title: Mg2+ sensitizes KATP channels to inhibition by DIDS: dependence on the sulphonylurea receptor subunit.
abstract: 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.
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology*; D018528:ATP-Binding Cassette Transporters; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008274:Magnesium / Q000494:pharmacology*; D015221:Potassium Channels / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying / Q000037:antagonists & inhibitors* / Q000502:physiology; D021122:Protein Subunits / Q000037:antagonists & inhibitors* / Q000502:physiology*; D011955:Receptors, Drug / Q000037:antagonists & inhibitors* / Q000502:physiology*; D064233:Sulfonylurea Receptors
chemical_list: D018528:ATP-Binding Cassette Transporters; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D008274:Magnesium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid