1 The binding of modulators of the ATP-sensitive K + channel (K ATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular K ATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 378C using the tritiated K ATP channel opener, .4 mM at 3 mM and 1 mM free Mg 2+ , respectively. Glibenclamide enhanced the dissociation of the [ 3 H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas. 6 It is concluded that an MgATP site on SUR2B with mM a nity must be occupied to allow opener binding whereas Mg 2+ concentrations 510 mM decrease the a nities for openers and glibenclamide. The properties of the [ 3 H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

ATP-dependent K(+) channels are composed of pore-forming subunits of the Kir6.x family and of sulfonylurea receptors (SURs). SUR1, expressed in pancreatic beta-cells, has a higher affinity for sulfonylureas, such as glibenclamide, than SUR2B, expressed in smooth muscle. This difference is mainly caused by serine 1237 in SUR1 corresponding to tyrosine 1206 in SUR2B. To increase the affinity of SUR2B for glibenclamide, the mutant SUR2B(Y1206S) was constructed. In whole-cell patch-clamp experiments, glibenclamide inhibited the channel formed by coexpression of mutant SUR2B with Kir6.1 or 6.2 in human embryonic kidney cells with IC(50) values of 2.7 and 13 nM, respectively (wild-type, 43 and 167 nM). In intact cells, [(3)H]glibenclamide bound to mutant SUR2B with a K(D) value of 4.7 nM (wild-type, 32 nM); coexpression with Kir6.1 or 6.2 increased affinity by 4- and 8-fold, respectively. Binding of the opener [(3)H]P1075 to SUR2B(Y1206S) was the same as to wild-type and was unaffected by coexpression. In cells, the ratio of glibenclamide:P1075 sites was approximately 1:1; in membranes, it varied with the MgATP concentration. Heterologous competition curves were generally biphasic; the shape of the curve depended on the Kir-subtype. The effects of coexpression were weakened or abolished when binding assays were conducted in membranes. It is concluded that the mutation Y1206S increases the affinity of SUR2B for and the channel sensitivity toward glibenclamide by 7- to 15-fold. The interaction of glibenclamide (but not opener) with mutant SUR2B is modified by coexpression with Kir6.x in a manner depending on the Kir subtype and on the integrity of the cell.

1 ATP-sensitive K + channels are composed of pore-forming subunits Kir6.2 and of sulphonylurea receptors (SURs); the latter are the target of the hypoglycaemic sulphonylureas like glibenclamide. Here, we report on the negative allosteric modulation by MgATP and MgADP of glibenclamide binding to SUR1 and to SUR2 mutants with high glibenclamide anity, SUR2A(Y1206S) and SUR2B(Y1206S). 2 ATP, in the presence of an ATP-regenerating system to oppose hydrolysis during incubation, inhibited glibenclamide binding to SUR1 and SUR2B(Y1206S) by *60%, to SUR2A(Y1206S) by 21%). Inhibition curves for the SUR2(Y1206S) isoforms were monophasic with IC 50 values of 5 ± 10 mM; the curve for SUR1 was biphasic (IC 50 values 4.7 and 1300 mM). 3 Glibenclamide inhibition curves for ADP, performed in the presence of an ATP-consuming system to oppose ATP formation from ADP, were generally shifted rightwards and showed positive cooperativity, in particular with the SUR2(Y1206S) isoforms. 4 In the absence of the coupled enzyme systems, inhibition curves of MgATP or MgADP were generally shifted leftwards. This indicated synergy of MgATP and MgATP in acting together. 5 Coexpression of SUR1 and SUR2B(Y1206S) with Kir6.2 reduced both potency and ecacy of ATP in inhibiting glibenclamide binding; this was particularly marked for Kir6.2/SUR1. 6 The data show (a) that the inhibitory eects of ATP and ADP on glibenclamide binding dier from one another, (b) that they depend on the SUR subtype, and (c) that they are weakened by coexpression with Kir6.2.

ATP-sensitive K ϩ channels are closed by the hypoglycemic sulfonylureas like glibenclamide (GBC) and activated by a class of vasorelaxant compounds, the K ϩ channel openers. These channels are octamers of Kir6.x and sulfonylurea receptor (SUR) subunits with 4:4 stoichiometry. The properties of the opener-sensitive K ϩ channel in the vasculature are well matched by the SUR2B/Kir6.1 channel; however, the GBC sensitivity of the recombinant channel is unknown. In binding experiments we have determined the affinity of GBC for SUR2B and the SUR2B/Kir6.1 channel and compared the results with the channel blocking potency of GBC. All experiments were performed in whole transfected human embryonic kidney cells at 37°C. The equilibrium dissociation constants (K D ) of GBC binding to SUR2B and to the SUR2B/Kir6.1 complex were determined to be 32 and 6 nM, respectively; the K D value of the opener P1075 (N-cyano-NЈ-(1,1-dimethylpropyl)-NЈЈ-3-pyridylguanidine) (Ϸ5 nM) was, however, not affected by cotransfection. In whole cell voltage-clamp experiments, GBC inhibited the SUR2B/Kir6.1 channel with IC 50 Ϸ 43 nM. The data show that, in the intact cell: 1) SUR2B, previously considered to be a low-affinity SUR, has a rather high affinity for GBC; 2) coexpression with the inward rectifier Kir6.1 increases the affinity of SUR2B for GBC; 3) the recombinant channel exhibits the same GBC affinity as the opener-sensitive K ϩ channel in vascular tissue; and 4) the K D value of GBC binding to the octameric channel is 7 times lower than the IC 50 value for channel inhibition. The latter finding suggests that occupation of all four GBC sites per channel is required for channel closure.

