syn-1A on K(ATP) channels in rat cardiac myocytes and HEK293 cells expressing SUR2A/Kir6.2. Together with our recent report, this study suggests that Syn-1A binds both NBFs of SUR1 and SUR2A but appears to exhibit distinct interactions with NBF2 of these SUR proteins in modulating the KATP channels in islet beta cells and cardiac myocytes.     

Review. SUR1: a unique ATP-binding cassette protein that functions as an ion channel regulator

SUR1 is an ATP-binding cassette (ABC) transporter with a novel function. In contrast to other ABC proteins, it serves as the regulatory subunit of an ion channel. The ATP-sensitive (KATP) channel is an octameric complex of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits, and it links cell metabolism to electrical activity in many cell types. ATPase activity at the nucleotide-binding domains of SUR results in an increase in KATP channel open probability. Conversely, ATP binding to Kir6.2 closes the channel. Metabolic regulation is achieved by the balance between these two opposing effects. Precisely how SUR1 talks to Kir6.2 remains unclear, but recent studies have identified some residues and domains that are involved in both physical and functional interactions between the two proteins. The importance of these interactions is exemplified by the fact that impaired regulation of Kir6.2 by SUR1 results in human disease, with loss-of-function SUR1 mutations causing congenital hyperinsulinism and gain-of-function SUR1 mutations leading to neonatal diabetes. This paper reviews recent data on the regulation of Kir6.2 by SUR1 and considers the molecular mechanisms by which SUR1 mutations produce disease.     

Regulation of mitochondrial KATP channel by redox agents

The ATP-dependent K+ channel (KATP) was purified from the inner mitochondrial membrane and reconstituted into lipid bilayer membranes. KATP activity was inhibited by high concentrations of ATP and ADP, but activated by low concentrations (up to 200 microM) of ADP. p-Diethylaminoethylbenzoate (DEB) acted as a KATP opener: at micromolar concentrations, it reversed inhibition by ATP and ADP and it also prevented KATP rundown. Pelargonidine, extracted from flowers of Pelargonium, reduced spontaneous activity of KATP channels and diminished their potentiation by DEB. Their opposite action on KATP corresponded with their opposite redox properties in reactions with free radicals: DEB behaved as an electron donor, whereas pelargonidine acted as an electron acceptor. We hypothesize that thiol groups on mitoKATP are targets for redox-active ligans.     

The anchoring protein SAP97 retains Kv1.5 channels in the plasma membrane of cardiac myocytes

Membrane- associated guanylate kinase proteins (MAGUKs) are important determinants of localization and organization of ion channels into specific plasma membrane domains. However, their exact role in channel function and cardiac excitability is not known. We examined the effect of synapse-associated protein 97 (SAP97), a MAGUK abundantly expressed in the heart, on the function and localization of Kv1.5 subunits in cardiac myocytes. Recombinant SAP97 or Kv1.5 subunits tagged with green fluorescent protein (GFP) were overexpressed in rat neonatal cardiac myocytes and in Chinese hamster ovary (CHO) cells from adenoviral or plasmidic vectors. Immunocytochemistry, fluorescence recovery after photobleaching, and patch-clamp techniques were used to study the effects of SAP97 on the localization, mobility, and function of Kv1.5 subunits. Adenovirus-mediated SAP97 overexpression in cardiac myocytes resulted in the clustering of endogenous Kv1.5 subunits at myocyte-myocyte contacts and an increase in both the maintained component of the outward K(+) current, I(Kur) (5.64 +/- 0.57 pA/pF in SAP97 myocytes vs. 3.23 +/- 0.43 pA/pF in controls) and the number of 4-aminopyridine-sensitive potassium channels in cell-attached membrane patches. In live myocytes, GFP-Kv1.5 subunits were mobile and organized in clusters at the basal plasma membrane, whereas SAP97 overexpression reduced their mobility. In CHO cells, Kv1.5 channels were diffusely distributed throughout the cell body and freely mobile. When coexpressed with SAP97, Kv subunits were organized in plaquelike clusters and poorly mobile. In conclusion, SAP97 regulates the K(+) current in cardiac myocytes by retaining and immobilizing Kv1.5 subunits in the plasma membrane. This new regulatory mechanism may contribute to the targeting of Kv channels in cardiac myocytes.     

