Introduction

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ATP-dependent K⁺ channels (K_ATP channels) are a class of K⁺ channels that are gated by the intracellular ATP/ADP ratio; functionally, these channels couple the metabolic state of the cell to membrane potential and excitability (Ashcroft and Ashcroft, 1990; Edwards and Weston, 1993). Pharmacologically, K_ATP channels are closed by the hypoglycemic sulfonylureas (SUs) used in the treatment of diabetes type II; they are activated by the K⁺ channel openers, a chemically heterogenous group of compounds that induce hypotension (Ashcroft and Ashcroft, 1990; Edwards and Weston, 1993).

K_ATP channels are heteromeric complexes composed of pore-forming -subunits belonging to the family of inwardly rectifying K⁺ channels (Kir6.x) and sulfonylurea receptors (SURs) as -subunits (Inagaki et al., 1995; Sakura et al., 1995; for reviews, see Ashcroft and Gribble, 1998; Aguilar-Bryan and Bryan, 1999). SUR is a member of the ATP-binding cassette protein superfamily (Aguilar-Bryan et al., 1995; Sakura et al., 1995). It carries binding sites for nucleotides (Ueda et al., 1999), sulfonylureas, and openers (Aguilar-Bryan et al., 1995; Hambrock et al., 1998; Schwanstecher et al., 1998). The K_ATP channels in different tissues combine different subtypes of SUR and Kir6.x. The SUR in the pancreatic -cell (SUR1) exhibits high affinity for the SUs and a low affinity for the openers, whereas the SUR found in the various muscle types (SUR2) shows high affinity for the openers but lower affinity for the SUs (Hambrock et al., 1998; Schwanstecher et al., 1998; Dörschner et al., 1999; Hambrock et al., 1999; Russ et al., 1999). Two isoforms of SUR2 have been characterized, SUR2A and SUR2B, that differ only in the last carboxyl-terminal exon. SUR2A is expressed in skeletal and heart muscle; SUR2B in smooth muscle (Inagaki et al., 1996; Isomoto et al., 1996). SUR2B combines with Kir6.1 to form the K_ATP channel in vascular smooth muscle and with Kir6.2 in nonvascular smooth muscle (Isomoto et al., 1996; Yamada et al., 1997). Kir6.2 carries a high-affinity site for ATP that mediates channel block by micromolar concentrations of ATP (Tucker et al., 1997) whereas Kir6.1-containing K_ATP channels are inhibited by ATP only in the millimolar concentration range (Yamada et al., 1997).

Essential parts of the binding sites for SUs and openers are located on the third domain of SUR (Ashfield et al., 1999; Uhde et al., 1999; Babenko et al., 2000). Uhde et al. (1999) have shown that the binding site for the opener N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine) (P1075) consists of two stretches of amino acids directly flanking the binding site for glibenclamide. Ashfield et al. (1999), comparing the SU binding domains of SUR1 and SUR2A, have identified a critical amino acid in the intracellular loop connecting transmembrane segments 15 and 16 that is important in determining SU-sensitivity: if serine 1237 in rat SUR1 is replaced by tyrosine, which is the corresponding residue in SUR2, the high affinity for tolbutamide and glibenclamide is lost. Conversely, one might expect that replacement of Tyr 1206 in SUR2 (mouse numbering) by Ser would increase the SU affinity of SUR2. Indeed, in a preliminary communication, it has been reported that rat SUR2B(Y1205S) has a 25-fold higher affinity for glibenclamide than for wild-type SUR2B (Toman et al., 2000).

The new mutant is useful because it exhibits high affinity for both openers and glibenclamide. This allows [³H]glibenclamide and [³H]P1075 binding to be performed with the same SUR and thereby a more precise analysis of the relationship between sulfonylurea and opener binding than previously possible. Using wild-type SUR2B, [³H]glibenclamide binding studies in cells were difficult to interpret because of the high level of intrinsic (non-SUR2B) glibenclamide binding (Russ et al., 1999); in membranes, they were impossible to quantify (Dörschner et al., 1999; Russ et al., 1999). There is agreement that sulfonylurea and opener binding is mutually exclusive by a negative allosteric coupling of the two binding sites (Bray and Quast, 1992; Schwanstecher et al., 1992); however, details of this interaction remain unknown. In addition, several aspects of the interaction of glibenclamide with SUR2, such as the equilibrium dissociation constant of the radioligand (K_D) are still in doubt and there is disagreement whether or not the affinity of glibenclamide binding to SUR can be determined quantitatively by inhibition of opener binding (Dörschner et al., 1999; Russ et al., 1999). We have made use of the SUR2B(Y1206S) mutant to clarify these questions. In addition, we compare here for the first time the differential effect of coexpression of (mutant) SUR with Kir6.1 and Kir6.2 on the interaction with glibenclamide and P1075.

