Role of Derlin-1 protein in proteostasis regulation of ATP-sensitive potassium channels

ATP-sensitive potassium (K(ATP)) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.2 regulate insulin secretion by linking glucose metabolism with membrane potential. The number of K(ATP) channels in the plasma membrane affects the sensitivity of β-cells to glucose. Aberrant surface channel expression leads to insulin secretion disease. Previously, we have shown that K(ATP) channel proteins undergo endoplasmic reticulum (ER)-associated degradation (ERAD) via the ubiquitin-proteasome pathway, and inhibition of proteasome function results in an increase in channel surface expression. Here, we investigated whether Derlin-1, a protein involved in retrotranslocation of misfolded or misassembled proteins across the ER membrane for degradation by cytosolic proteasomes, plays a role in ERAD and, in turn, biogenesis efficiency of K(ATP) channels. We show that both SUR1 and Kir6.2 form a complex with Derlin-1 and an associated AAA-ATPase, p97. Overexpression of Derlin-1 led to a decrease in the biogenesis efficiency and surface expression of K(ATP) channels. Conversely, knockdown of Derlin-1 by RNA interference resulted in increased processing of SUR1 and a corresponding increase in surface expression of K(ATP) channels. Importantly, knockdown of Derlin-1 increased the abundance of disease-causing misfolded SUR1 or Kir6.2 proteins and even partially rescued surface expression in a mutant channel. We conclude that Derlin-1, by being involved in ERAD of SUR1 and Kir6.2, has a role in modulating the biogenesis efficiency and surface expression of K(ATP) channels. The results suggest that physiological or pathological changes in Derlin-1 expression levels may affect glucose-stimulated insulin secretion by altering surface expression of K(ATP) channels.     

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.     

Glucagon-like peptide-1 inhibits pancreatic ATP-sensitive potassium channels via a protein kinase A- and ADP-dependent mechanism

Glucagon-like peptide-1 (GLP-1) elicits a glucose-dependent insulin secretory effect via elevation of cAMP and activation of protein kinase A (PKA). GLP-1-mediated closure of ATP-sensitive potassium (K(ATP)) channels is involved in this process, although the mechanism of action of PKA on the K(ATP) channels is not fully understood. K(ATP) channel currents and membrane potentials were measured from insulin-secreting INS-1 cells and recombinant beta-cell K(ATP) channels. 20 nM GLP-1 depolarized INS-1 cells significantly by 6.68 +/- 1.29 mV. GLP-1 reduced recombinant K(ATP) channel currents by 54.1 +/- 6.9% in mammalian cells coexpressing SUR1, Kir6.2, and GLP-1 receptor clones. In the presence of 0.2 mM ATP, the catalytic subunit of PKA (cPKA, 20 nM) had no effect on SUR1/Kir6.2 activity in inside-out patches. However, the stimulatory effects of 0.2 mM ADP on SUR1/Kir6.2 currents were reduced by 26.7 +/- 2.9% (P < 0.05) in the presence of cPKA. cPKA increased SUR1/Kir6.2 currents by 201.2 +/- 20.8% (P < 0.05) with 0.5 mM ADP present. The point mutation S1448A in the ADP-sensing region of SUR1 removed the modulatory effects of cPKA. Our results indicate that PKA-mediated phosphorylation of S1448 in the SUR1 subunit leads to K(ATP) channel closure via an ADP-dependent mechanism. The marked alteration of the PKA-mediated effects at different ADP levels may provide a cellular mechanism for the glucose-sensitivity of GLP-1.     

Sulfonylureas correct trafficking defects of ATP-sensitive potassium channels caused by mutations in the sulfonylurea receptor

