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Published as doi: 10.1096/fj.06-6730hyp.
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(The FASEB Journal. 2007;21:18-25.)
© 2007 FASEB

Defining a binding pocket for sulfonylureas in ATP-sensitive potassium channels

Wanda H. Vila-Carriles*,1, Guiling Zhao{dagger} and Joseph Bryan{dagger}

* Departments of Molecular Physiology and Biophysics and

{dagger} Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA

1Correspondence: Department of Physiology and Biophysics, The University of Alabama at Birmingham, 1918 University Blvd., MCLM 705, Birmingham, AL, USA 35294-0005. E-mail: carriles{at}uab.edu

ABSTRACT

Sulfonylurea receptors SUR1 and SUR2 are the regulatory subunits of KATP channels. Their differential affinity for hypoglycemic sulfonylureas provides a basis for the selectivity of these compounds for different KATP channel isoforms. Sulfonylureas have a 100- to 1000-fold greater affinity for SUR1 vs. SUR2. Structure-activity studies suggested a bipartite binding pocket. Chimeric SUR1~SUR2 receptors have shown TMD2, the third bundle of transmembrane helices, to be part of an "A" site that confers SUR1 selectivity for sulfonylureas. The purpose of this study is to determine the position of the "B" site. Previous photoaffinity labeling studies have placed the B site on the amino-terminal third of SUR and colabeled the associated KIR. In our study, deletion of TMD0, the first bundle of transmembrane helices, did not compromise labeling. Further deletions into the cytoplasmic linker, L0, eliminated binding and labeling. Alanine substitutions in L0 identified a limited number of conserved residues, Y230 and W232, important for affinity labeling. A fragment of KIR6.2, missing M2 and the entire carboxyl terminal, assembles with SUR1 and is affinity labeled, while deletion of 10 or more amino-terminal residues compromises labeling. These studies indicate that the B site involves L0 and the KIR amino terminus, elements that are critical for control of channel gating.—Vila-Carriles, W. H., Zhao, G., Bryan, J. Defining a binding pocket for sulfonylureas in ATP-sensitive potassium channels.


Key Words: ATP-binding cassette proteins • potassium inward rectifier • glibenclamide • hypoglycemia

IN PANCREATIC ß-CELLS, ATP-SENSITIVE K+ (KATP) channels set the resting membrane potential. Increases in ATP/ADP or oral hypoglycemic agents reduce their activity resulting in membrane depolarization. The subsequent activation of voltage-gated Ca2+ channels leads to an increase in cytosolic Ca2+, the triggering signal for insulin release. The neuroendocrine type KATP channels are composed of KIR6.2, a member of the inward rectifier family, which forms the channel pore and, SUR1, a member of the ATP-binding cassette (ABC) superfamily that regulates channel activity. SURs have two transmembrane domains (TMD1 and TMD2), each containing six helices and two nucleotide-binding folds that comprise the ABC cassette. SUR1 and SUR2, as well as four other ABC proteins, have an additional hydrophobic N-terminal extension, which adds a 5 helix bundle, TMD0, which is tethered to the core by an extended cytoplasmic segment termed L0 (1 2 3) . KIR6.2 and SUR1 associate rapidly in the ER to form a heterodimer (4) , which then assembles into the octameric channel, (KIR6.2/SUR1)4 (5 6 7) . Recent work (8 , 9) demonstrates that TMD0 can interact with and activate the KIR pore, while segments of L0 confer bidirectional control of gating (9) .

Sulfonylurea-based compounds have been used as oral hypoglycemic agents in the treatment of type 2 diabetes for over five decades (10) , and SUR1 is recognized as the primary target for these compounds. Extensive structure-activity studies are consistent with a simple pharmacophore model (11) with tolbutamide as the prototype first-generation compound (Fig. 1 ). The substitution of lipophilic aromatic or heterocyclic groups at position iii (Fig. 1) increased the potency of compounds like glibenclamide by 100- to 1000-fold (12) in accord with their affinities for SUR1, ~50 µM vs. 0.5 nM (13 , 14) . Studies with half molecules like meglitinide (15 16 17 18) suggested that the increase in affinity was a result of interactions with two overlapping binding sites, termed the A and B sites (11 , 19) . The A site recognizes the lipophilic center (i) adjacent to the sulfonylurea group, while the B site recognizes the benzamido (iii) and adjacent CONH–group (Fig. 1) .