	Experimental Procedures

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Cell Culture and Transfections. Human embryonic kidney (HEK) 293 cells were cultured as described previously (Hambrock et al., 1998, 1999) in minimum essential medium containing glutamine, supplemented with 10% fetal bovine serum and 20 µg/ml gentamycin. Cells were transfected with mammalian expression vector pcDNA3.1 (Invitrogen, Karlsruhe, Germany) containing the coding sequence of murine SUR2B (GenBank accession number D86038; Isomoto et al., 1996), mutant SUR2B (see below), murine Kir6.1 (D88159; Yamada et al., 1997) or Kir6.2 (D50581; Inagaki et al., 1996). As a control they were transfected with pcDNA3.1 vector alone. Cotransfection of SUR2B (wild-type or mutant) with Kir6.1 or Kir6.2 was done transiently at a molar plasmid ratio of 1:1, if not stated otherwise, and using LipofectAMINE and OptiMEM (Invitrogen) as described previously (Hambrock et al., 1998). In cotransfections used for electrophysiological experiments, the pEGFP-C1 vector (CLONTECH, Palo Alto, CA), encoding for green fluorescent protein, was added for easy identification of transfected cells. Two to 4 days after transfection, cells were used for binding studies and electrophysiological experiments. Cells stably transfected with wild-type or mutant SUR2B were isolated in the presence of 700 µg of geneticin/ml of medium within the first 3 weeks and 300 µg of geneticin/ml of medium thereafter; 1 week before experiments, the antibiotic was withdrawn.

Site-Directed Mutagenesis. The mutant SUR2B(Y1206S) was constructed using the QuikChange Site-Directed Mutagenesis System (Stratagene, Amstersdam, The Netherlands). Murine SUR2B-cDNA, inserted into pcDNA3.1, was used as the template. Two completely complementary primers (31-mer oligonucleotides) were designed, containing the desired mutation (TAC to TCC) in the middle region. For each reaction, 125 ng of forward and reverse mutagenic primers was combined with 100 ng of wild-type murine SUR2B-cDNA in pcDNA3.1 and 2.5 U of Pfu Turbo DNA polymerase in a 18 cycle PCR reaction (denaturation, 30 s at 95°C; annealing, 60 s at 55°C; extension, 29 min at 68°C). Parental methylated DNA was destroyed by digestion with DpnI and the newly synthesized DNA was transformed into Max Efficiency DH5 competent cells (Invitrogen). The presence of the desired mutation was confirmed by nucleotide sequencing of the relevant DNA region.

Patch-Clamp Experiments. The patch-clamp technique was used in the whole-cell configuration as described in detail in Russ et al. (1999). Bath solution was 142 mM NaCl, 2.8 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 11 mM D-(+)-glucose, and 10 mM HEPES, titrated to pH 7.4 with NaOH at 37°C. Patch pipettes were filled with 132 mM K-glutamate, 10 mM NaCl, 2 mM MgCl₂, 10 mM HEPES, 1 mM EGTA, 1 mM Li₂GDP, and 0.3 mM Na₂ATP, titrated to pH 7.2 with NaOH, and had a resistance of 3 to 5 M. Data were recorded with an EPC 9 (HEKA, Lambrecht, Germany) amplifier using the "Pulse" software (HEKA). Series resistance was compensated by 70%. Isolated cells showing green fluorescence were clamped to 60 mV; every 12 s, seven square pulses ranging from 110 to 10 mV (0.5 s each) were applied (Fig. 1). For evaluation of the inhibition by glibenclamide (GBC), the current at 60 mV was used and traces were individually corrected for run down.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Whole-cell current recordings from HEK 293 cells transfected with Kir6.2 or Kir6.1 together with SUR2B(Y1206S). Top, time course of the current at 60 mV and 37°C from a cell expressing Kir6.2 + SUR2B(Y1206S). After establishing the whole-cell configuration, the KATP current developed within some minutes; thereafter, GBC was applied cumulatively to the bath and washed out. Block by 1 µM GBC was fully reversible if the rundown of current ( 3%/min) is taken into account. Center and bottom, currents at different test potentials. Cells were clamped at 60 mV and the pulse protocol shown in the lower right corner was started every 12 s. The control traces (left) were recorded at  7 min; middle traces, GBC at 3 µM (Kir6.2/SUR) or 0.3 µM (Kir6.1/SUR) produced an almost complete block of the KATP current; right, currents after washout for  15 min.