The pancreatic ATP-sensitive potassium (K(ATP)) channel, a complex of four sulfonylurea receptor 1 (SUR1) and four potassium channel Kir6.2 subunits, regulates insulin secretion by linking metabolic changes to beta-cell membrane potential. Sulfonylureas inhibit K(ATP) channel activities by binding to SUR1 and are widely used to treat type II diabetes. We report here that sulfonylureas also function as chemical chaperones to rescue K(ATP) channel trafficking defects caused by two SUR1 mutations, A116P and V187D, identified in patients with congenital hyperinsulinism. Sulfonylureas markedly increased cell surface expression of the A116P and V187D mutants by stabilizing the mutant SUR1 proteins and promoting their maturation. By contrast, diazoxide, a potassium channel opener that also binds SUR1, had no effect on surface expression of either mutant. Importantly, both mutant channels rescued to the cell surface have normal ATP, MgADP, and diazoxide sensitivities, demonstrating that SUR1 harboring either the A116P or the V187D mutation is capable of associating with Kir6.2 to form functional K(ATP) channels. Thus, sulfonylureas may be used to treat congenital hyperinsulinism caused by certain K(ATP) channel trafficking mutations.     

Syntaxin-1A actions on sulfonylurea receptor 2A can block acidic pH-induced cardiac K(ATP) channel activation

During cardiac ischemia, ATP stores are depleted, and cardiomyocyte intracellular pH lowers to <7.0. The acidic pH acts on the Kir6.2 subunit of K(ATP) channels to reduce its sensitivity to ATP, causing channel opening. We recently reported that syntaxin-1A (Syn-1A) binds nucleotide binding folds (NBF)-1 and NBF2 of sulfonylurea receptor 2A (SUR2A) to inhibit channel activity (Kang, Y., Leung, Y. M., Manning-Fox, J. E., Xia, F., Xie, H., Sheu, L., Tsushima, R. G., Light, P. E., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 47125-47131). Here, we examined Syn-1A actions on SUR2A to influence the pH regulation of cardiac K(ATP) channels. K(ATP) channel currents from inside-out patches excised from Kir6.2/SUR2A expressing HEK293 cells and freshly isolated cardiac myocytes were increased by reducing intracellular pH from 7.4 to 6.8, which could be blocked by increasing concentrations of Syn-1A added to the cytoplasmic surface. Syn-1A had no effect on C-terminal truncated Kir6.2 (Kir6.2-deltaC26) channels expressed in TSA cells without the SUR subunit. In vitro binding and co-immunoprecipitation studies show that Syn-1A binding to SUR2A or its NBF-1 and NBF-2 domain proteins increased progressively as pH was reduced from 7.4 to 6.0. The enhancement of Syn-1A binding to SUR2A by acidic pH was further regulated by Mg2+ and ATP. Therefore, pH regulates Kir.6.2/SUR2A channels not only by its direct actions on the Kir6.2 subunit but also by modulation of Syn-1A binding to SUR2A. The increased Syn-1A binding to the SUR2A at acidic pH would assert some inhibition of the K(ATP) channels, which may serve as a "brake" to temper the fluctuation of low pH-induced K(ATP) channel opening that could induce fatal reentrant arrhythmias.     

A role of the sulfonylurea receptor 1 in endocytic trafficking of ATP-sensitive potassium channels

The ATP-sensitive potassium (K(ATP) ) channel consisting of sulfonylurea receptor 1 (SUR1) and inward-rectifier potassium channel 6.2 (Kir6.2) has a well-established role in insulin secretion. Mutations in either subunit can lead to disease due to aberrant channel gating, altered channel density at the cell surface or a combination of both. Endocytic trafficking of channels at the plasma membrane is one way to influence surface channel numbers. It has been previously reported that channel endocytosis is dependent on a tyrosine-based motif in Kir6.2, while SUR1 alone is unable to internalize. In this study, we followed endocytic trafficking of surface channels in real time by live-cell imaging of channel subunits tagged with an extracellular minimal α-bungarotoxin-binding peptide labeled with a fluorescent dye. We show that SUR1 undergoes endocytosis independent of Kir6.2. Moreover, mutations in the putative endocytosis motif of Kir6.2, Y330C, Y330A and F333I are unable to prevent channel endocytosis. These findings challenge the notion that Kir6.2 bears the sole endocytic signal for K(ATP) channels and support a role of SUR1 in this trafficking process.     

