Mg2+ and ATP dependence of KATP channel modulator binding to the recombinant sulphonylurea receptor,SUR2B |
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Authors: | Annette Hambrock Cornelia L?ffler-Walz Yoshihisa Kurachi Ulrich Quast |
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Institution: | 1.Department of Pharmacology, University of Tübingen, Wilhelmstr. 56, D-72074 Tübingen, Germany;2.Department of Pharmacology II, Faculty of Medicine, Osaka University, Yamada-oka 2-2, Suita, Osaka 565, Japan |
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Abstract: | - The binding of modulators of the ATP-sensitive K+ channel (KATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular KATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37°C using the tritiated KATP channel opener, [3H]-P1075.
- Binding of [3H]-P1075 required the presence of Mg2+ and ATP. MgATP activated binding with EC50 values of 10 and 3 μM at free Mg2+ concentrations of 3 μM and 1 mM, respectively. At 1 mM Mg2+, binding was lower than at 3 μM Mg2+.
- [3H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg2+ concentrations of 3 μM and 1 mM, gave KD values of 1.8 and 3.4 nM and BMAX values of 876 and 698 fmol mg−1, respectively.
- In competition experiments, openers inhibited [3H]-P1075 binding with potencies similar to those determined in rings of rat aorta.
- Glibenclamide inhibited [3H]-P1075 binding with Ki values of 0.35 and 2.4 μM at 3 μM and 1 mM free Mg2+, respectively. Glibenclamide enhanced the dissociation of the [3H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas.
- It is concluded that an MgATP site on SUR2B with μM affinity must be occupied to allow opener binding whereas Mg2+ concentrations ⩾10 μM decrease the affinities for openers and glibenclamide. The properties of the [3H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.
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Keywords: | Vascular sulphonylurea receptor SUR2B KATP channel openers [ 3H] -P1075 binding levcromakalim minoxidil sulphate aprikalim diazoxide glibenclamide AZ-DF 265 MgATP dependence |
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