Toward Linking Structure With Function in ATP-Sensitive K؉ Channels Joseph Bryan,1 Wanda H. Vila-Carriles,2 Guiling Zhao,1 Audrey P. Babenko,1 and Lydia Aguilar-Bryan1,3

ϩ Advances in understanding the overall structural fea- sensitive K channels (KATP channels), has been an tures of inward rectifiers and ATP-binding cassette effective target, based on the observations by Janbon and (ABC) transporters are providing novel insight into the colleagues that treatment with a sulfonamide, 2254 RP, ؉ architecture of ATP-sensitive K channels (KATP chan- produced severe hypoglycemia, as well as subsequent nels) (K 6.0/SUR) . The structure of the K pore has IR 4 ؉ IR studies by Loubatie`res that the RP compound stimulated been modeled on bacterial K channels, while the lipid-A ␤ release (rev. in 1–3). In pancreatic -cells, KATP exporter, MsbA, provides a template for the MDR-like channels are part of the ionic triggering mechanism that core of receptor (SUR)-1. TMD0, an NH2- terminal bundle of five ␣-helices found in SURs, binds to increases insulin secretion in response to increased glu- cose metabolism. The activity of K channels is modu- and activates KIR6.0. The adjacent cytoplasmic L0 linker ATP serves a dual function, acting as a tether to link the lated by changes in the ATP/ADP ratio. In the absence of MDR-like core to the KIR6.2/TMD0 complex and exerting nucleotides, these channels are spontaneously active, but bidirectional control over channel gating via interactions binding of ATP to the KIR subunits (the half-maximal with the NH -terminus of the K . Homology modeling of ϳ ␮ 2 IR inhibitory concentration [IC50] for ATP is 10 mol/l) the SUR1 core offers the possibility of defining the gliben- inhibits activity. This inhibition can be antagonized by clamide/sulfonylurea binding pocket. Consistent with 30- MgADP acting through the sulfonylurea receptor (SUR). year-old studies on the pharmacology of hypoglycemic agents, the pocket is bipartite. Elements of the COOH- The coupling of membrane potential with cellular metab- terminal half of the core recognize a hydrophobic group in olism provides a means to adjust the activity of voltage- 2ϩ 2ϩ glibenclamide, adjacent to the sulfonylurea moiety, to gated Ca channels and, thus, modulate Ca -dependent provide selectivity for SUR1, while the benzamido group insulin exocytosis. KATP channels are known targets for appears to be in proximity to L0 and the KIR NH2-termi- hypoglycemic like tolbutamide and gliben- nus. Diabetes 53 (Suppl. 3):S104–S112, 2004 clamide and for nonsulfonylureas like nateglinide and repaglinide, which increase insulin output by reducing Kϩ channel activity, thus modulating intracellular free Ca2ϩ ([Ca2ϩ] ) (4). he regulation of insulin secretion from pancre- i The physiologic importance of the ionic mechanism is atic ␤-cells in the islets of Langerhans is under well illustrated by the dramatic changes in glucose ho- the orchestration of multiple conductors. While meostasis that can result from genetic alterations, which glucose metabolism and changes in cellular en- T affect the PO—the probability that KATP channels are in the ergy status ultimately drive insulin output, a variety of open state. This is shown conceptually in Fig. 1, which inputs converge to modify the rate of secretion and thus ␤ maintain blood glucose levels within normal limits. Under- relates the PO to -cell membrane potential and insulin secretion. Depolarization activates voltage-gated Ca2ϩ standing the molecular basis of these inputs provides 2ϩ multiple routes for therapeutic intervention to both aug- channels, induces oscillation of cytosolic [Ca ]i, and thus ment and diminish insulin secretion in disorders of glu- pulsatile release of insulin (5,6). In humans, the loss of cose homeostasis. The ionic pathway, particularly ATP- SUR1/KIR6.2 KATP channel activity is the most common cause of persistent hyperinsulinemic hypoglycemia when ␤-cells are persistently depolarized and oversecrete insulin From the 1Department of Molecular and Cellular Biology, Baylor College of (rev. in 7,8). A mild dominant form of hypoglycemia due to Medicine, Houston, Texas; the 2Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas; and the 3Department a mutation in SUR1, Glu1507Lys, produces hyperinsulin- of Medicine, Baylor College of Medicine, Houston, Texas. ism at an early age and then progresses to decreased Address correspondence and reprint requests to Joseph Bryan, PhD, Department of Molecular and Cellular Biology, Baylor College of Medicine, insulin secretory capacity in early adulthood and diabetes Houston, TX 77030. E-mail: [email protected]. in middle age (9). At the other extreme, overexpression of Received for publication 13 March 2004 and accepted in revised form a mutant pore subunit, engineered to activate channels by 12 May 2004. This article is based on a presentation at a symposium. The symposium and decreasing their sensitivity to inhibitory ATP, resulted in the publication of this article were made possible by an unrestricted educa- transgenic mice with neonatal diabetes (10). These results tional grant from Servier. 2ϩ 2ϩ implied that “gain-of-activity” mutations were candidates ABC, ATP-binding cassette; [Ca ]i, intracellular Ca concentration; ER, endoplasmic reticulum; ICD, intracellular coupling domain; KATP channel, for producing neonatal diabetes, and a genetic screen of ATP-sensitive Kϩ channel; K channel, inwardly rectifying Kϩ channel; MDR, IR patients identified mutations at two NH2-terminal and two multidrug resistance protein; NBD, nucleotide-binding fold; PO, open proba- bility; SUR, sulfonylurea receptor; TMD, transmembrane domain. COOH-terminal positions (Q52, V59, R201, and I296) in © 2004 by the American Diabetes Association. KIR6.2 associated with permanent neonatal diabetes (11).