Endosomal KATP channels as a reservoir after myocardial ischemia: a role for SUR2 subunits

ATP-sensitive K(+) (K(ATP)) channels, composed of inward rectifier K(+) (Kir)6.x and sulfonylurea receptor (SUR)x subunits, are expressed on cellular plasma membranes. We demonstrate an essential role for SUR2 subunits in trafficking K(ATP) channels to an intracellular vesicular compartment. Transfection of Kir6.x/SUR2 subunits into a variety of cell lines (including h9c2 cardiac cells and human coronary artery smooth muscle cells) resulted in trafficking to endosomal/lysosomal compartments, as assessed by immunofluorescence microscopy. By contrast, SUR1/Kir6.x channels efficiently localized to the plasmalemma. The channel turnover rate was similar with SUR1 or SUR2, suggesting that the expression of Kir6/SUR2 proteins in lysosomes is not associated with increased degradation. Surface labeling of hemagglutinin-tagged channels demonstrated that SUR2-containing channels dynamically cycle between endosomal and plasmalemmal compartments. In addition, Kir6.2 and SUR2 subunits were found in both endosomal and sarcolemmal membrane fractions isolated from rat hearts. The balance of these K(ATP) channel subunits shifted to the sarcolemmal membrane fraction after the induction of ischemia. The K(ATP) channel current density was also increased in rat ventricular myocytes isolated from hearts rendered ischemic before cell isolation without corresponding changes in subunit mRNA expression. We conclude that an intracellular pool of SUR2-containing K(ATP) channels exists that is derived by endocytosis from the plasma membrane. In cardiac myocytes, this pool can potentially play a cardioprotective role by serving as a reservoir for modulating surface K(ATP) channel density under stress conditions, such as myocardial ischemia.     

Functional localization of single active ion channels on the surface of a living cell

The spatial distribution of ion channels in the cell plasma membrane has an important role in governing regional specialization, providing a precise and localized control over cell function. We report here a novel technique based on scanning ion conductance microscopy that allows, for the first time, mapping of single active ion channels in intact cell plasma membranes. We have mapped the distribution of ATP-regulated K+ channels (KATP channels) in cardiac myocytes. The channels are organized in small groups and anchored in the Z-grooves of the sarcolemma. The distinct pattern of distribution of these channels may have important functional implications.     

KATP channels gated by intracellular nucleotides and phospholipids.

The KATP channel is a heterooctamer composed of two different subunits, four inwardly rectifying K+ channel subunits, either Kir6. 1 or Kir6.2, and four sulfonylurea receptors (SUR), which belong to the family of ABC transporters. This unusual molecular architecture is related to the complex gating behaviour of these channels. Intracellular ATP inhibits KATP channels by binding to the Kir6.x subunits, whereas Mg-ADP increases channel activity by a hydrolysis reaction at the SUR. This ATP/ADP dependence allows KATP channels to link metabolism to excitability, which is important for many physiological functions, such as insulin secretion and cell protection during periods of ischemic stress. Recent work has uncovered a new class of regulatory molecules for KATP channel gating. Membrane phospholipids such as phosphoinositol 4, 5-bisphosphate and phosphatidylinositiol 4-monophosphate were found to interact with KATP channels resulting in increased open probability and markedly reduced ATP sensitivity. The membrane concentration of these phospholipids is regulated by a set of enzymes comprising phospholipases, phospholipid phosphatases and phospholipid kinases providing a possible mechanism for control of cell excitability through signal transduction pathways that modulate activity of these enzymes. This review discusses the mechanisms and molecular determinants that underlie gating of KATP channel by nucleotides and phospholipids and their physiological implications.     

Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of {beta}-cell ATP-sensitive potassium channels