Figure 1
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  		Figure 1. Sulfonylureas used as oral antidiabetic drugs. Pharmacophore model taken from Grell et al. (11) . Three lipophilic or hydrophobic centers separated by an amide and an anionic linker are proposed to interact with the A and B sites in SUR1. Structure of tolbutamine and nateglinide, which interact with the A site, glibenclamide that interacts with both the A and B site, and meglitinide and repaglinide, which interact with the B site, are shown.

The challenge has been to locate the A and B sites within the receptor. Previous studies with chimeric receptor, SUR1~SUR2A, showed that part of TMD2 is required for the high affinity, SUR1-selective action of tolbutamide (20 , 21) . Initial localization of the B site in SUR1 came from affinity-labeling studies with [125I]-labeled glibenclamide, an iodinated derivative of glibenclamide (22) , which labeled a ~50 kDa amino-terminal fragment including TMD0-L0 (23) . However, the B site has yet to be elucidated. Here, we identify the importance of a limited number of amino acids in L0 for labeling with [125I]-azido-glibenclamide (24) , a photosensitive derivative of glibenclamide that binds with high affinity to SUR1 (25) . Photolysis generates a nitrene radical at the position of the azido group that covalently labels both the receptor and KIR6.2 in KATP channels (5 , 26) . Coexpression of half fragments of SUR1, neither of which displayed high-affinity, reconstituted glibenclamide binding activity (27) consistent with a requirement for the amino-terminal half of the receptor. The deletion of TMD0 had little effect on binding, while complete truncation of L0 abolished binding (27) . The results were consistent with a bipartite binding pocket and position part of the B-site near L0.

Cophotolabeling of KIR6.2 with [125I]-azido-glibenclamide in the presence of SUR1, suggests that part of KIR6.2 might lie close to the sulfonylurea binding site on SUR1 (5) . Previous studies have shown that coexpression of SUR with KIR6.x enhances [3H]-glibenclamide binding affinity 3- to 4-fold, supporting the idea that KIR6.x may be part of the sulfonylurea binding pocket (28) . In this article, we have used SUR1 deletions and alanine substitutions to show that a segment of L0 is important, and a limited number of amino acids in the L0 linker and the N terminus of KIR6.2 is critical for affinity labeling. Our results identified residues within the SUR1 and KIR6.2 subunits that are essential structural determinants of the sulfonylurea binding pocket.

MATERIALS AND METHODS

Molecular biology
The hamster SUR1 cDNA, encoding 1582 amino acids, in the pECE vector (29) , with two copies of the myc-epitope (30) was truncated at its amino terminus by introducing start codons at positions 198, 205, 207, 208, 209, 210, 220, 232 using polymerase chain reaction (PCR). SUR1 point mutations were created by PCR overlap extension using primers containing the mutation. Reactions were done using the QuikChange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) following the manufacturer’s directions. The presence of mutations was confirmed by sequencing. The TMD0-L0-SUR2~SUR1 chimera was constructed as described earlier as Chim II (31) . The human KIR6.2 cDNA, encoding 390 amino acids, in the pECE vector was introduced a myc-epitope at position 100 by ligating complementary oligonucleotides with appropriate overhangs. N-terminal deletions ({Delta}5, {Delta}10, {Delta}20, {Delta}32, and {Delta}44) in Myc-KIR6.2 were constructed as described previously (21) . For truncations of M1 helix in above N-terminal deletions, a stop codon was introduced at position 128.

Cell culture and transfections
COSm6 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS. Transient transfections were done using lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer’s directions or PEI. When PEI was used, COSm6 cells were grown in 150-mm culture dishes to 70–90% confluency, 21 µl of a stock solution of PEI (25 kDa, branched form) at a concentration of 4.3 mg/ml (0.1 M in nitrogen) in PBS (32) were diluted in 5 ml PBS. The DNA (70 µg) was then added, mixed, and incubated at room temperature for 45 min before the addition of 15 ml of culture medium.

Whole-cell membrane preparation
Whole-cell membranes were prepared from cells seeded on 150-mm dishes 72 h after transfection. Transfected cells were washed twice with PBS, pH 7.4. A hypotonic buffer (5 mM Tris, pH 7.4, 2 mM EDTA) containing Complete protease inhibitors (Roche Applied Science, Indianapolis, IN, USA) was added, and the cells were kept on ice for 45 min before scraping and collecting. The cells were homogenized and centrifuged at 1000 g for 20 min at 4°C to remove nuclei and unbroken cells. Membranes were collected by ultracentrifugation at 100,000 g for 1 h. The membranes were resuspended in membrane buffer (50 mM Tris-HCL, pH 7.4, 5 mM EDTA) with protease inhibitors and stored frozen at –80°C. The protein concentration, typically ~2 mg/ml, was measured using the bicinchoninic acid (BCA) Protein Assay (Pierce, Rockford, IL, USA).