S104 DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 J. BRYAN AND ASSOCIATES

FIG. 1. A conceptual view of the relation-

ship between the PO of KATP channels, ␤-cell membrane potential, and insulin secretion. A spectrum of mutations that affect channel open probability producing altered glucose homeostasis is illus- trated. E23K, Glutamate23Lysine; GFG3 AAA, GlycinePhenylalanineGlycine in the selectivity filter substituted with Ala- nineAlanineAlanine; HI, hyperinsulinism. Interestingly, R201 had earlier been reported to be critical for brane helix pore forming subunit, and its regulatory sub- ATP-inhibitory gating (12), while V59 and Q52 are in, or near, unit, the ATP-binding cassette (ABC) protein SUR1 (rev. in a helix argued to be critical for SUR/KIR stimulatory coupling 40). KIR6.2 is a member of the potassium inward rectifier (13 and below). In addition, the E23K polymorphism, re- family, channels that in physiological solutions conduct ϩ ported to increase KATP channel activity at physiologic nu- K better in the inward than outward direction (rev. in 41). cleotide levels (14–16) and increase the channel sensitivity to Work on the structure(s) of KIR6.0 pores necessary to stimulation by acyl CoAs (17), appears to be a predisposing understand their weak inward rectification and ATP-inhib- factor for (18–23; see also 24,25). itory gating has been limited. Eukaryotic ion channels are This review is focused on recent structure-function difficult to express and purify in the quantities needed for studies that give insight into the SUR/KIR coupling mech- structural work. Thus, insight has come mainly from X-ray anism and emphasizes the contributions the sulfonylurea crystallographic studies on the cytoplasmic pore of mouse receptor makes to KATP channel functionality. This distinc- Kir3.1 (42), on the truncated bacterial channels KcsA (43) tion is somewhat artificial—neither subunit has been shown and KirBac1.1 (44), and on the use of these templates to to be important physiologically in the absence of its part- generate homology models for the KATP channel (13) or ner—but it serves to highlight the tight integration of the various domains (45,46). KcsA provided the archetypal ϩ KIR6.2 pore and SUR1 regulatory subunits at multiple levels, tetrameric K pore assembled from subunits with three including assembly, trafficking, and control of gating. A helical elements: the outer M1, pore, and inner M2 helices. recent report (26) shows, for example, that rapid assembly of The selectivity filter, at the external face, is formed pri- SUR and KIR subunits in the endoplasmic reticulum (ER) marily from the main chain carbonyls of the GYG (GlyTyr- protects the pore subunit from degradation and implies that Gly) sequence between the M1 and pore helixes (47), SUR/KIR heterodimers assemble the octameric, (SUR1/ whereas the bundle of four M2 helices at the internal face ␤ KIR6.2)4 KATP channels found in -cells. Several studies have is thought to form the ligand-sensitive gate. An additional emphasized that subunit coexpression and correct assembly amphipathic helix, termed the slide helix, precedes M1 and into the octameric channel is essential to pass quality-control appears to be lipid-anchored in KcsA (48). The slide helix checkpoints and reach the cell surface (27–29). The effec- is positioned perpendicular to the pore axis in the Kir- tiveness of this control is supported by the failure of KIR6.2 Bac1.1 structure (44). The KIR3.1 cytoplasmic pore and subunits to contribute to membrane hyperpolarization in KirBac1.1 pore have provided insight into the organization knockout mice lacking SUR1 (30,31). In addition to assembly of the long Mg2ϩ and polyamine binding permeation path and trafficking, other studies have emphasized the role of that confers inward rectification. SUR1 in “activating” the pore in the absence of ligands and The sulfonylurea receptors, SURs, are in the ABC trans- increasing its sensitivity to inhibitory ATP (32,33); in the porter family (49). They have a modular structure (Fig. 2) stimulatory action of MgADP (34,35); in the action of phar- with an MDR-like core consisting of two bundles of macologic agents (both openers and inhibitors) (36,37); and, transmembrane domains (TMDs) linked to cytoplasmic ␤ in the case of cardiac and -cell KATP channels, which share nucleotide-binding folds (NBDs) by helical extensions of a common KIR6.2 pore, in determining the isoform differ- the TMDs termed intracellular coupling domains (ICDs) ences in slow gating and its modulation by inhibitory and (50). Similar to eukaryotic channels, structural studies on stimulatory nucleotides (38,39). eukaryotic transporters are limited. Insight has come from the structures of several bacterial ABC transporters in- K CHANNELS ARE COMPOSED OF A WEAK ATP cluding EC- and VC-MsbA, which are lipid-A exporters ؉ INWARDLY RECTIFYING K PORE AND A QUARTET OF from E. coli (50) and Vibrio cholera (50), respectively, and ATP-BINDING CASSETTE REGULATORY PROTEINS a vitamin B12 importer, BtuCD (51). The VC-MsbA dimer ␤ -Cell KATP channels are hetero-octamers composed of (52) is currently the best structural template available for two disparate subunits (Fig. 2): KIR6.2, a two transmem- homology modeling of the multidrug-resistance protein

DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 S105 STRUCTURE-FUNCTION IN KATP CHANNELS

FIG. 2. Summary of structures and mini-KATP channel data. The top panel illustrates the topology of SUR1 and KIR6.2. The SUR NH2-terminal domains ؉ that interact with and stabilize or destabilize the KIR open state are in red and blue, respectively. Glycosylation sites (trees), “ ” exit, and RKR (Arg Lys Arg) ER retention signals are important for surface expression. The positions where SUR1 was cut to generate Nt and complementary Ct fragments are shown. The right middle panel summarizes the biphasic effect of progressively longer NH2-terminal segments on ligand-independent channel activity. Macrocurrents and single-channel recordings are shown. The red segments, single-channel recordings on an expanded time scale, illustrate differences in the open-state stability. See ref. 13 for details of the analysis. The left middle panel illustrates the interactions critical

S106 DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 J. BRYAN AND ASSOCIATES

(MDR)-like core of SUR1 (13), based on sequence homol- KIR6.2, but we have been unable to identify stable com- ogy, overall topology, and interacting NBDs. SURs, as well plexes between KIR6.2 and the SUR1 core or to demon- as several other ABCC family members (49), differ from strate the core has a functional effect on the KIR. The the canonical MDR structure in having an additional results imply that these additional interactions, presum- NH2-terminal helical bundle, TMD0, and a long cytoplas- ably regulatory in nature, are transient and that L0 serves mic L0 linker connecting TMD0 with the core (Fig. 2). as a de facto “tether” linking the ABC core to the TMD0/KIR Although TMD0 and L0 of MRPs are critical for apical- complex. Additional work is necessary to define how the basolateral routing and substrate transport (53–57), a core is “docked” to the KIR6.2/TMD0-L0 complex. functional role for the TMD0-L0 module in SURs remained unknown until recently (13,58). THE L0 LINKER BIDIRECTIONALLY CONTROLS PO SUR1 TMD0 “ACTIVATES” K 6.2 AND ANCHORS IT TO Analysis of channels assembled from KIR6.2 and chimeric IR receptors SUR1ϳSUR2A demonstrated that TMD0-L0 was THE ABCC8 CORE a determinant of the isoform differences in slow gating While the basic octameric stoichiometry of KATP channels (38). The importance of L0 in the control of the slow is well established (59–61), a basic structural problem has component of gating was strongly reinforced by the ob- been understanding what parts of the SUR and KIR sub- servation that including parts of L0 in mini-KATP channels units interact to activate a fully functional channel. In the dramatically altered their interburst kinetics. Inclusion of absence of inhibitory ligands, KATP channels exhibit spon- the initial third of L0 dramatically reduced the rate of the taneous activity characterized by bursts of openings and burst termination, resulting in “hyperactivation” seen in brief closings. Bursts are separated by interburst intervals, Nt232/K 6.2⌬C35 channels (Fig. 2). At a mechanistic ϳ IR so that channels conduct 65–70% of the time (POmax up level, this submembrane part of L0, predicted to contain a ϳ to 0.7). Expressed in the absence of SUR, homomeric helix, appears to interact with a submembrane part of the ⌬ (KIR6.2 C)4 pores, lacking their ER retention signals, pore to lock mini-K channels in the burst state. Inclu- ϳ ATP display profoundly destabilized burst states—an 10-fold sion of additional sequence attenuated this activation, Ͼ reduction in their POmax and a 30-fold reduction in their implying L0 has a bidirectional effect and that the two sensitivity to ATP. Therefore, we asked which domain(s) actions, saturating versus attenuating the POmax, require of SUR1 could convert the spiky activity of the pore to structurally distinct segments of the linker. Existing data Ͻ ␮ normal bursting inhibited with an IC50(ATP) 10 mol/l. provide insight into which parts of K 6.2 are involved; Both the structure of the conserved Kϩ pore (43,62) and IR deletion of the distal KIR6.2 NH2-terminus similarly locks work using chimeras of KIR6.2 and KIR2.1, a subunit that ⌬ NKIR6.2/SUR1 channels in the burst state (65–67). The does not associate with SUR1, implied the outer M1 helix results imply that an interaction of the peripheral K was needed for coassembly (28). While there have been IR NH2-terminus with a cytoplasmic segment of L0 attenuates conflicting models of SUR/KIR6.0 coupling (28,63,64), two the POmax. This hypothesis is supported by the observation recent studies have shown that TMD0 is the principal SUR that a synthetic, hydrophilic peptide, Ntp, based on the coassembly domain (13,58). SUR1 TMD0 assembled stable first 33 K 6.2 NH -terminal residues, can compete with the ⌬ IR 2 complexes, or “mini-KATP channels,” with the (KIR6.2 C)4 endogenous NH2-terminus for a putative inhibitory linker pores resulting in restoration of full KATP-like bursting in SUR1 and increase the POmax by reducing transitions activity with about a fivefold increase in the POmax in from the burst state (64). The proposed interactions ligand-free solutions (13,58) (Fig. 2). between TMD0-L0 and the pore are color-coded in Fig. 2: The sensitivity to inhibitory ATP was not increased by inhibitory contacts (in blue) between the cytoplasmic TMD0 or TMD0-L0, implying that interaction with another domains of the two subunits counterbalance the stimula- part of SUR will be required to account for the increased tory interactions (in red) between the submembrane L0 affinity observed in the full channel. As expected, in the helix and the KIR “slide” helix (44), which saturate the absence of the drug-binding SUR core, mini-KATP channels P . are not stimulated by ADP and their activity is not modu- Omax lated by sulfonylureas or KATP openers. The approach demonstrated that TMD0 is a major SUR domain that TOWARD UNDERSTANDING THE ATP-INHIBITORY MACHINERY OF K CHANNELS interacts with KIR6.2. The interaction activates the pore ATP and restores bursting, presumably via contacts of trans- Site-directed mutagenesis reduced the candidate residues membrane helices. One expects that other parts of the involved in ATP binding, or its coupling to the ATP receptor might interact with cytoplasmic domains of inhibition gate, to Lys185 and Gly334 in the COOH-domain

for assembly and gating of (SUR/KIR6.0)4 channels. TMD1-NBD1 and TMD2-NBD2 of one SUR1 (gray parts on the left) were modeled using the VC-MsbA structure (52) (see structural alignments in ref. 13). The KIR6.2 pore was modeled using the KirBac1.1 structure (44) (adjacent subunits are colored using differential tones). Loops that had to be built de novo are not shown; these include the linker between the inner helix

and the COOH-terminal domain, which is longer in eukaryotic KIRs. Ile182, Lys185, and Gly334 are colocalized with Arg50 from the adjacent subunit (shown in yellow and light yellow residues). Phe133 of the GlyPheGly-based selectivity filter is in green. The TMS important for KIR/SUR recognition points outward from the M1 helix (red). The horizontal slide helix (red or light red), preceding M1, is accessible for interaction with

the submembrane L0 domain (red cylinder). The first 44 residues of the KIR (the blue NH2 terminus, missing in the template, is shown for one KIR subunit) are not required for KATP assembly (65) and are at the periphery of the cytoplasmic pore accessible to parts of L0 (in blue). Residue ૾ 23 in KIR6.2 (*) and 116, an HI-SUR1 mutation in TMD0 ( ) are indicated. The lower left panel provides an enlarged view, from the top, of one of the predicted inhibitory ATP-binding pockets shown in the figure above. ATP, in an extended conformation, is shown for illustrative purposes only. The exact coordination of ATP is unknown. The lower right panel shows a truncated view of the SUR1 core at the level of the ICDs. The NBDs have been cut off. The cyan residue in the lower left is S1237 and is pointed to by the cyan arrow. The russet colored residues are T1286 and M1290 on TM17, and are pointed to by the russet arrow. For illustrative purposes, the potassium channel opener diazoxide is shown to the left; glibenclamide in extended and compact low-energy configurations is at the right.

DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 S107 STRUCTURE-FUNCTION IN KATP CHANNELS

and Arg50 in the NH2-terminus of KIR6.2. Substitutions at of this work is being rediscovered, and structure-function these positions decreased the IC50(ATP) without affecting studies on SUR1 and SUR2 have begun to provide struc- the POmax of homomeric KIR pores and KATP channels in tural information about the nature of the “A” and “B” sites ligand-free solutions (65,68,69). Substitutions at other res- (rev. in 81). Chimeric receptors SUR1ϳSUR2A were used to idues that affect ATP-binding, e.g., I182 (69,70), can alter exploit the pharmacological differences between ␤-cell the POmax. The first double mutant, KIR6.2 Arg50Gln/ and cardiomyocyte channels and identify the importance Lys185Gln (65), demonstrated the additive contribution of of the TMD2 domain for high-affinity recognition of tolbu- NH2- and COOH-terminal residues to ATP-induced stabili- tamide (73,82). A detailed consideration of the available zation of the closed state, consistent with the possibility structure-activity data suggests that TMD2 forms part of that these two residues cooperate in the coordination of the “A” site and that SUR1 is able to accommodate the separate negatively charged phosphate groups of ATP in lipophilic center adjacent to the anionic group on the SUR1-containing channels. The GIRK1-based homology sulfonylurea moiety (i.e., the butyl side chain in tolbut- models of the KIR6.0 C-domain (13,71) support the idea amide, cyclohexane substituted with a propyl group in that Ile182, Lys185, and Gly334 are colocalized and that nateglinide, or the cyclohexyl ring in glibenclamide) while Lys185 and Gly334 probably contribute to the surface of SUR2A is not. The implication is that SUR1 has a hydro- ␤ the adenine- and -phosphate-selective ATP-binding pocket phobic cavity able to accommodate these side chains and that we have argued is conserved in both KIR6.0 subunits that this cavity is missing or occluded in SUR2. Substitu- (39,72). Our modeling of the complete pore indicated an tion of Ser1237 with Tyr, the analogous residue in SUR2, additional key structural aspect of the ATP-inhibitory reduced the apparent affinity of SUR1 for tolbutamide and machinery. Consistent with the data on the R50Q/K185Q glibenclamide (82). One possibility is that Ser1237 is in double-mutant channels (65), we proposed that the COOH- close proximity to the lipophilic center and that Tyr1206 terminal binding pocket on one K 6.0 subunit is comple- IR occludes binding in SUR2A; the reverse substitution, mented by Arg50 from the neighboring K 6.0 subunit, IR Tyr1206Ser in SUR2B, does increase the affinity for gli- producing four intersubunit ATP-binding pockets (inset, benclamide several fold but not to the level of SUR1 (83). Fig. 2). The key feature of this structural model is that Arg50 immediately precedes (by three residues) the slide The interpretational difficulty lies in not being able to helix that we hypothesize contacts the submembrane helix distinguish experimentally between a direct interaction of of L0 and moves with it during interburst transitions. Ser1237 with the drug versus an indirect effect of the Repositioning of these elements thus has a dual effect: substitution at a more distant drug binding site. In the Movement of the activating (red) domains away from the homology model of the SUR1 core, Ser1237 is at the end of ␣ pore axis opens the gate and will also move Arg50 away one of the -helices, which form the ICD (see Fig. 2). The from the nucleotide-binding cavities, effectively opening ICDs are proposed to play a critical role in coupling the the intersubunit pockets and thus increasing the rate of binding of stimulatory nucleotides to the NBD dimer with dissociation (K ) for inhibitory ATP. This mechanism can conformational changes in the substrate binding cavity of d MsbA (52) or the maltose transporter (84). It is tempting to explain the marked increase in the IC50(ATP) by Nt232, which effectively mimics the action of full-length SUR speculate that the