H3 domain of syntaxin 1A inhibits KATP channels by its actions on the sulfonylurea receptor 1 nucleotide-binding folds-1 and -2

The ATP-sensitive potassium (K(ATP)) channel in pancreatic islet beta cells consists of four pore-forming (Kir6.2) subunits and four regulatory sulfonylurea receptor (SUR1) subunits. In beta cells, the K(ATP) channel links intracellular metabolism to the dynamic regulation of the cell membrane potential that triggers insulin secretion. Syntaxin 1A (Syn-1A) is a SNARE protein that not only plays a direct role in exocytosis, but also binds and modulates voltage-gated K(+) and Ca(2+) channels to fine tune exocytosis. We recently reported that wild type Syn-1A inhibits rat islet beta cell K(ATP) channels and binds both nucleotide-binding folds (NBF-1 and NBF-2) of SUR1. However, wild type Syn-1A inhibition of rat islet beta cell K(ATP) channels seems to be mediated primarily via NBF-1. During exocytosis, Syn-1A undergoes a conformational change from a closed form to an open form, which would fully expose its active domain, the C-terminal H3 domain. Here, we show that the constitutively open form Syn-1A mutant (L165A/E166A) has a similar affinity to NBF-1 and NBF-2 as wild type Syn-1A and was equally effective in inhibiting the K(ATP) channels of rat pancreatic beta cells and a cell line (BA8) stably expressing SUR1/Kir6.2. Although dialysis of NBF-1 into BA8 and islet beta cells effectively blocked wild type and open form Syn-1A inhibition of the K(ATP) current, NBF-2 was also effective in blocking the open form Syn-1A inhibition. This prompted us to examine the specific domains within Syn-1A that would mediate its action on the K(ATP) channels. The C-terminal H3 domain of Syn-1A (Syn-1A-H3), but not the N-terminal H(ABC) domain (Syn-1A-H(ABC)), binds the SUR1 protein of BA8 cells, causing an inhibition of K(ATP) currents, and this inhibition was mediated via both NBF-1 and NBF-2. It therefore appears that the H3 domain of Syn-1A is the putative domain, which binds SUR1, but its distinct actions on the NBFs may depend on the conformation of Syn-1A occurring during exocytosis.     

Engineered interaction between SUR1 and Kir6.2 that enhances ATP sensitivity in KATP channels

The ATP-sensitive potassium (K(ATP)) channel consisting of the inward rectifier Kir6.2 and SUR1 (sulfonylurea receptor 1) couples cell metabolism to membrane excitability and regulates insulin secretion. Inhibition by intracellular ATP is a hallmark feature of the channel. ATP sensitivity is conferred by Kir6.2 but enhanced by SUR1. The mechanism by which SUR1 increases channel ATP sensitivity is not understood. In this study, we report molecular interactions between SUR1 and Kir6.2 that markedly alter channel ATP sensitivity. Channels bearing an E203K mutation in SUR1 and a Q52E in Kir6.2 exhibit ATP sensitivity ～100-fold higher than wild-type channels. Cross-linking of E203C in SUR1 and Q52C in Kir6.2 locks the channel in a closed state and is reversible by reducing agents, demonstrating close proximity of the two residues. Our results reveal that ATP sensitivity in K(ATP) channels is a dynamic parameter dictated by interactions between SUR1 and Kir6.2.     

Destabilization of ATP-sensitive potassium channel activity by novel KCNJ11 mutations identified in congenital hyperinsulinism

The inwardly rectifying potassium channel Kir6.2 is the pore-forming subunit of the ATP-sensitive potassium (K(ATP)) channel, which controls insulin secretion by coupling glucose metabolism to membrane potential in beta-cells. Loss of channel function because of mutations in Kir6.2 or its associated regulatory subunit, sulfonylurea receptor 1, causes congenital hyperinsulinism (CHI), a neonatal disease characterized by persistent insulin secretion despite severe hypoglycemia. Here, we report a novel K(ATP) channel gating defect caused by CHI-associated Kir6.2 mutations at arginine 301 (to cysteine, glycine, histidine, or proline). These mutations in addition to reducing channel expression at the cell surface also cause rapid, spontaneous current decay, a gating defect we refer to as inactivation. Based on the crystal structures of Kir3.1 and KirBac1.1, Arg-301 interacts with several residues in the neighboring Kir6.2 subunit. Mutation of a subset of these residues also induces channel inactivation, suggesting that the disease mutations may cause inactivation by disrupting subunit-subunit interactions. To evaluate the effect of channel inactivation on beta-cell function, we expressed an alternative inactivation mutant R301A, which has equivalent surface expression efficiency as wild type channels, in the insulin-secreting cell line INS-1. Mutant expression resulted in more depolarized membrane potential and elevated insulin secretion at basal glucose concentration (3 mm) compared with cells expressing wild type channels, demonstrating that the inactivation gating defect itself is sufficient to cause loss of channel function and hyperinsulinism. Our studies suggest the importance of Kir6.2 subunit-subunit interactions in K(ATP) channel gating and function and reveal a novel gating defect underlying CHI.     