Cells and whole-cell membranes photolabeling
Cells were grown in six-well plates and transfected at 80% confluence; experiments were performed in triplicate. After 48 h, cells were washed twice with PBS containing Ca2+ and Mg2+ (PBSCM) pH 6.8. Cells were incubated at 22°C in the dark with 1 nM [125I]-azidoglibenclamide (24) . After 30 min, the cells were irradiated in a UV crosslinker (FB-UVXL-1000, Fisher Scientific, Pittsburgh, PA) with 312-nm bulbs at a setting of 200 mJ/cm2. The unbound drug was removed by washing twice with PBSCM. The whole-cell membrane photolabeling was carried out as described previously (22 , 33) .

Immunoprecipitation and Western blot analysis
After photolabeling, transfected cells were solubilized with 1% Triton X-100 or 1% digitonin (Sigma Chemical, St. Louis, MO) in PBS and 1 mM EDTA, pH 7.4, containing complete protease inhibitors (Roche Applied Science, Indianapolis, IN). Lysates were clarified at 9000 g for 30 min at 4°C. The supernatants were incubated with mouse monoclonal antimyc agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) or rat anti-hemagglutinin antibody (Ab) (Roche Applied Science) for 6 h followed by the addition of Protein G plus (Santa Cruz Biotechnology) overnight at 4°C. The beads were washed five times with either 0.1% Triton X-100 or 0.1% digitonin in PBS and solubilized in 2X SDS sample buffer (34) . Proteins were separated on 7.5% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 10% nonfat milk in Tris-buffered saline (10 mM Tris, pH 7.5, 150 mM NaCl, and 2 mM EDTA) and incubated overnight at 4°C with antimyc Ab or anti-hemagglutinin Ab. After washing, blots were reacted with either a peroxidase-conjugated secondary Ab or IRDye 680-conjugated Ab (Molecular Probes, Eugene, OR) and visualized using either a chemiluminescent substrate or the Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE). The membranes containing photolabeled samples were put on an intensifying screen for quantification with a StormTM PhosphorImager (Amersham Biosciences, Piscataway, NJ).

RESULTS

SUR1 L0 is critical for sulfonylurea binding and affinity labeling
SUR1 deletion constructs were generated to analyze the requirement of TMD0-L0 for [125I]-azido-glibenclamide labeling. Deletion of TMD0, to generate {Delta}198, did not affect photolabeling. Further truncation of L0 ({Delta}220 and {Delta}232) resulted in the progressive loss of affinity labeling (Fig. 2 C). A quantitative measure of loss was obtained by dividing the radioactivity in the labeled bands, estimated using a phosphoimager, by the level of expression obtained from Western blot analysis. The data showed a decline in labeling between residues 205 and 210, and no labeling once amino acids 220 or more were deleted (Fig. 2D ). The predicted secondary structure includes a small loop preceding a sequence with a high hydrophobic moment (Fig. 2B ). Truncations that completely abrogate [125I]-azido-glibenclamide labeling (e.g., {Delta}232) did not impair expression or labeling with 8-azido-[{alpha}-32P]ATP, indicating that the SUR core is intact and able to bind ATP (data not shown). Also, SUR1 {Delta}232 did not bind glibenclamide in binding assays with [3H]-labeled glibenclamide (data not shown).


Figure 2
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  		Figure 2. Truncation of SUR1 L0 impairs photolabeling. A) Schematic diagram of SUR1 shows TMD0, TMD1, and TMD2. Gray arrow marks the beginning of the L0 linker, amino acid 198. B) The SUR1-L0 sequence and the predicted secondary structure. COSm6 cells were transfected with the truncated myc-tagged SUR1 plasmids as described. After 48 h, cells were photolabeled with [125I]-azido-glibenclamide (1 nM), solubilized with 1% digitonin in PBS, and immunoprecipitated using mouse antimyc Ab agarose beads conjugated. The beads were washed five times with 0.1% digitonin in PBS, solubilized with 2x SDS sample buffer and resolved on 7.5% SDS-PAGE. C) Gel containing photolabeled samples with [125I]-azido-glibenclamide was dried and exposed to an X-ray film. Radioactivity was quantified using a phosphoimager. D) Gel was transferred to a nitrocellulose membrane and blotted with mouse antimyc Ab. The bands were visualized using chemiluminescence to estimate protein expression. E) The films were scanned using a Molecular Dynamics densitometer. Bands were analyzed using Iquant software (Molecular Dynamics). The data are plotted as the ratio of label-to-protein in arbitrary units.