ATP-sensitive potassium (K(ATP)) channels of pancreatic beta-cells mediate glucose-induced insulin secretion by linking glucose metabolism to membrane excitability. The number of plasma membrane K(ATP) channels determines the sensitivity of beta-cells to glucose stimulation. The K(ATP) channel is formed in the endoplasmic reticulum (ER) on coassembly of four inwardly rectifying potassium channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits. Little is known about the cellular events that govern the channel's biogenesis efficiency and expression. Recent studies have implicated the ubiquitin-proteasome pathway in modulating surface expression of several ion channels. In this work, we investigated whether the ubiquitin-proteasome pathway plays a role in the biogenesis efficiency and surface expression of K(ATP) channels. We provide evidence that, when expressed in COS cells, both Kir6.2 and SUR1 undergo ER-associated degradation via the ubiquitin-proteasome system. Moreover, treatment of cells with proteasome inhibitors MG132 or lactacystin leads to increased surface expression of K(ATP) channels by increasing the efficiency of channel biogenesis. Importantly, inhibition of proteasome function in a pancreatic beta-cell line, INS-1, that express endogenous K(ATP) channels also results in increased channel number at the cell surface, as assessed by surface biotinylation and whole cell patch-clamp recordings. Our results support a role of the ubiquitin-proteasome pathway in the biogenesis efficiency and surface expression of beta-cell K(ATP) channels.     

Physiological development of insulin secretion, calcium channels, and GLUT2 expression of pancreatic rat beta-cells

Insulin secretion in mature beta-cells increases vigorously when extracellular glucose concentration rises. Glucose-stimulated insulin secretion depends on Ca(2+) influx through voltage-gated Ca(2+) channels. During fetal development, this structured response is not well established, and it is after birth that beta-cells acquire glucose sensitivity and a robust secretion. We compared some elements of glucose-induced insulin secretion coupling in beta-cells obtained from neonatal and adult rats and found that neonatal cells are functionally immature compared with adult cells. We observed that neonatal cells secrete less insulin and cannot sense changes in extracellular glucose concentrations. This could be partially explained because in neonates Ca(2+) current density and synthesis of mRNA alpha1 subunit Ca(2+) channel are lower than in adult cells. Interestingly, immunostaining for alpha1B, alpha1C, and alpha1D subunits in neonatal cells is similar in cytoplasm and plasma membrane, whereas it occurs predominantly in the plasma membrane in adult cells. We also observed that GLUT2 expression in adult beta-cells is mostly located in the membrane, whereas in neonatal cells glucose transporters are predominantly in the cytoplasm. This could explain, in part, the insensitivity to extracellular glucose in neonatal beta-cells. Understanding neonatal beta-cell physiology and maturation contributes toward a better comprehension of type 2 diabetes physiopathology, where alterations in beta-cells include diminished L-type Ca(2+) channels and GLUT2 expression that results in an insufficient insulin secretion.     

Surface expression of Kv1 channels is governed by a C-terminal motif

Voltage-gated K(+) channel subunits must reach the plasma membrane to repolarize action potentials. Yet the efficiency of cell surface targeting varies among Kv subunits with some requiring auxiliary subunits for optimal expression. Here we identify a conserved motif located in the variable C-terminal region of Kv1 channels that controls the efficiency of functional channel expression. Variations among wild type channels in the optimal sequence VXXSL produce differences in distribution and the requirement for auxiliary subunits. Furthermore, deletion of this motif decreases subunit glycosylation and surface localization but does not prohibit subunit multimerization. Finally, the action of the essential sequence is shown to be independent of the chaperone effect of Kvbeta subunits. Thus, the newly identified C-terminal motif governs processing and cell surface expression of Kv1 voltage-gated K(+) channels.     

Gene delivery of Kir6.2/SUR2A in conjunction with pinacidil handles intracellular Ca2+ homeostasis under metabolic stress.

Metabolic injury is a complex process affecting various tissues, with intracellular Ca2+ loading recognized as a common precipitating event leading to cell death. We have recently observed that cells overexpressing recombinant ATP-sensitive K+ (KATP) channel subunits may acquire resistance against metabolic stress. To examine whether, under metabolic challenge, intracellular Ca2+ homeostasis can be maintained by an activator of channel proteins, we delivered Kir6.2 and SUR2A genes, which encode KATP channel subunits, into a somatic cell line lacking native KATP channels. Hypoxia-reoxygenation was simulated by application and removal of the mitochondrial poison 2,4 dinitrophenol. Under such metabolic stress, Ca2+ loading was induced by Ca2+ influx during hypoxia and release of Ca2+ from intracellular stores during reoxygenation. Delivery of Kir6.2/SUR2A genes, in conjunction with the KATP channel activator pinacidil, prevented intracellular Ca2+ loading irrespective of whether the channel opener was applied throughout the duration of hypoxia-reoxygenation or transiently during the hypoxic or reoxygenation stage. In all stages of injury, the effect of pinacidil was inhibited by the selective antagonist of KATP channel, 5-hydroxydecanoate. The present study provides evidence that combined use of gene delivery and pharmacological targeting of recombinant proteins can handle intracellular Ca2+ homeostasis under hypoxia-reoxygenation irrespective of the stage of the metabolic insult.     

KCNE1 subunits require co-assembly with K+ channels for efficient trafficking and cell surface expression

KCNE peptides are a class of type I transmembrane beta subunits that assemble with and modulate the gating and ion conducting properties of a variety of voltage-gated K(+) channels. Accordingly, mutations that disrupt the assembly and trafficking of KCNE-K(+) channel complexes give rise to disease. The cellular mechanisms responsible for ensuring that KCNE peptides assemble with voltage-gated K(+) channels have yet to be elucidated. Using enzymatic deglycosylation, immunofluorescence, and quantitative cell surface labeling experiments, we show that KCNE1 peptides are retained in the early stages of the secretory pathway until they co-assemble with specific K(+) channel subunits; co-assembly mediates KCNE1 progression through the secretory pathway and results in cell surface expression. We also address an apparent discrepancy between our results and a previous study in human embryonic kidney cells, which showed wild type KCNE1 peptides can reach the plasma membrane without exogenously expressed K(+) channel subunits. By comparing KCNE1 trafficking in three cell lines, our data suggest that the errant KCNE1 trafficking observed in human embryonic kidney cells may be due, in part, to the presence of endogenous voltage-gated K(+) channels in these cells.     

A novel KCNJ11 mutation associated with congenital hyperinsulinism reduces the intrinsic open probability of beta-cell ATP-sensitive potassium channels

The beta-cell ATP-sensitive potassium (KATP) channel controls insulin secretion by linking glucose metabolism to membrane excitability. Loss of KATP channel function due to mutations in ABCC8 or KCNJ11, genes that encode the sulfonylurea receptor 1 or the inward rectifier Kir6.2 subunit of the channel, is a major cause of congenital hyperinsulinism. Here, we report identification of a novel KCNJ11 mutation associated with the disease that renders a missense mutation, F55L, in the Kir6.2 protein. Mutant channels reconstituted in COS cells exhibited a wild-type-like surface expression level and normal sensitivity to ATP, MgADP, and diazoxide. However, the intrinsic open probability of the mutant channel was greatly reduced, by approximately 10-fold. This low open probability defect could be reversed by application of phosphatidylinositol 4,5-bisphosphates or oleoyl-CoA to the cytoplasmic face of the channel, indicating that reduced channel response to membrane phospholipids and/or long chain acyl-CoAs underlies the low intrinsic open probability in the mutant. Our findings reveal a novel molecular mechanism for loss of KATP channel function and congenital hyperinsulinism and support the importance of phospholipids and/or long chain acyl-CoAs in setting the physiological activity of beta-cell KATP channels. The F55L mutation is located in the slide helix of Kir6.2. Several permanent neonatal diabetes-associated mutations found in the same structure have the opposite effect of increasing intrinsic channel open probability. Our results also highlight the critical role of the Kir6.2 slide helix in determining the intrinsic open probability of KATP channels.     

Kir6.2ΔC26 Channel Traffics to Plasma Membrane by Constitutive Exocytosis

Adenosine triphosphate （ATP）-sensitive K^＊ （KATP） channels regulate many cellular functions by coupling the metabolic state of the cell to the changes in membrane potential. Truncation of C-terminal 26 amino acid residues of Kir6.2 protein （Kir6.2ΔC26） deletes its endoplasmic reticulum retention signal, allowing functional expression of Kit6.2 in the absence of sulfonylurea receptor subunit, pEGFP-Kir6.2ΔC26 and pKir6.2ΔC26-IRES2-EGFP expression plasmids were constructed and transfected into HEK293 cells. We identified that Kir6.2ΔC26 was localized on the plasma membrane and trafficked to the plasmalemma by means of constitutive exocytosis of Kir6.2ΔC26 transport vesicles, using epi-fluorescence and total intemal reflection fluorescence microscopy. Our electrophysiological data showed that Kir6.2ΔC26 alone expressed KATP currents, whereas EGFP-Kir6.2ΔC26 fusion protein displayed no KATP channel activity.