Numerous mutations have been identified in SUR1 (ABCC8) subunit of the neuroendocrine type KATP channel in subjects with neonatal diabetes, neonatal diabetes plus epilepsy and/or other neurological features, maturity-onset diabetes of young, and later-onset diabetes. Patch-clamping, single-channel kinetics analysis, affinity photolabeling and molecular modeling were used to clarify how diabetogenic mutations in different parts of SUR1 affect open probability and sulfonylurea inhibition of SUR1/Kir6.2 KATP channel. Essentially all tested diabetogenic mutations in the canonical TMD1-NBD1-TMD2-NBD2 ABC exporter core of SUR1 hyperactivated KATP by stabilizing the stimulatory Mg-nucleotide bound (outward facing) state of SUR1 without affecting the intrinsic gating of KATP channel or its sensitivity to inhibitory nucleotides. Hyperstimulated mutant channels showed attenuated sulfonylurea inhibition in the presence, but not the absence, of stimulatory MgATP/ADP, indicating that KATP with SUR1 in the inward facing state has the lowest Kd for sulfonylureas. Diabetogenic mutations in the non-canonical TMD0-L0 part of SUR1 hyperactivated KATP by destabilizing its long-lived closed state with the highest affinity to inhibitory ATP or by strengthening the functional coupling between the MgATP/ADP-bound SUR1 core and the active (burst) state of the Kir pore. Mutations destabilizing the long-lived closed state compromised sulfonylurea inhibition of KATP in the absence of nucleotides but not the drug-induced release of stimulatory nucleotides. The findings support the mechanistic model (FEBS Letters, 585:3555-9) in which the TMD0-L0 module couples the SUR1 core with the KATP pore, define the most common ABCC8-associated mechanisms of KATP hyperactivity, and largely explain why the majority of diabetic subjects with mutant SUR1 require body-weight normalized doses of sulfonylureas exceeding those recommended by the FDA for treatment of common type 2 diabetes.

Neonatal diabetes (ND) mutations in different domains of either KATP subunit overactivate KATP via different mechanisms. Mutations in the ABC core of SUR1 augment its Mg-nucleotide-dependent stimulation of Kir6.2. Mutations at the putative ATP site of Kir6.2 supposedly compromise the inhibitory nucleotide binding. Mutations at the putative gate reportedly stabilize its open state. Many ND mutations map to the putative L0 helix preceding the SUR1 core and to the M0 ('slide') helix of Kir6.2. We discovered that L0 controls spontaneous bursting and introduced a model in which L0 and M0 partner in KATP gating (Babenko & Bryan 2003). The model predicted that ND mutations in either amphipathic helix at the membrane-cytosol interface hyperactivate KATP by altering its intrinsic gating kinetics. Here we tested the idea. We compared the effects of severe ND mutations in the middle of either interface helix on spontaneous single-channel kinetics, ATP-inhibition, and Mg-nucleotide stimulation of KATP. We found that each of these mutations decreases the rate of burst termination while increasing the rate of burst initiation, thereby reducing the availability of the closed state with the lowest Kd for inhibitory ligands. This mechanism attenuates ATP inhibition but not Mg-nucleotide stimulation, thereby hyperactivating KATP in vivo, and uncouples sulfonylurea binding from Kir6.2 closure. Thus, in support of our model, perturbing either interface helix disrupts SUR1/Kir6.2 inhibitory coupling. Affinity photolabeling with I-azidoglibenclamide indicated that none of the ND mutations altered the proximity of L0 to M0. But each of these mutations changed the helical hydrophobic moment, implying that rotation of either helix along its axis disrupts L0/M0 interactions. We propose that the two interface helices have been optimized to interact and slide together to stabilize the long-lived closed state of SUR1/Kir6.2 complex.