Sulfonylureas correct trafficking defects of disease-causing ATP-sensitive potassium channels by binding to the channel complex

ATP-sensitive potassium (K(ATP)) channels mediate glucose-induced insulin secretion by coupling metabolic signals to beta-cell membrane potential and the secretory machinery. Reduced K(ATP) channel expression caused by mutations in the channel proteins: sulfonylurea receptor 1 (SUR1) and Kir6.2, results in loss of channel function as seen in congenital hyperinsulinism. Previously, we reported that sulfonylureas, oral hypoglycemic drugs widely used to treat type II diabetes, correct the endoplasmic reticulum to the plasma membrane trafficking defect caused by two SUR1 mutations, A116P and V187D. In this study, we investigated the mechanism by which sulfonylureas rescue these mutants. We found that glinides, another class of SUR-binding hypoglycemic drugs, also markedly increased surface expression of the trafficking mutants. Attenuating or abolishing the ability of mutant SUR1 to bind sulfonylureas or glinides by the following mutations: Y230A, S1238Y, or both, accordingly diminished the rescuing effects of the drugs. Interestingly, rescue of the trafficking defects requires mutant SUR1 to be co-expressed with Kir6.2, suggesting that the channel complex, rather than SUR1 alone, is the drug target. Observations that sulfonylureas also reverse trafficking defects caused by neonatal diabetes-associated Kir6.2 mutations in a way that is dependent on intact sulfonylurea binding sites in SUR1 further support this notion. Our results provide insight into the mechanistic and structural basis on which sulfonylureas rescue K(ATP) channel surface expression defects caused by channel mutations.     

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.     

ATP modulates interaction of syntaxin-1A with sulfonylurea receptor 1 to regulate pancreatic beta-cell KATP channels

ATP-sensitive potassium (K(ATP)) channels are regulated by a variety of cytosolic factors (adenine nucleotides, Mg(2+), phospholipids, and pH). We previously reported that K(ATP) channels are also regulated by endogenous membrane-bound SNARE protein syntaxin-1A (Syn-1A), which binds both nucleotide-binding folds of sulfonylurea receptor (SUR)1 and 2A, causing inhibition of K(ATP) channel activity in pancreatic islet β-cells and cardiac myocytes, respectively. In this study, we show that ATP dose-dependently inhibits Syn-1A binding to SUR1 at physiological concentrations, with the addition of Mg(2+) causing a decrease in the ATP-induced inhibitory effect. This ATP disruption of Syn-1A binding to SUR1 was confirmed by FRET analysis in living HEK293 cells. Electrophysiological studies in pancreatic β-cells demonstrated that reduced ATP concentrations increased K(ATP) channel sensitivity to Syn-1A inhibition. Depletion of endogenous Syn-1A in insulinoma cells by botulinum neurotoxin C1 proteolysis followed by rescue with exogenous Syn-1A showed that Syn-1A modulates K(ATP) channel sensitivity to ATP. Thus, our data indicate that although both ATP and Syn-1A independently inhibit β-cell K(ATP) channel gating, they could also influence the sensitivity of K(ATP) channels to each other. These findings provide new insight into an alternate mechanism by which ATP regulates pancreatic β-cell K(ATP) channel activity, not only by its direct actions on Kir6.2 pore subunit, but also via ATP modulation of Syn-1A binding to SUR1.     

