Article date: October 2002
By: Ljiljana Gojkovic‐Bukarica, Annette Hambrock, Cornelia Löffler‐Walz, Ulrich Quast, Ulrich Russ in Volume 137, Issue 4, pages 429-440
ATP‐sensitive potassium channels (KATP channels) consist of pore‐forming Kir6.x subunits and of sulphonylurea receptors (SURs). In the absence of Mg2+, the stilbene disulphonate, DIDS, irreversibly inhibits KATP channels by binding to the Kir subunit. Here, the effects of Mg2+ on the interaction of DIDS with recombinant KATP channels were studied in electrophysiological and [3H]‐glibenclamide binding experiments.
In inside‐out macropatches, Mg2+ (0.7 mM) increased the sensitivity of KATP channels towards DIDS up to 70 fold (IC50=2.7 μM for Kir6.2/SUR2B). Inhibition of current at DIDS concentrations 10 μM was irreversible.
Mg2+ sensitized the truncated Kir6.2Δ26 channel towards inhibition by DIDS only upon coexpression with a SUR subunit (SUR2B). The effect of Mg2+ did not require the presence of nucleotides.
[3H]‐glibenclamide binding to SUR2B(Y1206S), a mutant with improved affinity for glibenclamide, was inhibited by DIDS. The potency of inhibition was increased by Mg2+ and by coexpression with Kir6.2.
In the presence of Mg2+, DIDS inhibited binding of [3H]‐glibenclamide to Kir6.2/SUR2B(Y1206S) with IC50=7.9 μM by a non‐competitive mechanism. Inhibition was fully reversible.
It is concluded that the binding site of DIDS on SUR that is sensed by glibenclamide does not mediate channel inhibition. Instead, Mg2+ binding to SUR may allosterically increase the accessibility and/or reactivity of the DIDS site on Kir6.2. The fact that the Mg2+ effect does not require the presence of nucleotides underlines the importance of this ion in modulating the properties of the KATP channel.
ATP‐sensitive potassium channels (KATP channels) consist of pore‐forming Kir6.x subunits and of sulphonylurea receptors (SURs). In the absence of Mg2+, the stilbene disulphonate, DIDS, irreversibly inhibits KATP channels by binding to the Kir subunit. Here, the effects of Mg2+ on the interaction of DIDS with recombinant KATP channels were studied in electrophysiological and [3H]‐glibenclamide binding experiments.
In inside‐out macropatches, Mg2+ (0.7 mM) increased the sensitivity of KATP channels towards DIDS up to 70 fold (IC50=2.7 μM for Kir6.2/SUR2B). Inhibition of current at DIDS concentrations 10 μM was irreversible.
Mg2+ sensitized the truncated Kir6.2Δ26 channel towards inhibition by DIDS only upon coexpression with a SUR subunit (SUR2B). The effect of Mg2+ did not require the presence of nucleotides.
[3H]‐glibenclamide binding to SUR2B(Y1206S), a mutant with improved affinity for glibenclamide, was inhibited by DIDS. The potency of inhibition was increased by Mg2+ and by coexpression with Kir6.2.
In the presence of Mg2+, DIDS inhibited binding of [3H]‐glibenclamide to Kir6.2/SUR2B(Y1206S) with IC50=7.9 μM by a non‐competitive mechanism. Inhibition was fully reversible.
It is concluded that the binding site of DIDS on SUR that is sensed by glibenclamide does not mediate channel inhibition. Instead, Mg2+ binding to SUR may allosterically increase the accessibility and/or reactivity of the DIDS site on Kir6.2. The fact that the Mg2+ effect does not require the presence of nucleotides underlines the importance of this ion in modulating the properties of the KATP channel.
British Journal of Pharmacology (2002) 137, 429–440. doi:10.1038/sj.bjp.0704905
DOI: 10.1038/sj.bjp.0704905
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