A site lies at the level of the ICDs and saturated with stimulatory ligands. Conversely, movement that tolbutamide, or other insulin secretagogues, can “in- of the inhibitory region (blue) of L0 is hypothesized to sert” between the halves of the core and restrict movement. This tentative structural picture provides a framework push the KIR NH2-terminus, thus repositioning Arg50 and increasing the affinity for nucleotides, while stabilizing a with which to interpret the abolishment of the “basal,” straight, closed KcsA- and KirBac1.1-like conformation of Mg-nucleotide–dependent stimulation of SUR1 by oral the M2 helical bundle, thereby reducing the mean PO. This hypoglycemic agents (73,85,86), which is their principal attenuation of the POmax by the inhibitory segments of L0 mechanism of action in vivo. (see Nt288 in Fig. 2) mimics the effect of full-length SUR1 Insight into the location of the B site comes from affinity saturated with a hypoglycemic compound when tested in labeling studies and efforts to define the glibenclamide nucleotide-free solutions (73). binding site. Analysis of peptides affinity labeled by 125I- iodoglibenclamide, presumably by photoactivation of the carbonyl group in the benzamido moiety, placed the SUR1 HAS A MULTIFACETED BINDING POCKET labeling site in the TMD0-L0 segment (87). When SUR1 and The main action of oral hypoglycemic agents, both sulfo- KIR6.2 are in KATP channels, both subunits are labeled by nylurea and nonsulfonylurea compounds, is to stimulate 125I-azidoglibenclamide, where the active group is a ni- insulin secretion by inhibiting KATP channels via their trene on the benzamido ring generated by photolysis and binding to SUR1. Early studies on structure-activity rela- thus positioned to react with residues in the B site tionships indicated, over 30 years ago, that an effective (59,88,89). Mikhailov et al. (90) showed that TMD0 is not pharmacophore consisted of three lipophilic or hydropho- required for high-affinity glibenclamide binding to SUR1. bic centers separated by a –CONH– group and a negative Similarly, TMD0 is not required, whereas L0 is necessary, charge, respectively (Fig. 3). Recognition was posited to for affinity labeling (W.H.V.-C., unpublished data). Alanine occur through interaction with a multifaceted bipartite substitutions around Trp232, a conserved residue in L0, pocket with distinct “A” and “B” binding sites within a identify a cluster of amino acids critical for affinity labeling “sulfonylurea receptor” (75–77,80; rev. in 81). More recent (Fig. 3). Substitution of Trp232 with alanine results in loss work has made it clear that the sulfonylurea group is not of affinity labeling, whereas replacement with cysteine essential for hypoglycemic activity, but rather provides a retains labeling. The results are consistent with the nitrene properly positioned anionic group (78,79). The importance on the benzamido group being in close proximity to Trp232,

S108 DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 J. BRYAN AND ASSOCIATES

FIG. 3. Pharmacophores and affinity labels. A: The simple pharmacophore model in the upper panel is modified from Grell et al. (74) and is based on early structure-activ- ity relation studies (75–79). The dashed line symbolizes the surrounding protein pocket. The structures of glibenclamide and tolbutamide are shown; the position of the azido group in azidoglibenclamide is given in parentheses. B: Alanine substitu- tion of residues in L0 was used to identify residues important for affinity labeling. myc-tagged receptors were labeled with 125I-azidoglibenclamide, immunoprecipi- tated with anti-myc antibodies, and visual- ized by autoradiography. Results for the wild-type (WT) control, the Tyr230Ala (Y230A), and Trp231Ala (W232A) substitu- tions are shown. The results are summa- rized on a helical net for illustrative

purposes. C:Amyc-tagged NH2-terminal fragment of KIR6.2 containing M1 immuno- precipitates SUR1 and is affinity labeled.