Kir6.2 mutations causing neonatal diabetes provide new insights into Kir6.2-SUR1 interactions

ATP-sensitive K(+) (K(ATP)) channels, comprised of pore-forming Kir6.2 and regulatory SUR1 subunits, play a critical role in regulating insulin secretion. Binding of ATP to Kir6.2 inhibits, whereas interaction of MgATP with SUR1 activates, K(ATP) channels. We tested the functional effects of two Kir6.2 mutations (Y330C, F333I) that cause permanent neonatal diabetes mellitus, by heterologous expression in Xenopus oocytes. Both mutations reduced ATP inhibition and increased whole-cell currents, which in pancreatic beta-cells is expected to reduce insulin secretion and precipitate diabetes. The Y330C mutation reduced ATP inhibition both directly, by impairing ATP binding (and/or transduction), and indirectly, by stabilizing the intrinsic open state of the channel. The F333I mutation altered ATP binding/transduction directly. Both mutations also altered Kir6.2/SUR1 interactions, enhancing the stimulatory effect of MgATP (which is mediated via SUR1). This effect was particularly dramatic for the Kir6.2-F333I mutation, and was abolished by SUR1 mutations that prevent MgATP binding/hydrolysis. Further analysis of F333I heterozygous channels indicated that at least three SUR1 must bind/hydrolyse MgATP to open the mutant K(ATP) channel.     

PKA-mediated phosphorylation of the human K(ATP) channel: separate roles of Kir6.2 and SUR1 subunit phosphorylation.

ATP-sensitive potassium (K(ATP)) channels play important roles in many cellular functions such as hormone secretion and excitability of muscles and neurons. Classical ATP-sensitive potassium (K(ATP)) channels are heteromultimeric membrane proteins comprising the pore-forming Kir6.2 subunits and the sulfonylurea receptor subunits (SUR1 or SUR2). The molecular mechanism by which hormones and neurotransmitters modulate K(ATP) channels via protein kinase A (PKA) is poorly understood. We mutated the PKA consensus sequences of the human SUR1 and Kir6.2 subunits and tested their phosphorylation capacities in Xenopus oocyte homogenates and in intact cells. We identified the sites responsible for PKA phosphorylation in the C-terminus of Kir6.2 (S372) and SUR1 (S1571). Kir6.2 can be phosphorylated at its PKA phosphorylation site in intact cells after G-protein (Gs)-coupled receptor or direct PKA stimulation. While the phosphorylation of Kir6.2 increases channel activity, the phosphorylation of SUR1 contributes to the basal channel properties by decreasing burst duration, interburst interval and open probability, and also increasing the number of functional channels at the cell surface. Moreover, the effect of PKA could be mimicked by introducing negative charges in the PKA phosphorylation sites. These data demonstrate direct phosphorylation by PKA of the K(ATP) channel, and may explain the mechanism by which Gs-coupled receptors stimulate channel activity. Importantly, they also describe a model of heteromultimeric ion channels in which there are functionally distinct roles of the phosphorylation of the different subunits.     

Sulfonylurea receptors type 1 and 2A randomly assemble to form heteromeric KATP channels of mixed subunit composition

ATP-sensitive potassium (K(ATP)) channels play important roles in regulating insulin secretion, controlling vascular tone, and protecting cells against metabolic stresses. K(ATP) channels are heterooctamers of four pore-forming inwardly rectifying (Kir6.2) subunits and four sulfonylurea receptor (SUR) subunits. K(ATP) channels containing SUR1 (e.g. pancreatic) and SUR2A (e.g. cardiac) display distinct metabolic sensitivities and pharmacological profiles. The reported expression of both SUR1 and SUR2 together with Kir6.2 in some cells raises the possibility that heteromeric channels containing both SUR subtypes might exist. To test whether SUR1 can coassemble with SUR2A to form functional K(ATP) channels, we made tandem constructs by fusing SUR to either a wild-type (WT) or a mutant N160D Kir6.2 subunit. The latter mutation greatly increases the sensitivity of K(ATP) channels to block by intracellular spermine. We expressed, individually and in combinations, tandem constructs SUR1-Kir6.2 (S1-WT), SUR1-Kir6.2[N160D] (S1-ND), and SUR2A-Kir6.2[N160D] (S2-ND) in Xenopus oocytes, and studied the voltage dependence of spermine block in inside-out macropatches over a range of spermine concentrations and RNA mixing ratios. Each tandem construct expressed alone supported macroscopic K(+) currents with pharmacological properties indistinguishable from those of the respective native channel types. Spermine sensitivity was low for S1-WT but high for S1-ND and S2-ND. Coexpression of S1-WT and S1-ND generated current components with intermediate spermine sensitivities indicating the presence of channel populations containing both types of Kir subunits at all possible stoichiometries. The relative abundances of these populations, determined by global fitting over a range of conditions, followed binomial statistics, suggesting that WT and N160D Kir6.2 subunits coassemble indiscriminately. Coexpression of S1-WT with S2-ND also yielded current components with intermediate spermine sensitivities, suggesting that SUR1 and SUR2A randomly coassemble into functional K(ATP) channels. Further pharmacological characterization confirmed coassembly of not only S1-WT and S2-ND, but also of coexpressed free SUR1, SUR2A, and Kir6.2 into functional heteromeric channels.     