We discovered numerous congenital diabetes (CD), insulin release-inhibiting mutations in ABCC8/KCNJ11 genes coding for the pancreatic beta-cell (SUR1/Kir6.2)4 KATP channel (Diabetes 53:2719; N Engl J Med 355:456; Diabetes 56:1737; J Biol Chem 283:8778; reviewed in Endocr Rev 29:265). Based on FEBS Lett 445:131; 459:367 and subsequent structure-activity analyses and mechanistic models of KATP channels (reviewed in J Mol Cell Cardiol 39:79), Babenko predicted three possible mechanisms of KATP hyperactivation by mutations in ABCC8 and started testing the hypothesis (DK077827 R01 project ABCC8/KCNJ11 Mechanisms and Diabetes).

Activating mutations in different domains of the ABCC8 gene-coded sulfonylurea receptor 1 (SUR1) cause neonatal diabetes. Here we show that a diabetogenic mutation in an unexplored helix preceding the ABC core of SUR1 dramatically increases open probability of (SUR1/Kir6.2)(4) channel (KATP) by reciprocally changing rates of its transitions to and from the long-lived, inhibitory ligand-stabilized closed state. This kinetic mechanism attenuates ATP and sulfonylurea inhibition, but not Mg-nucleotide stimulation, of SUR1/Kir6.2. The results suggest a key role for L0 helix in KATP gating and together with previous findings from mutant KATP clarify why many patients with neonatal diabetes require high doses of sulfonylureas.

Many diabetogenic mutations (Endocrine Rev 2009;29:265) map to the canonical, N-terminal, submembrane (BBRC 1999;255:231) “slide” M0 helix of KCNJ11 (KIR6.2). To clarify the principal biophysical mechanism of their action, inhibiting insulin release, I analyzed effects of the first reported severe Neonatal Diabetes (ND with epilepsy and developmental delay) mutation and 18 other mutations, V59X, in M0 on macroscopic and unitary currents through ABCC8(SUR1)-containing ATP-sensitive potassium (KATP) channels, reconstituted in mammalian cells lacking endogenous SUR or KIR. Several V59X decreased, and no V59X increased or abolished, functional expression (N) of the neuroendocrine-type adenine nucleotide sensors. This a) indicated that the effect of any possible ND V59X-induced decrease in N on Vm in humans is overruled by the mean open channel probability(PO)-increasing effect, explaining insufficient insulin release due to hyperpolarization of insulin producing cells, and b) allowed complete analysis of relationships between the physical properties of the side chain in the middle of M0 and PO, its sensitivity to nucleotides, and single-channel gating kinetics. The established relationships are consistent with the results of molecular modeling and molecular dynamics simulation of severe ND KATP pores and strongly suggest that a ligand-independent stabilization of the active (burst) state with conformations without specific, micromolar affinity for inhibitory ATP, is the principal mechanism of pathogenic hyperactivity of KATP with mutations in M0, the small domain proposed to play a big role in gating of KIR6 and their relatives. I thank the National Institutes of Health for funding and G. Zhao for technical assistance.

Numerous mutations in ABCC8 (SUR1), the neuroendocrine-type regulatory subunit of KATP channels, cause Neonatal Diabetes (ND). Many of these mutations compromising insulin release cluster around the first ND mutation identified in the SUR1 L0-linker, L213R (NEJM 355:456). Here we show the ND-SUR1 increased the on-cell open channel probability, Po, not the density of Kir6.2-based channels. Single-channel kinetics analyses demonstrate that the increase in the Po is due to a ligand-independent stabilization of the active (burst) state. A similar diabetogenic effect was reproduced by deletions in the Kir6.2 N-terminus (BBRC 255:231; Cell 100:645) that partners with the L0-linker controlling the "slow" (inter-burst) gating transitions (JBC 278:41577). These findings illustrate the physiologic significance of our original model of SUR1/Kir6.2 coupling (JBC 278:41577) where the L0-linker plays a key role in controlling the maximal Po.