Sulfonylurea receptors (SURs) are subunits of ATP-sensitive K ϩ channels (K ATP channels); they mediate the channel-closing effect of sulfonylureas such as glibenclamide and the channelactivating effect of K ATP channel openers such as the pinacidil analog P1075. We investigated the inhibition by MgATP and P1075 of glibenclamide binding to SUR2B, the SUR subtype in smooth muscle. To increase specific binding, experiments were also performed using SUR2B(Y1206S), a mutant with higher affinity for glibenclamide than for the wild-type (K D ϭ 4 versus 22 nM, respectively) but otherwise exhibiting similar pharmacological properties. (1 mM), inhibition was biphasic with one K i value resembling the true affinity of P1075 for SUR2B (2-6 nM) and the other resembling K i in the absence of MgATP (Ϸ1 M). The data show that (1) MgATP induces heterogeneity in the glibenclamide sites; (2) the high-affinity glibenclamide sites remaining with MgATP are linked to two classes of P1075 sites; and (3) P1075 interacts specifically with SUR2B also in the absence of MgATP. The data are discussed with the assumption that SUR2B, expressed alone, forms tetramers; that MgATP induces allosteric interactions between the subunits; and that mixed SUR2B-glibenclamide-P1075 complexes can exist at equilibrium.

1 The binding of [ 3 H]-P1075, a potent opener of adenosine-5'-triphosphate-(ATP)-sensitive K + channels, was studied in a crude heart membrane preparation of the rat, at 378C.

Openers of ATP‐sensitive K+ channels (KATP channels) are thought to act by enhancing the ATPase activity of sulphonylurea receptors (SURs), the regulatory channel subunits. At higher concentrations, some openers activate KATP channels also in the absence of MgATP. Here, we describe binding and effect of structurally diverse openers in the absence of Mg2+ and presence of EDTA. Binding of openers to SUR2B was measured using a mutant with high affinity for [3H]glibenclamide ([3H]GBC). In the absence of Mg2+, ‘typical' openers (benzopyrans, cyanoguanidines and aprikalim) inhibited [3H]GBC binding with Ki values ∼200 × higher than in the presence of MgATP. Minoxidil sulphate and nicorandil were inactive, whereas binding of diazoxide was unaffected by MgATP. In the absence/presence of MgATP, N‐cyano‐N′‐(1,1‐dimethylpropyl)‐N″‐3‐pyridylguanidine (P1075) activated the Kir6.2/SUR2B channel in inside–out patches with EC50=2000/67nM and Emax=32/134%. In the absence of Mg2+, responses were variable with only a small part of the variability being explained by a decrease in channel responsiveness with time after patch excision and to differences in the ATP sensitivity between patches. The rank order of efficacy of the openers was P1075>rilmakalim ∼nicorandil>diazoxide>minoxidil sulphate. The data show that structurally diverse openers are able to bind to, and to activate the Kir6.2/SUR2B channel by a pathway independent of ATP hydrolysis. These effects are observed at concentrations used to define the biochemical mechanism of the openers in the presence of MgATP and allow the openers to be classified into ‘typical’ and ‘atypical’ KCOs with diazoxide standing apart. British Journal of Pharmacology (2003) 139, 368–380. doi:

ATP-dependent K؉ channels (K ATP channels) are composed of pore-forming subunits Kir6.x and sulfonylurea receptors (SURs). Cyanoguanidines such as pinacidil and P1075 bind to SUR and enhance MgATP binding to and hydrolysis by SUR, thereby opening K ATP channels. In the vasculature, openers of K ATP channels produce vasorelaxation. Some novel cyanoguanidines, however, selectively reverse opener-induced vasorelaxation, suggesting that they might be K ATP channel blockers. Here we have analyzed the interaction of the enantiomers of a racemic cyanoguanidine blocker, PNU-94750, with Kir6.2/SUR channels. In patch clamp experiments, the R-enantiomer (PNU-96293) inhibited Kir6.2/SUR2 channels (IC 50 ϳ50 nM in the whole cell configuration), whereas the S-enantiomer (PNU-96179) was a weak opener. Radioligand binding studies showed that the R-enantiomer was more potent and that it was negatively allosterically coupled to MgATP binding, whereas the S-enantiomer was weaker and positively coupled. Binding experiments also suggested that both enantiomers bound to the P1075 site of SUR. This is the first report to show that the enantiomers of a K ATP channel modulator affect channel activity and coupling to MgATP binding in opposite directions and that these opposite effects are apparently mediated by binding to the same (opener) site of SUR.