Alanine scanning identifies a limited number of amino acids important for affinity labeling with [125I]-azido-glibenclamide
Bakos et al. (2) identified the importance of a predicted amphipathic helix within L0 for MRP1 transport functions. Additionally, it was observed that GSH-dependent photoaffinity labeling of MRP1 requires the L0 linker and a double mutation, W261A and K267M, in L0 decreased labeling three-fold (35) . SUR1 has a unique insert in this region, so the sequence correspondence is not exact. However, alanine scanning over the comparable predicted amphipathic helix in SUR1 identifies residues Y230 and W232, which are important for affinity labeling (Fig. 3 A). A quantitative measure of loss of affinity labeling was obtained by dividing the radioactivity in the labeled bands (Fig. 3A ) by the protein expression levels obtained from Western blot analysis (Fig. 3B ). The graph shows a decrease in photoaffinity labeling for SUR1 Y230A and W232A (Fig. 3C ).


Figure 3
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  		Figure 3. Alanine scan in L0 linker. COSm6 cells were transfected with the mutant myc-tagged SUR1 plasmids as described. After 48 h, cells were photolabeled with 1 nM [125I]-azido-glibenclamide, solubilized with 1% digitonin in PBS, and immunoprecipitated with mouse antimyc Ab agarose beads conjugated. The beads were washed five times with 0.1% digitonin in PBS, solubilized with 2x SDS sample buffer, and resolved by 7.5% SDS-PAGE. A) Gels containing photolabeled samples with [125I]-azido-glibenclamide were dried and exposed to an X-ray film. Radioactivity was quantified using a phosphoimager (Molecular Dynamics). B) Gels were transferred to a nitrocellulose membrane and blotted with mouse antimyc Ab. The bands were visualized using chemiluminescence to estimate protein expression. C) The films were scanned using a Molecular Dynamics densitometer. Bands were analyzed using Iquant software (Molecular Dynamics). The data are plotted as the ratio of label-to-protein in arbitrary units ± SD.

Swapping TMD0-L0 from the low-affinity SUR2 receptor does not have an effect on binding or labeling
The pharmacophore model is based in part on the large increase in affinity associated with addition of a lipophilic group in position iii, e.g., the benzamido group in glibenclamide (Fig. 1) . We assessed the contribution of the source of L0 to this effect by constructing a chimeric receptor in which TMD0-L0 of SUR1 was replaced with the equivalent fragments from SUR2, which does not bind the hypoglycemic sulfonylurea compounds with high affinity. Human SUR1 and SUR2 have a considerable degree of sequence identity in L0, ~65% between positions 195 and 300. They have the conserved –TYWW–sequence identified by alanine substitution in SUR1 (Fig. 3) . The SUR2~SUR1 chimera photolabeled and have comparable affinities for [3H]-glibenclamide (Fig. 4 ).


Figure 4
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  		Figure 4. An L0 linker is required for labeling, but it does not appear to be specific for SUR1. A chimera was made by swapping TMD0-L0 from SUR2A into SUR1. COSm6 cells were transfected, and after 48 h, cells were photolabeled with 1 nM [125I]-azido-glibenclamide in the presence (+) or absence (–) of 1 µM glibenclamide and resolved by 7.5% SDS-PAGE. Autoradiograph shows that SUR1 and the chimera photolabeled and bind glibenclamide, while the parental SUR2A does not. Binding assays using 1 nM [3H]-glibenclamide were done with membranes isolated from COSm6 cells transfected with each of the constructs. Membranes were incubated in the dark at room temperature with increasing concentrations of unlabeled glibenclamide. After 1 h, the reaction was terminated by rapid filtration through GF/F glass fiber filters (Whatman International, Maidstone, UK) followed by two washes with ice-cold PBS at pH 7.4. Radioactivity was determined using a scintillation counter. The data were interpreted using a single-site binding model plus a nonspecific term and the nonlinear fitting routines in Ligand or Prism (GraphPad, San Diego, CA, USA). The dissociation constant KD and the number of glibenclamide binding sites per cell (Bmax) were estimated.