ATP/ADP-sensing (sulfonylurea receptor (SUR)/KIR6)4 KATP channels regulate the excitability of our insulin secreting and other vital cells via the differential MgATP/ADP-dependent stimulatory actions of their tissue-specific ATP-binding cassette regulatory subunits (sulfonylurea receptors), which counterbalance the nearly constant inhibitory action of ATP on the K+ inwardly rectifying pore. Mutations in SUR1 that abolish its stimulation have been found in infants persistently releasing insulin. Activating mutations in SUR1 have been shown to cause neonatal diabetes. Here, analyses of KIR6.2-based channels with diabetogenic receptors reveal that MgATP-dependent hyper-stimulation of mutant SUR can compromise the ability of KATP channels to function as metabolic sensors. I demonstrate that the channel hyperactivity rises exponentially with the number of hyperstimulating subunits, so small subpopulations of channels with more than two mutant SUR can dominate hyperpolarizing currents in heterozygous patients. I uncovered an attenuated tolbutamide inhibition of the hyperstimulated mutant, which is normally sensitive to the drug under non-stimulatory conditions. These findings show the key role of SUR in sensing the metabolic index in humans and urge others to (re)test mutant SUR/KIR6 channels from probands in physiologic MgATP.

BACKGROUND The ATP-sensitive potassium (K(ATP)) channel, composed of the beta-cell proteins sulfonylurea receptor (SUR1) and inward-rectifying potassium channel subunit Kir6.2, is a key regulator of insulin release. It is inhibited by the binding of adenine nucleotides to subunit Kir6.2, which closes the channel, and activated by nucleotide binding or hydrolysis on SUR1, which opens the channel. The balance of these opposing actions determines the low open-channel probability, P(O), which controls the excitability of pancreatic beta cells. We hypothesized that activating mutations in ABCC8, which encodes SUR1, cause neonatal diabetes. METHODS We screened the 39 exons of ABCC8 in 34 patients with permanent or transient neonatal diabetes of unknown origin. We assayed the electrophysiologic activity of mutant and wild-type K(ATP) channels. RESULTS We identified seven missense mutations in nine patients. Four mutations were familial and showed vertical transmission with neonatal and adult-onset diabetes; the remaining mutations were not transmitted and not found in more than 300 patients without diabetes or with early-onset diabetes of similar genetic background. Mutant channels in intact cells and in physiologic concentrations of magnesium ATP had a markedly higher P(O) than did wild-type channels. These overactive channels remained sensitive to sulfonylurea, and treatment with sulfonylureas resulted in euglycemia. CONCLUSIONS Dominant mutations in ABCC8 accounted for 12 percent of cases of neonatal diabetes in the study group. Diabetes results from a newly discovered mechanism whereby the basal magnesium-nucleotide-dependent stimulatory action of SUR1 on the Kir pore is elevated and blockade by sulfonylureas is preserved.

ATP-sensitive K+ channels, termed K(ATP) channels, provide a link between cellular metabolism and membrane electrical activity in a variety of tissues. Channel isoforms have been identified and are targets for compounds that both stimulate and inhibit their activity resulting in membrane hyperpolarization and depolarization, respectively. Examples include relaxation of vascular smooth muscle and stimulation of insulin secretion. This article reviews the cloning, molecular biology, and structure of K(ATP) channels, with particular focus on the SUR1/K(IR)6.2 neuroendocrine channels that are important for the regulation of insulin secretion. We integrate the extensive pharmacologic structure-activity-relationship data on these channels, which defines a bipartite drug binding pocket in the SUR (sulfonylurea receptor), with recent structure-function studies that identify domains of SUR and K(IR)6.2, the channel pore, which are critical for channel assembly, for gating, and for the ligand-receptor interactions that modulate channel activity. The atomic structure of a sulfonylurea in a protein pocket is used to develop insight into the recognition of these compounds. A homology model of K(ATP) channels, based on VC-MsbA, another member of the ABC protein family, is described and used to position amino acids important for the action of channel openers and blockers within the core of SUR. The model has a central chamber which could serve as a multifaceted binding pocket.

