The mutation Y1206S increases the affinity of the sulphonylurea receptor SUR2A for glibenclamide and enhances the effects of coexpression with Kir6.2.

1. ATP-sensitive K(+) channels (K(ATP) channels) are tetradimeric complexes of inwardly rectifying K(+) channels (Kir6.x) and sulphonylurea receptors (SURs). The SURs SUR2A (cardiac) and SUR2B (smooth muscle) differ only in the last 42 amino acids. In SUR2B, the mutation Y1206S, located at intracellular loop 8, increases the affinity for glibenclamide (GBC) about 10-fold. Here, we examined whether the mutation Y1206S in SUR2A had effects similar to those in SUR2B.2. GBC bound to SUR2A with K(D)=20 nM; the mutation increased affinity approximately 5 x. 3. In cells, coexpression of SUR2A with Kir6.2 increased the affinity for GBC approximately 3 x; with the mutant, the increase was 9 x. 4. The mutation did not affect the affinity of SUR2A for openers; coexpression with Kir6.2 reduced opener affinity of wild-type and mutant SUR2A by about 2 x. 5. The negative allosteric interaction between the opener, P1075, and GBC at wild-type and mutant SUR2A was markedly affected by the presence of MgATP and by coexpression with Kir6.2. 6. In inside-out patches, GBC inhibited the wild-type Kir6.2/SUR2A and 2B channels with IC(50) values of 27 nM; the mutation shifted the IC(50) values to approximately 1 nM. 7. The data show that the mutation Y1206S increased the affinity of SUR2A for GBC and modulated the effects of coexpression. Overall, the changes were similar to those observed with SUR2B(Y1206S), suggesting that the differences in the last 42 carboxy-terminal amino acids of SUR2A and 2B are of limited influence on the binding of GBC and P1075 to the SUR2 isoforms.

Interaction of a novel dihydropyridine K+ channel opener, A-312110, with recombinant sulphonylurea receptors and KATP channels: comparison with the cyanoguanidine P1075.

1. ATP-sensitive K(+) channels (K(ATP) channels) are composed of pore-forming subunits (Kir6.x) and of regulatory subunits, the sulphonylurea receptors (SURx). Synthetic openers of K(ATP) channels form a chemically heterogeneous class of compounds that are of interest in several therapeutic areas. We have investigated the interaction of a novel dihydropyridine opener, A-312110 ((9R)-9-(4-fluoro-3-iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thieno [2,3-e]pyridin-8(7H)-one-1,1-dioxide), with SURs and Kir6/SUR channels in comparison to the cyanoguanidine opener P1075. 2. In the presence of 1 mM MgATP, A-312110 bound to SUR2A (the SUR in cardiac and skeletal muscle) and to SUR2B (smooth muscle) with K(i) values of 14 and 18 nM; the corresponding values for P1075 were 16 and 9 nM, respectively. Decreasing the MgATP concentration reduced the affinity of A312110 binding to SUR2A significantly more than that to SUR2B; for P1075, the converse was true. At SUR1 (pancreatic beta-cell), both openers showed little binding up to 100 microM. 3. In the presence of MgATP, both openers inhibited [(3)H]glibenclamide binding to the SUR2 subtypes in a biphasic manner. In the absence of MgATP, the high-affinity component of the inhibition curves was absent. 4. In inside-out patches, the two openers activated the Kir6.2/SUR2A and Kir6.2/SUR2B channels with similar potency (approximately 50 nm). Both were almost 2 x more efficacious in opening the Kir6.2/SUR2B than the Kir6.2/SUR2A channel. 5. The results show that the novel dihydropyridine A-312110 is a potent K(ATP) channel opener with binding and channel-opening properties similar to those of P1075.

Binding and effect of K ATP channel openers in the absence of Mg2+.