Regulation of ATP-sensitive potassium channel function by protein kinase A-mediated phosphorylation in transfected HEK293 cells

ATP-sensitive potassium (K(ATP)) channels regulate insulin secretion, vascular tone, heart rate and neuronal excitability by responding to transmitters as well as the internal metabolic state. K(ATP) channels are composed of four pore-forming alpha-subunits (Kir6.2) and four regulatory beta-subunits, the sulfonylurea receptor (SUR1, SUR2A or SUR2B). Whereas protein kinase A (PKA) phosphorylation of serine 372 of Kir6.2 has been shown biochemically by others, we found that the phosphorylation of T224 rather than S372 of Kir6.2 underlies the catalytic subunits of PKA (c-PKA)- and the D1 dopamine receptor-mediated stimulation of K(ATP) channels expressed in HEK293 cells. Specific changes in the kinetic properties of channels treated with c-PKA, as revealed by single-channel analysis, were mimicked by aspartate substitution of T224. The T224D mutation also reduced the sensitivity to ATP inhibition. Alteration of channel gating and a decrease in the apparent affinity for ATP inhibition thus underlie the positive regulation of K(ATP) channels by PKA phosphorylation of T224 in Kir6.2, which may represent a general mechanism for K(ATP) channel regulation in different tissues.     

Structure-function relationships in the beta-cell K(ATP) channel

The ATP-sensitive potassium (K(ATP)) channel plays a key role in controlling beta-cell membrane potential and insulin secretion. The channels are composed of two subunits, Kir6.2, which forms the channel pore, and SUR1, which contains binding sites for nucleotides and sulphonylureas and acts as a channel regulator. Our current studies are aimed at delineating the molecular interactions involved in assembly and ligand binding by K(ATP) channel proteins. We have employed a complementation approach in which SUR1 half-molecules are co-expressed in insect cells using a baculovirus system. Together with data from truncated SUR1 molecules and a fusion protein in which SUR1 is linked to Kir6.2, we have interpreted our findings in terms of a model for the structure of the K(ATP) channel. The main features of the model are: (i) the C-terminal end of SUR1 is close to the N-terminus of Kir6.2; (ii) the two nucleotide binding domains (NBDs) of SUR1--NBD1 and NBD2--are in proximity; (iii) transmembrane helix 12 of SUR1 is orientated in such a way that it can make contact with Kir6.2; (iv) formation of the glibenclamide binding site requires that the two cytosolic loops (CLs) CL3 and CL8 are located close to each other; (v) there are homomeric interactions between the NBD1 domains of neighbouring subunits. We suggest that binding of glibenclamide leads to conformational changes in CL3 and CL8 leading to rearrangement of transmembrane helices. These effects are transmitted to Kir6.2 to result in channel closure.     