Equilibrium Binding Experiments in Cells. Experiments were conducted at 37°C with an incubation time of 30 min as described previously (Hambrock et al., 1998; Russ et al., 1999). Cells were suspended by rinsing with a HEPES-buffered physiological salt solution containing 139 mM NaCl, 5 mM KCl, 1.2 mM MgCl₂, 1.25 mM CaCl₂, 11 mM D-(+)-glucose; 5 mM HEPES, gassed with 95% O₂/5% CO₂, and titrated to pH 7.4 with NaOH at 37°C. Binding experiments were started by addition of cells (final concentration, 0.8-2 × 10⁶ cells/ml corresponding to 0.2-0.5 mg of protein/ml) to physiological salt solution containing the radiolabel (for competition studies, 2 to 4 nM [³H]GBC/1 to 3 nM [³H]P1075 and the inhibitor of interest). After 30 min, incubation was stopped by diluting 0.3-ml aliquots in triplicate into 8 ml of ice-cold quench solution (50 mM Tris-(hydroxymethyl)-aminomethane, 154 mM NaCl, pH 7.4) and rapid filtration under vacuum over Whatman GF/C filters. Filters were washed twice with 8 ml of ice-cold quench solution and counted for ³H in the presence of 6 ml of scintillant (Ultima Gold; Packard, Meriden, CT). Nonspecific binding of [³H]GBC/[³H]P1075 binding was determined in the presence of 100/10 µM P1075. P1075 completely and specifically inhibits [³H]P1075 and [³H]GBC binding to (mutant and wild-type) SUR2B, whereas GBC also binds to nontransfected HEK 293 cells with low affinity (Russ et al., 1999).

Equilibrium Binding Experiments in Membranes. Membranes were prepared as described previously (Hambrock et al., 1998). Briefly, cells at  80% confluence (16 million cells per dish) were suspended by rinsing with medium and centrifuged for 6 min at 500 g at 37°C. The pelleted cells were lysed by addition of 5 ml (per dish) of ice-cold hypotonic buffer containing, 10 mM HEPES, 1 mM EGTA, pH 7.4, and the lysate centrifuged at 100,000g and 4°C for 60 min. The resulting membrane pellet was resuspended in a buffer containing 5 mM HEPES, 5 mM KCl, 139 mM NaCl; 0 or 2.2 mM MgCl₂ at pH 7.4 and 4°C at a protein concentration of  0.7 mg/ml and frozen at 80°C. In the binding assay, membranes (final protein concentration, 0.2-0.5 mg/ml) were added to the incubation buffer (139 mM NaCl, 5 mM KCl, 5 mM HEPES, 2.2 mM MgCl₂) supplemented with 1 mM Na₂ATP, the radiolabel (for competition, [³H]GBC  3 nM or [³H]P1075 = 1-3 nM), and the inhibitor of interest at 37°C. At equilibrium (15 min for [³H]GBC and 25 min for [³H]P1075 binding), incubation was stopped as described above and aliquots were filtered over Whatman GF/B filters.

Data Analysis, Modeling and Statistics. In saturation experiments, nonspecific binding (B_NS) was proportional to the free label concentration, L, and was fitted to the equation, B_NS = a × L, where a denotes the proportionality constant. Total binding (B_TOT) was then analyzed as the sum of specific and nonspecific binding and was fitted to the equation,

(1)

to estimate K_D and B_max (fmol/mg of protein) values by the least-squares method. Experiments were performed over a large range of radioligand concentrations so that all parameters including B_NS, could be determined from the B_TOT curve. B_NS was determined independently in the presence of 100 µM P1075 giving the same result and thus validating this approach.