1 Openers of ATP-sensitive K(+) channels (K(ATP) channels) are thought to act by enhancing the ATPase activity of sulphonylurea receptors (SURs), the regulatory channel subunits. At higher concentrations, some openers activate K(ATP) channels also in the absence of MgATP. Here, we describe binding and effect of structurally diverse openers in the absence of Mg(2+) and presence of EDTA. 2 Binding of openers to SUR2B was measured using a mutant with high affinity for [(3)H]glibenclamide ([(3)H]GBC). In the absence of Mg(2+), 'typical' openers (benzopyrans, cyanoguanidines and aprikalim) inhibited [(3)H]GBC binding with K(i) values approximately 200 x higher than in the presence of MgATP. Minoxidil sulphate and nicorandil were inactive, whereas binding of diazoxide was unaffected by MgATP. 3 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 EC(50)=2000/67nM and E(max)=32/134%. In the absence of Mg(2+), 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. 4 The rank order of efficacy of the openers was P1075>rilmakalim approximately nicorandil>diazoxide>minoxidil sulphate. 5 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.

Mg2+ sensitizes KATP channels to inhibition by DIDS: dependence on the sulphonylurea receptor subunit.

1. ATP-sensitive potassium channels (K(ATP) channels) consist of pore-forming Kir6.x subunits and of sulphonylurea receptors (SURs). In the absence of Mg(2+), the stilbene disulphonate, DIDS, irreversibly inhibits K(ATP) channels by binding to the Kir subunit. Here, the effects of Mg(2+) on the interaction of DIDS with recombinant K(ATP) channels were studied in electrophysiological and [(3)H]-glibenclamide binding experiments. 2. In inside-out macropatches, Mg(2+) (0.7 mM) increased the sensitivity of K(ATP) channels towards DIDS up to 70 fold (IC(50)=2.7 micro M for Kir6.2/SUR2B). Inhibition of current at DIDS concentrations > or =10 micro M was irreversible. 3. Mg(2+) sensitized the truncated Kir6.2Delta26 channel towards inhibition by DIDS only upon coexpression with a SUR subunit (SUR2B). The effect of Mg(2+) did not require the presence of nucleotides. 4. [(3)H]-glibenclamide binding to SUR2B(Y1206S), a mutant with improved affinity for glibenclamide, was inhibited by DIDS. The potency of inhibition was increased by Mg(2+) and by coexpression with Kir6.2. 5. In the presence of Mg(2+), DIDS inhibited binding of [(3)H]-glibenclamide to Kir6.2/SUR2B(Y1206S) with IC(50)=7.9 micro M by a non-competitive mechanism. Inhibition was fully reversible. 6. It is concluded that the binding site of DIDS on SUR that is sensed by glibenclamide does not mediate channel inhibition. Instead, Mg(2+) binding to SUR may allosterically increase the accessibility and/or reactivity of the DIDS site on Kir6.2. The fact that the Mg(2+) effect does not require the presence of nucleotides underlines the importance of this ion in modulating the properties of the K(ATP) channel.

The stereoenantiomers of a pinacidil analog open or close cloned ATP-sensitive K+ channels.

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) approximately 50 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.

Glibenclamide binding to sulphonylurea receptor subtypes: dependence on adenine nucleotides.

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 affinity, 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 approximately 60%, to SUR2A(Y1206S) by 21%). Inhibition curves for the SUR2(Y1206S) isoforms were monophasic with IC(50) values of 5-10 microM; the curve for SUR1 was biphasic (IC(50) values 4.7 and 1300 microM). 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 efficacy of ATP in inhibiting glibenclamide binding; this was particularly marked for Kir6.2/SUR1. 6: The data show (a) that the inhibitory effects of ATP and ADP on glibenclamide binding differ from one another, (b) that they depend on the SUR subtype, and (c) that they are weakened by coexpression with Kir6.2.

Interaction of K(ATP) channel modulators with sulfonylurea receptor SUR2B: implication for tetramer formation and allosteric coupling of subunits.

Sulfonylurea receptors (SURs) are subunits of ATP-sensitive K(+) channels (K(ATP) channels); they mediate the channel-closing effect of sulfonylureas such as glibenclamide and the channel-activating 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 )= 4 versus 22 nM, respectively) but otherwise exhibiting similar pharmacological properties. In the absence of MgATP, [(3)H]glibenclamide binding to both SURs was homogenous. MgATP inhibited [(3)H]glibenclamide binding to both SURs to 25% by reducing the apparent number of glibenclamide binding sites, leaving the affinity unchanged. In the absence of MgATP, P1075 inhibited [(3)H]glibenclamide binding in a monophasic manner with K(i) approximately 1 microM. In the presence of MgATP (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 (approximately 1 microM). 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.