The KIR6.2 amino terminus is critical for KIR cophotolabeling
We reported that [125I]-azido-glibenclamide will affinity label KIR6.2 assembled with SUR1, while KIR6.2 alone is unlabeled (5) . Deletion of approximately half of the N terminus compromised the labeling of N{Delta}33KIR6.2 (36) and uncoupled the inhibitory effect of sulfonylurea binding to SUR1 on spontaneous {Delta}NKIR6.2/SUR channel activity in the absence of nucleotides (37) Also, in a N{Delta}14KIR6.2/SUR1 channel high-affinity sulfonylurea’s block was abolished (38) , suggesting that the N terminus of KIR6.2 may be involved in conferring sulfonylurea sensitivity to the KATP channel. To define the labeling segment better, we constructed amino-terminal fragments of KIR6.2 tagged with myc epitope, containing the outer M1 helix, a total of 100 amino acids. The KIR6.2 N-terminal fragment containing the M1 helix photolabels (Fig. 5 A). Coexpression of SUR1 with {Delta}NKIR6.2 amino-terminal constructs ({Delta}5, {Delta}10, {Delta}20, {Delta}32, and {Delta}44) followed by labeling, detergent solubilization, and immunoprecipitation with antimyc antibodies showed that removing 10 residues from the KIR6.2 N-terminal reduces cophotolabeling, while removing 20 amino acids or more (N{Delta}32, N{Delta}44) eliminate it without impairing the labeling of SUR1 (Fig. 5A ). A quantitative measure for the {Delta}NKIR6.2M1 loss of affinity labeling was obtained by dividing the labeled bands (Fig. 5A ) by the protein expression levels obtained from the Western blot analysis (Fig. 5B ). The graph shows a decrease in labeling once 10 or more amino acids are deleted from KIR6.2 N terminus (Fig. 5C ).


Figure 5
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  		Figure 5. Deletion of KIR6.2 N-terminal abolishes affinity-labeling. COSm6 cells were cotransfected with untagged SUR1 and either Myc-KIR6.2 M1 or Myc-{Delta}NKIR6.2 M1. Cells were photolabeled with 1 nM [125I]-azido-glibenclamide, solubilized, and immunoprecipitated with mouse antimyc Ab agarose beads conjugated. Proteins were separated by 7.5–15% SDS-PAGE and detected using a phosphoimager and Western blot analysis. A) Autoradiograph of 125I-azido-glibenclamide cophotolabeling of SUR1, Myc-KIR6.2 M1, and Myc-{Delta}NKIR6.2 M1. B) Western blot analysis of Myc-KIR6.2 M1 and Myc-{Delta}NKIR6.2 M1. C) Graph shows percent of the ratio of photolabeling-to-protein expression based on immunoprecipitation of Myc-KIR6.2 M1 and Myc-{Delta}NKIR6.2 M1. The data are an average from 3 experiments ± SD.

Coexpression of KIR6.2 with SUR1 mutants increases their affinity for glibenclamide
Assembly with KIR6.2 increases the affinity of SUR1 for glibenclamide ~3- to 4-fold (28) . We tested whether coexpression with KIR6.2 affected affinity labeling of the SUR1, Y230A, and W232A substitutions. Both SUR1 and KIR6.2 in KATP channels are affinity-labeled by [125I]-azido-glibenclamide and coexpression of SUR1, Y230A, and W232A with KIR6.2 resulted in labeling of the KIR (data not shown) and also in an increase of the mutant SUR1 labeling (Fig. 6 ). We increased the drug concentration from 0.3 nM, 1 nM, 3 nM, and 10 nM to test whether a higher [125I]-azido-glibenclamide amount would give a good labeling of the SUR1 point mutations without the KIR; but even in the presence of 10 nM [125I]-azido-glibenclamide the SUR1 mutants did not label (data not shown for 3 nM and 10 nM). When SUR1 Y230A, and W232A were coexpressed with N{Delta}33KIR6.2, a KIR6.2 construct that does not photolabel, none of the SUR1 mutants labeled (Fig. 6) , indicating that the KIR6.2 N terminus play a role in the sulfonylurea binding pocket.


Figure 6
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  		Figure 6. The presence of the KIR6.2 N terminus increase labeling of SUR1. Wild-type SUR1, SUR1, Y230A, and W232A were expressed or coexpressed with either KIR6.2 or N{Delta}33KIR6.2 in COSm6 cells. After photolabeling with either 0.3 or 1 nM [125I]-azido-glibenclamide, samples were separated by 10% SDS-PAGE. Autoradiograhs show that assembly with KIR6.2 increases the affinity of SUR1 and the alanine point mutations for glibenclamide. However, coexpression of SUR1, Y230A, and W232A with N{Delta}33KIR6.2 did not increase labeling of SUR1 or the mutants.

DISCUSSION

Sulfonylureas are widely used in the treatment of noninsulin-dependent diabetes mellitus, but there is little detailed information available on the sulfonylurea binding pocket. High-affinity binding of sulfonylureas is a hallmark of SUR1. [125I]-glibenclamide was synthesized and used to purify SUR1, the high-affinity receptor (23) . Several labeled peptides, generated by V8 protease cleavage, all included the amino terminus of SUR1. The sizes of the labeled peptides were consistent with a labeling site within TMD0-L0. In contrast to the labeling results, studies on SUR1~SUR2A chimeric receptors showed that a second domain of SUR1 was functionally important. SUR2 receptors have a lower affinity for sulfonylureas. Chimeric KIR6.2/SUR1~SUR2A channels demonstrated that TMD2 was critical for high-affinity inhibition of channel activity (21) . At first, the chimera data appear to be at odds with the labeling studies. However, tolbutamide does not have the meglitinide portion of glibenclamide (Fig. 1) proposed to be recognized by the B-site. The initial argument was that TMD2 recognized the sulfonylurea group. However, the structure-activity relationship data on hypoglycemic compounds suggest that TMD2 recognizes the lipophilic group adjacent to the sulfonylurea group. This makes sulfonylurea and nonsulfonylurea compounds good hypoglycemic drugs because they bind selectively to SUR1 vs. SUR2.

Over 40 years ago, pharmacologists developing hypoglycemic therapeutic agents proposed a pharmacopore model, which can be used to explain some of the current affinity-labeling and functional studies. This pharmacopore model proposes a multifaceted binding pocket with two binding sites (11 , 19) . The A site recognizes the lipophilic group adjacent to the negative charge supplied by the sulfonylurea group, while the B site recognizes a lipophilic group adjacent to the amide. Tolbutamide lacks the elements needed to interact with the B site; therefore, it is ~1000-fold less potent than glibenclamide and other second-generation sulfonylureas. Previous studies demonstrated that switching a single amino acid, serine 1237, between transmembrane helices (TM) 15 and 16 of SUR1 to the tyrosine residue found at the equivalent position in SUR2 abolished the high-affinity interaction of SUR1 with tolbutamide, glibenclamide (20) and nateglinide (39 , 40) . However, this switch had little effect on the action of meglitinide (20) and repaglinide (39) . The reciprocal substitution, Y1206S, in SUR2B increased the affinity for glibenclamide ~10-fold (41) . The results imply S1237 is either part of the sulfonylurea binding pocket or that substitution at this position alters the conformation of the pocket (39 , 42) . In a Vibrio cholera MsbA transporter-based SUR1 homology model, S1237 is in the submembrane helical extensions of TM 15 and 16, part of the intracellular coupling domain (ICD) proposed to transmit nucleotide-driven changes in the ATP-binding cassette to the transmembrane helical bundles (43) . Overall consideration of the pharmacologic data is consistent with a critical part of the A-site being in TMD2 in the ICD between TM15 and TM16 (44) .

In the present study, we positioned the B site. It has been previously shown that while removing TMD0 did not affect [3H]-glibenclamide binding, further truncations, which remove L0, abolished binding (27) . The data presented here both support and expand on those earlier results. We showed that deleting almost one-third of the L0 linker, reduced [125I]-azido-glibenclamide photolabeling, while deletion of 232 amino acids completely eliminated glibenclamide labeling and binding. The lack of photolabeling in SUR1 {Delta}N232 is not due to improper folding because the fragments are competent to bind 8-azido-[{alpha}-32P]ATP. The predicted secondary structure in this region, 202 through 207, shows a small loop and a highly hydrophobic segment. Truncation of the small loop may affect the local structure of L0 important for the B site conformation, causing a decrease in photoaffinity labeling. Another structural feature is a predicted amphipathic helix in L0 between residues 222 and 240, which is conserved in all members of the SUR and MRP family. This alpha helix within the L0 region is absolutely required for the function of MRP1 (2) . We hypothesized that this helix has hydrophobic interactions with the sulfonylurea. Alanine scanning showed that amino acids Y230 and W232 are required for high affinity [125I]-azido-glibenclamide photolabeling. These residues may be labeled themselves or may position part of the receptor for labeling.