(2)

(3)

(4)

Materials. [³H]P1075 [specific activity, 4.5 TBq (117 Ci)/mmol] was purchased from Amersham Buchler (Braunschweig, Germany) and [³H]GBC [specific activity, 1.85 TBq (50 Ci)/mmol] from PerkinElmer Life Science Products (Bad Homburg, Germany). The reagents and media used for cell culture and transfection were from Invitrogen. Na₂ATP and Li₂GDP were from Roche Molecular Biochemicals (Mannheim, Germany), glibenclamide from Sigma (Deisenhofen, Germany) and cytochalasin D from Fluka (Neu-Ulm, Germany). The following drugs were kind gifts of the pharmaceutical companies indicated in parentheses: AZ-DF 265 (Thomae, Biberach, Germany), levcromakalim (SmithKline-Beecham, Harlow, UK), meglitinide (Aventis, Frankfurt, Germany), and P1075 (Leo Pharmaceuticals, Ballerup, Denmark). K_ATP channel modulators were dissolved in dimethyl sulfoxide/ethanol [50/50 (v/v)] and further diluted with the same solvent or with incubation buffer. In binding studies, the final solvent concentration in the assays was always below 0.3%, in electrophysiological studies  0.1%.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Whole-Cell Current Measurements. Fig. 1 shows whole-cell currents from HEK 293 cells transfected with SUR2B(Y1206S) and Kir6.2 or 6.1. After establishing the whole-cell patch-clamp configuration and dialyzing the cell with GDP (1 mM) + ATP (0.3 mM) in the presence of Mg²⁺, an outward current developed which was sensitive to inhibition by glibenclamide. No such current was seen in control cells. These observations and the reversal of the slowly developing currents in transfected cells at  100 mV (Fig. 1) identified the currents as typical K_ATP currents.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent inhibition by glibenclamide of whole cell currents from HEK 293 cells transfected with Kir6.1 (a) or Kir6.2 (b) together with SUR2B(Y1206S) or SUR2B. Experiments were performed as in Fig. 1, n = 3 to 12 per point; data for Kir6.1 + SUR2B are from Russ et al. (1999). Regression curves represent the fit of the logistic equation (eq. 2) to the data with the following parameters [SUR2B(Y1206S)/SUR2B wild-type]: a, pK = 8.56 ± 0.05/7.37 ± 0.05, nH = 1.03 ± 0.14/1.14 ± 0.16; b, pK = 7.88 ± 0.17/6.78 ± 0.11, nH = 0.73 ± 0.18/0.68 ± 0.15, respectively.

Affinity of SUR2B(Y1206S) for Glibenclamide and P1075 in Intact Cells. Fig. 3a shows a [³H]glibenclamide saturation binding experiment in intact HEK 293 cells expressing mutant SUR2B. A mean K_D value of 4.7 nM was obtained from four such experiments (for confidence intervals, see Table 1); B_max was 574 ± 39 fmol/mg protein. In homologous competition experiments, glibenclamide displaced its tritiated analog in a multiphasic manner (Fig. 4). It is known from earlier experiments that control HEK 293 cells (Russ et al., 1999) and HEK 293 cells transfected only with pcDNA3.1 vector lacking the insert (A. Piehl, C. Loffler-Walz, and A. Hambrock, unpublished observations) have endogenous glibenclamide binding components with K_D values around 300 nM and 16 µM. If we keep the value of 300 nM constant and analyze for three otherwise free floating components, a high-affinity component is obtained with 52 ± 2% of total binding and a K_i value of 5.4 nM. This K_i value is in excellent agreement with the result of the saturation binding experiments. Without keeping the K_D value of the first endogenous component fixed, similar parameters were obtained; however, the errors in the parameters were much larger because of the overlap of the binding sites (not shown). In heterologous competition experiments, the two carboxamido-benzoate analogs of the SUs (`glitinides'), AZ-DF 265 and meglitinide, inhibited [³H]glibenclamide binding in a triphasic manner (Fig. 4); for the specific component (i.e., binding to mutant SUR2B,  50% B_TOT) K_i values of 0.63 (0.60, 0.66) and 6.8 (6.2, 7.7) µM were obtained, respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   [3H]glibenclamide saturation binding experiments in HEK 293 cells expressing mutant SUR2B (Y1206S) (a), Kir6.2/SUR2B(Y1206S) (b), and membranes containing Kir6.2/SUR2B(Y1206S) in the presence of MgATP (1 mM) (c). Total binding (BTOT, ) and nonspecific binding (BNS, ) are shown; BNS was determined in the presence of P1075 (100 µM; see Experimental Procedures). Specific binding (BS), calculated as BTOT  BNS is represented by the dotted curves. The fit of eq. (1) in Experimental Procedures gave KD values of 6.3, 0.5, and 2.7 nM and Bmax values of 694, 628, and 400 fmol/mg protein in a, b, and c, respectively. For mean values from at least three experiments, see Tables 1 and 2.