Progressive deletion of its NH2-terminus diminishes affinity labeling of the KIR frag- ment without significantly affecting label- ing or coimmunoprecipitation of SUR1. D: The equivalent deletions in full-length ⌬ NKIR6.2/SUR1 channels compromise cou- pling between binding of the inhibitory sulfonylurea tolbutamide (Tlb) and atten-

uation of the PO in the nucleotide-free solutions (73). The lower dashed line gives

the NPO value for the full-length SUR1/ KIR6.2 channel; the upper dashed line gives the mean NPO (channel number times open probability) for tolbutamide-insensitive

cardiac SUR2A/KIR6.2 channels. The dotted lines are the standard deviations. but again conformational change with action at a more the absence of nucleotides (65). The results support the distant site cannot be ruled out completely. A conservative idea that the outer helix of KIR6.0 subunits specifically interpretation is that the benzamido group of gliben- interact with SUR (28), as well as the conservative inter- clamide lies near the Trp232 region of L0, which forms part pretation that the principal affinity-labeling site is either in of the B site. the first half of the KIR6.2 NH2-terminus or that the Deletion of the NH2-terminus of KIR6.2 reduced its peripheral NH2-terminus is essential for positioning more affinity labeling with 125I-azidoglibenclamide (64). Data in proximal residues near the nitrene (64). Fig. 3 show that a fragment of KIR6.2 containing M1 but lacking M2 and the COOH-terminal cytoplasmic domain makes a stable complex with SUR1 and is affinity labeled. LOCATION OF THE POTASSIUM CHANNEL OPENER SITE Progressive deletion of residues from the NH2-terminus of this M1 fragment abolished affinity labeling without affect- Chimeric receptors were also used to localize regions ing its association with SUR1. Short NH2-terminal dele- important for recognition of several channel openers, tions of KIR6.2 also impaired the coupling of tolbutamide including diazoxide, cromakalim, and pinacidil. The action binding to SUR1 and consequent attenuation of the PO in of diazoxide required TMD1 and part of the first nucleotide

DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 S109 STRUCTURE-FUNCTION IN KATP CHANNELS binding domain, NBD1 (91), a localization supported by a 7. Aguilar-Bryan L, Bryan J: Molecular biology of - study on the novel opener NNC 55-9216 (92). A region of sensitive potassium channels. Endocr Rev 20:101–135, 1999 8. Bryan J, Aguilar-Bryan L: Sulfonylurea receptors, ATP-sensitive potassium TMD2 was shown to be critical for the action of cromaka- channels and insulin secretion. In Diabetes Mellitus. 3rd ed. LeRoith D, lim and pinacidil (91,93,94), and Moreau et al. (94) identi- Taylor SI, Olefsky JM, Eds. Philadelphia, Lippincott Williams & Wilkins, fied two residues in the last transmembrane helix of 2004, p. 69–98 SUR—Leu1249 and Thr1253 in SUR2 and Thr1286 and 9. Huopio H, Otonkoski T, Vauhkonen I, Reimann F, Ashcroft FM, Laakso M: Met1290 in SUR1. Swapping these two residues conferred A new subtype of autosomal dominant diabetes attributable to a mutation sensitivity to cromakalim and pinacidil on SUR1 and in the gene for sulfonylurea receptor 1. Lancet 361:301–307, 2003 10. Koster JC, Marshall BA, Ensor N, Corbett JA, Nichols CG: Targeted abolished sensitivity in SUR2A. In the homology model of overactivity of KATP channels induces profound neonatal diabe- the SUR1 core (13,81), Met1290 is near the top of TMD2 tes. Cell 100:645–654, 2000 facing the putative drug-binding cavity (see Fig. 2). The 11. Gloyn AL, Pearson ER, Antcliff JF, Proks P, Bruining GJ, Slingerland AS, results are consistent with the conclusion that both halves Howard N, Srinivasan S, Silva JM, Molnes J, Edghill EL, Frayling TM, of the SUR core are required for specific interaction with Temple IK, Mackay D, Shield JP, Sumnik Z, van Rhijn A, Wales JK, Clark P, Gorman S, Aisenberg J, Ellard S, Njolstad PR, Ashcroft FM, Hattersley KATP channel openers (91). The positioning of Met1290 in AT: Activating mutations in the gene encoding the ATP-sensitive potassi- central cavity, as well as earlier data indicating that the um-channel subunit Kir6.2 and permanent neonatal diabetes. N Engl J Med action of the potassium channel openers depends on the 350:1838–1849, 2004 presence of hydrolysable MgATP (95–97; however, see 98), 12. Ribalet B, John SA, Weiss JN: Molecular basis for Kir6.2 channel inhibition suggests a parallel with transport mechanisms in which by adenine nucleotides. Biophys J 84:266–276, 2003 13. Babenko AP, Bryan J: SUR domains that associate with and gate KATP site accessibility is dependent on nucleotide hydrolysis pores define a novel gatekeeper. J Biol Chem 278:41577–41580, 2003 and implies that binding of an opener in the central cavity 14. Schwanstecher C, Neugebauer B, Schulz M, Schwanstecher M: The com- can trap a specific conformation of the core and thus mon single nucleotide polymorphism E23K in KIR6.2 sensitizes pancreatic potentiate channel activity. beta-cell ATP-sensitive potassium channels toward activation through nucleoside diphosphates. Diabetes 51 (Suppl. 3):S363–S367, 2002 15. Schwanstecher C, Schwanstecher M: Nucleotide sensitivity of pancreatic SUMMARY ATP-sensitive potassium channels and type 2 diabetes. Diabetes 51 (Suppl. Current research is providing an increasingly detailed 3):S358–S362, 2002 ␤ 16. Schwanstecher C, Meyer U, Schwanstecher M: K 6.2 polymorphism picture of -cell KATP channel structure, including homol- IR ogy models of the ion-conducting pore and the nucleotide- predisposes to type 2 diabetes by inducing overactivity of pancreatic beta-cell ATP-sensitive Kϩ channels. Diabetes 51:875–879, 2002 and drug-binding SUR1 core. A role for TMD0-L0 in the 17. Riedel MJ, Boora P, Steckley D, de Vries G, Light PE: Kir6.2 polymor- coupling of SUR1 to KIR6.2 has been demonstrated, and phisms sensitize beta-cell ATP-sensitive potassium channels to activation the importance of these interactions for control of gating by acyl CoAs: a possible cellular mechanism for increased susceptibility to has been emphasized. This structural picture provides an type 2 diabetes? Diabetes 52:2630–2635, 2003 18. Hani EH, Boutin P, Durand E, Inoue H, Permutt MA, Velho G, Froguel P: increasingly sophisticated framework with which to inter- ϩ pret mutations and polymorphisms that affect glucose Missense mutations in the pancreatic islet beta cell inwardly rectifying K channel gene (KIR6.2/BIR): a meta-analysis suggests a role in the polygenic homeostasis by altering the normal coupling mechanism basis of type II diabetes mellitus in Caucasians. Diabetologia 41:1511– to produce both inactive and hyperactive channels. The 1515, 1998 spectrum of diseases linked with KATP channels, from 19. Gloyn AL, Hashim Y, Ashcroft SJ, Ashfield R, Wiltshire S, Turner RC: neonatal to adult diabetes through congenital hyperinsu- Association studies of variants in promoter and coding regions of beta-cell linism, is broad and of considerable social, medical, and ATP-sensitive K-channel genes SUR1 and Kir6.2 with type 2 diabetes mellitus (UKPDS 53). Diabet Med 18:206–212, 2001 economic importance. Increased understanding of the 20. t Hart LM, van Haeften TW, Dekker JM, Bot M, Heine RJ, Maassen JA: structure and coupling mechanism(s) in KATP channels Variations in insulin secretion in carriers of the E23K variant in the KIR6.2 will lead to improved pharmaceuticals directed at these subunit of the ATP-sensitive Kϩ channel in the beta-cell. Diabetes 51:3135– disorders. 3138, 2002 21. Gloyn AL, Weedon MN, Owen KR, Turner MJ, Knight BA, Hitman G, Walker M, Levy JC, Sampson M, Halford S, McCarthy MI, Hattersley AT, ACKNOWLEDGMENTS Frayling TM: Large-scale association studies of variants in genes encoding

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