Advances in understanding the overall structural features of inward rectifiers and ATP-binding cassette (ABC) transporters are providing novel insight into the architecture of ATP-sensitive K+ channels (KATP channels) (KIR6.0/SUR)4. The structure of the K(IR) pore has been modeled on bacterial K+ channels, while the lipid-A exporter, MsbA, provides a template for the MDR-like core of sulfonylurea receptor (SUR)-1. TMD0, an NH2-terminal bundle of five alpha-helices found in SURs, binds to and activates KIR6.0. The adjacent cytoplasmic L0 linker serves a dual function, acting as a tether to link the MDR-like core to the KIR6.2/TMD0 complex and exerting bidirectional control over channel gating via interactions with the NH2-terminus of the KIR. Homology modeling of the SUR1 core offers the possibility of defining the glibenclamide/sulfonylurea binding pocket. Consistent with 30-year-old studies on the pharmacology of hypoglycemic agents, the pocket is bipartite. Elements of the COOH-terminal half of the core recognize a hydrophobic group in glibenclamide, adjacent to the sulfonylurea moiety, to provide selectivity for SUR1, while the benzamido group appears to be in proximity to L0 and the KIR NH2-terminus.

Structure-function analyses of K+ channels identify a common pore architecture whose gating depends on diverse signal sensing elements. The "gatekeepers" of the long, ATP-inhibited KIR6.0 pores of KATP channels are ABC proteins, SURs, receptors for channel opening and closing drugs. Several competing models for SUR/KIR coupling exist. We show that SUR TMD0, the N-terminal bundle of five transmembrane helices, specifically associates with KIR6.2, forcing nearly silent pores to burst like native KATP channels and enhancing surface expression. Inclusion of adjacent submembrane residues of L0, the linker between TMD0 and the stimulatory nucleotide- and drug-binding ABC core, generates constitutively active channels, whereas additional cytoplasmic residues counterbalance this activation establishing a relationship between the mean open and burst times of intact pores. SUR fragments, lacking TMD0, fail to modulate KIR. TMD0 is thus the domain that anchors SUR to the KIR pore. Consistent with data on chimeric ABCC/KIRs and a modeled channel structure, we propose that interactions of TMD0-L0 with the outer helix and N terminus of KIR bidirectionally modulate gating. The results explain and predict pathologies associated with alteration of the 5' ends of clustered ABCC8 (9)/KCNJ11 (8) genes.

Ntp and Ctp, synthetic peptides based on the N- and C-terminal sequences of K(IR)6.0, respectively, were used to probe gating of K(IR)6.0/SUR K(ATP) channels. Micromolar Ntp dose-dependently increased the mean open channel probability in ligand-free solution (P(O(max))) and attenuated the ATP inhibition of K(IR)6.2/SUR1, but had no effect on homomeric K(IR)6.2 channels. Ntp (up to approximately 10(-4) m) did not affect significantly the mean open or "fast," K(+) driving force-dependent, intraburst closed times, verifying that Ntp selectively modulates the ratio of mean burst to interburst times. Ctp and Rnp, a randomized Ntp, had no effect, indicating that the effects of Ntp are structure specific. Ntp opened K(IR)6.1/SUR1 channels normally silent in the absence of stimulatory Mg(-) nucleotide(s) and attenuated the coupling of high-affinity sulfonylurea binding with K(ATP) pore closure. These effects resemble those seen with N-terminal deletions (DeltaN) of K(IR)6.0, and application of Ntp to DeltaNK(ATP) channels decreased their P(O(max)) and apparent IC(50) for ATP in the absence of Mg(2+). The results are consistent with a competition between Ntp and the endogenous N terminus for a site of interaction on the cytoplasmic face of the channel or with partial replacement of the deleted N terminus by Ntp, respectively. The K(IR) N terminus and the TMD0-L0 segment of SUR1 are known to control the P(O(max)). The L0 linker has been reported to be required for glibenclamide binding, and DeltaNK(IR)6.2/SUR1 channels exhibit reduced labeling of K(IR) with (125)I-azidoglibenclamide, implying that the K(IR) N terminus and L0 of SUR1 are in proximity. We hypothesize that L0 interacts with the K(IR) N terminus in ligand-inhibited K(ATP) channels and put forward a model, based on the architecture of BtuCD, MsbA, and the KcsA channel, in which TMD0-L0 links the MDR-like core of SUR with the K(IR) pore.