View this table: [in this window] [in a new window]   TABLE 1 Binding experiments with [3H]GBC and [3H]P1075 in intact cells KD/Ki values are geometric means from n independent experiments; A denotes the amplitudes in percentage of specific binding. Experiments were performed as described in Figs. 3 to 5. Data for SUR2B wild-type are from Russ et al. (1999).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Inhibition of [3H]glibenclamide binding in HEK 293 cells expressing SUR2B(Y1206S) by glibenclamide, AZ-DF265, and meglitinide. The data, expressed as percentage of total binding (BTOT) are means from three experiments; [3H]glibenclamide was  3 nM. To account for [3H]glibenclamide binding to proteins in addition to SUR2B(Y1206S), a logistic equation with three components (eq. 2 under Experimental Procedures) was fitted to the data (see text) and gave the parameters (glibenclamide/AZ-DF 265/meglitinide) A1 (% BTOT), 52 ± 2/52/52; pIC50,1, 8.05 ± 0.04/5.97 ± 0.02/4.94 ± 0.03; A2 (% BTOT), 24 ± 3/24/24; pIC50,2, 6.51/4.9 ± 0.1/3.8 ± 0.1; A3 (% BTOT), 12 ± 5/12/0; pIC50,3, 4.2 ± 0.5; 4.4 ± 0.2/not determined (due to solubility problems). The broken curves represent binding to SUR2B(Y1206S) (high-affinity component).

Interaction of Glibenclamide and P1075 Sites. In HEK 293 cells transfected with mutant SUR2B, P1075 inhibited total [³H]glibenclamide binding to  50% (i.e., exactly the amount of glibenclamide binding to SUR; see above); no inhibition was found in nontransfected HEK 293 cells. Figure 5a shows the inhibition curve in transfected cells expressed as percentage of specific binding. The curve was biphasic with the high-affinity component making up about two thirds of the total amplitude and a having a K_i value of 2.6 nM; the low-affinity component was in the micromolar range. A similarly biphasic curve was found for the opener, levcromakalim (not illustrated). Coexpression with Kir6.2 left the high-affinity component of the [³H]glibenclamide-P1075 curve unchanged and shifted the low-affinity component to the left by a factor of 7, thus reducing the biphasic nature of the curve (Table 1 and Fig. 5c). Coexpression with Kir6.1 led to the disappearance of the low-affinity component, rendering the curve monophasic. The K_i value of 5.5 nM corresponded to the true affinity of P1075 to SUR2B(Y1206S) (Fig. 5b and Table 1).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Inhibition of [3H]glibenclamide binding by P1075 (left) and [3H]P1075 by glibenclamide in cells () and membranes (). a and d, SUR2B(Y1206S) alone. b and e, coexpression with Kir6.1 c and f, coexpression with Kir6.2. Data are expressed as percentage of specific binding (% BS); n = 3 to 6. The parameters listed in Table 1 and 2 were obtained from the fit of a two-component logistic function with Hill coefficients of 1 to the data. Experiments in membranes were conducted in the presence of MgATP (1 mM); [3H]glibenclamide concentration was 1.5 to 3.0 nM and [3H]P1075 was 2.7 to 3.0 nM.

Experiments in the Presence of Cytochalasin D. We have shown previously that disruption of the actin cytoskeleton abolished high affinity binding of [³H]glibenclamide binding to the vascular K_ATP channel in rat aortic rings (Löffler-Walz and Quast, 1998).

Binding Assays in Membranes. These experiments are summarized in Table 2. Nucleotides strongly affect glibenclamide and opener binding to SURs (Hambrock et al., 1998, 1999; Schwanstecher et al., 1998) and K_ATP channels (for review, see Ashcroft and Ashcroft, 1992). To facilitate comparison with the studies in intact cells, experiments were performed in the presence of 1 mM MgATP; at the end of the incubation period, 50 to 100 µM ADP was also present (Hambrock et al., 1999). Binding of [³H]glibenclamide to mutant SUR2B gave a K_D value of 3.4 nM, similar to that in cells. The [³H]glibenclamide-P1075 inhibition curve was again strongly biphasic, with the K_i value of the high-affinity component (5.6 nM) in perfect agreement with the result of the [³H]P1075 experiments in membranes (see below and Table 2). On order to examine whether or not the biphasic nature of this curve depended on the radioligand concentration, experiments were performed at [³H]glibenclamide concentrations ranging from 0.6 to 6 nM. No change in amplitudes and K_i values was observed (data not shown). Coexpression with Kir6.1 and 6.2 did not increase the affinity of glibenclamide for SUR in membranes (see Fig. 3c for Kir6.2/SUR2B(Y1206S); however, it increased the amplitude of the high-affinity component (see Fig. 5, b and c; Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Binding experiments with [3H]GBC and [3H]P1075 in membranes KD/Ki values are geometric means from n independent experiments; A denotes the amplitudes in percentage of specific binding. Experiments were performed as described in Fig. 5. Experiments on SUR2B are from Hambrock et al. (1998).

	Discussion

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Properties of SUR2B(Y1206S). In this study, we have investigated the impact of the substitution Y1206S in SUR2B on the interaction with sulfonylureas and openers and have assessed the effects of coexpression of mutant SUR2B with Kir6.1 and 6.2.

Coexpression of SUR2B(Y1206S) with Kir6.1 or 6.2. Upon coexpression of mutant SUR2B with the Kir6.x subtypes, functional K_ATP channels were formed. Glibenclamide inhibited these channels with approximately 15-fold higher potency than the corresponding wild-type channels; this fits reasonably well with the 7-fold difference in affinity of glibenclamide binding. Interestingly, the glibenclamide sensitivity depended on the Kir6.x subtype: under otherwise identical conditions, glibenclamide inhibited Kir6.1/SUR2B or Kir6.1/SUR2B(Y1206S) channels with 4-fold higher potency than those formed with Kir6.2. These results predict that the vascular K_ATP channel (Kir6.1/SUR2B) exhibits a higher sensitivity toward glibenclamide than that in nonvascular smooth muscle (Kir6.2/SUR2B). Furthermore and in contrast to Kir6.1, the glibenclamide sensitivity of the Kir6.2 containing channels was highly variable in intact cells but not in isolated patches, and the corresponding concentration-response curves showed a low Hill coefficient. This points to the modulation of glibenclamide sensitivity by an additional component that was poorly controlled in our experiments and that affected Kir6.2-containing channels more than those with Kir6.1. Possible candidates are phospholipids such as phosphatidylinositol-4,5-bisphosphate (PIP₂; Krauter et al., 2001) or nucleotides that might be incompletely clamped by dialysis of the cell with the pipette solution.

Relationship between Opener and Sulfonylurea Binding. A first result from this study is that in cells expressing SUR2B(Y1206S) alone, the stoichiometry of glibenclamide to P1075 sites is  1:1. This holds true also in membranes, if the maximum number of glibenclamide sites at 0 MgATP is compared with the P1075 sites at 1 mM MgATP. If both radioligands are measured in the presence of 1 mM MgATP, however, the ratio is 0.38 ± 0.03. The fact that MgATP reduces B_max but does not affect the affinity K_D value of glibenclamide rules out the possibility that SUR2B(Y1206S), expressed alone, exists predominantly as a monomer with a single binding site for glibenclamide, which is negatively allosterically linked to MgATP binding. In this case, MgATP should reduce only the affinity of glibenclamide binding. Instead, MgATP, at saturating concentrations (C. Löffler-Walz, A. Hambrock, and U. Quast, in preparation), induces a nonequivalence of the glibenclamide sites leaving about one third of them unaffected and shifting the other ones to very low affinity or rendering them inaccessible. Therefore, the glibenclamide sites are coupled. Assuming one site per monomer, SUR2B(Y1206S) expressed alone must then form homomultimers. Because K_ATP channels are octamers [(Kir/SUR)₄; Clement et al., 1997; Shyng and Nichols, 1997] and NBF1 of SUR1, expressed alone, forms tetramers (Mikhailov and Ashcroft, 2000), one may speculate that SUR alone also forms tetramers.