ATP modulation of ATP-sensitive potassium channel ATP sensitivity varies with the type of SUR subunit

ATP-sensitive potassium (K(ATP)) channels comprise Kir and SUR subunits. Using recombinant K(ATP) channels expressed in Xenopus oocytes, we observed that MgATP (100 microm) block of Kir6.2/SUR2A currents gradually declined with time, whereas inhibition of Kir6.2/SUR1 or Kir6.2DeltaC36 currents did not change. The decline in Kir6.2/SUR2A ATP sensitivity was not observed in Mg(2+) free solution and was blocked by the phosphatidylinositol (PI) 3-kinase inhibitors LY 294002 (10 microm) and wortmannin (100 microm), and by neomycin (100 microm). These results suggest that a MgATP-dependent synthesis of membrane phospholipids produces a secondary decrease in the ATP sensitivity of Kir6.2/SUR2A. Direct application of the phospholipids PI 4,5-bisphosphate and PI 3,4,5-trisphosphate in the presence of 100 microm MgATP activated all three types of channel, but the response was faster for Kir6.2/SUR2A. Chimeric studies indicate that the different responses of Kir6.2/SUR2A and Kir6.2/SUR1 are mediated by the first six transmembrane domains of SUR. The MgATP-dependent loss of ATP sensitivity of Kir6.2/SUR2A was enhanced by the actin filament disrupter cytochalasin and blocked by phalloidin (which stabilizes the cytoskeleton). Phalloidin did not block the effect of PI 3,4,5-trisphosphate. This suggests that MgATP may cause disruption of the cytoskeleton, leading to enhanced membrane phospholipid levels (or better targeting to the K(ATP) channel) and thus to decreased channel ATP sensitivity.     

Assembly limits the pharmacological complexity of ATP-sensitive potassium channels

ATP-sensitive potassium channels (K(ATP) channels) are formed from an octameric complex of an inwardly rectifying K(+) channel (Kir6.1, Kir6.2) and a sulfonylurea receptor (SUR1, SUR2A, and SUR2B). In this study we have attempted to address the question of whether SUR heteromultimers can form using a combination of biochemical and electrophysiological approaches. We have constructed monoclonal stable lines in HEK293 cells co-expressing Kir6.2 with SUR1 and SUR2A. Using coimmunoprecipitation analysis with SUR isotype-specific antibodies two biochemical populations are distinguished, one containing SUR1 and the other SUR2A. It is not possible to detect immune complexes containing both SUR1 and SUR2A. Functional studies were undertaken and whole cell membrane currents were studied using the patch clamp. Concentrations of sulfonylureas and potassium channel openers were determined that selectively inhibited or activated SUR1/Kir6.2 and SUR2A/Kir6.2. In the cell line expressing SUR1/SUR2AKir6.2 we were unable to demonstrate a population of channels with unique pharmacological properties. Thus we conclude from these studies that heteromultimeric channel complexes containing both SUR1 and SUR2A are not formed, suggesting an incompatibility between different SUR subtypes. This incompatibility limits the pharmacological complexity of K(ATP) channels that may be observed in native tissues.     

Identification of a familial hyperinsulinism-causing mutation in the sulfonylurea receptor 1 that prevents normal trafficking and function of KATP channels

Mutations in the pancreatic ATP-sensitive potassium (K(ATP)) channel subunits sulfonylurea receptor 1 (SUR1) and the inwardly rectifying potassium channel Kir6.2 cause persistent hyperinsulinemic hypoglycemia of infancy. We have identified a SUR1 mutation, L1544P, in a patient with the disease. Channels formed by co-transfection of Kir6.2 and the mutant SUR1 in COS cells have reduced response to MgADP ( approximately 10% that of the wild-type channels) and reduced surface expression ( approximately 19% that of the wild-type channels). However, the steady-state level of the SUR1 protein is unaffected. Treating cells with lysosomal or proteasomal inhibitors did not improve surface expression of the mutant channels, suggesting that increased degradation of mutant channels by either pathway is unlikely to account for the reduced surface expression. Removal of the RKR endoplasmic reticulum retention/retrieval trafficking motif in either SUR1 or Kir6.2 increased the surface expression of the mutant channel by approximately 35 and approximately 20%, respectively. The simultaneous removal of the RKR motif in both channel subunits restored surface expression of the mutant channel to the wild-type channel levels. Thus, the L1544P mutation may interfere with normal trafficking of K(ATP) channels by causing improper shielding of the RKR endoplasmic reticulum retention/retrieval trafficking signals in the two channel subunits.