When SUR1 and the SUR1 alanine point mutations Y230A and W232A were coexpressed with KIR, this resulted in an increase in [125I]-azido-glibenclamide labeling and KIR cophotolabeling, consistent with KIR forming part of the B site. Also, when point mutations were coexpressed with N{Delta}33KIR6.2, [125I]-azido-glibenclamide labeling was not recovered, suggesting that the KIR N-terminal 33 amino acids form part of the B-site. The KIR N terminus may be either positioning SUR1 point mutations, allowing them to be labeled by [125I]-azido-glibenclamide or positioning [125I]-azido-glibenclamide, allowing it to label the SUR1 point mutations. We determined the minimum number of residues on the KIR N terminus required for affinity labeling with [125I]-azido-glibenclamide, deleting 10 amino acids from KIR6.2 N terminus reduced photoaffinity labeling, while deleting 20 eliminated it. The results correlate with the experiments with N{Delta}33KIR6.2, which did not rescue photoaffinity labeling of the SUR1 alanine point mutations, most likely because of the lack of the first 20 amino acids from the N terminus. In the N{Delta}20KIR6.2M1/SUR1 channels, only SUR1 is labeled implying that the KIR6.2 N terminus itself may be the labeling site or required to position some other part of KIR6.2, which gets photolabeled. The results confirm a report that M1 is critical for KIR/SUR interactions (45) and show that M2 and the carboxyl terminal cytoplasmic domain are not essential for labeling. The results constrain the site of labeling to the first ~100 amino acids of KIR6.2 and demonstrate the importance of the distal KIR amino terminus.

We assessed whether SUR1-TMD0-L0 contributed to the specificity or affinity of binding and labeling by swapping the TMD0-L0 segment from SUR2 into a SUR1 background. This chimeric receptor showed high-affinity binding and photolabeling with [125I]-azido-glibenclamide, suggesting that while L0 is important, binding and labeling appear to be relatively tolerant of changes in its sequence. This is consistent with the observation that meglitinide (the nonsulfonylurea portion of glibenclamide) has lower affinity, but is an equally effective inhibitor of SUR1 and SUR2-based KATP channels (20) . The results also suggest either the interactions of the benzamido group are nonspecific, but good enough to bind meglitinide with ~10 µM affinity or that the –TYWW–motif is critical, as suggested by the alanine substitutions in Fig. 4 , consistent with an L0 being required for binding but not determining specificity of binding. The results support the idea that glibenclamide, meglitinide, and repaglinide interact with a site common to all types of SURs leading to inhibition of KATP channels and imply that the B site within SUR1 and SUR2 has similar chemical specificities.

In conclusion, KATP channel activity can be inhibited by isolated occupation of either the binding site A or binding site B. Both binding sites can be occupied simultaneously, as in the case when glibenclamide is present, resulting in the high potency of this drug. It is believed that the two binding sites are localized at a distance recognizable in the structure of glibenclamide. Accordingly, the data suggest that SUR1 has a hydrophobic pocket, which can accommodate lipophilic side chains, and that SUR2 is less able to accommodate those side chains because the A site in SUR2 is different from the one in SUR1. The deletion constructs and point mutation experiments imply that the reactive nitrene generated during photolysis of [125I]-azido-glibenclamide is positioned near L0. These studies support the premise of a multifaceted sulfonylurea binding pocket previously reviewed by us (46 , 47) . This binding pocket is found where part of SUR1-TMD2 forms the A site, and the B site involves both SUR-L0 and the KIR N terminus, located in proximity to or on the binding site of the nitrene-generating azido group on the benzamido part of glibenclamide.

ACKNOWLEDGMENTS

We thank Dr. Xue-Nong Zhang and Maria Janecki for help with plasmid construction. This work was supported by grants from the National Institutes of Health (DK44311, to J.B. and GM66478, to W.H.V.)

Received for publication June 22, 2006. Accepted for publication August 21, 2006.

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