Coexpression with the Inward Rectifier K+ Channel Kir6.1 Increases the Affinity of the Vascular Sulfonylurea Receptor SUR2B for Glibenclamide
- Ulrich Russ1,
- Annette Hambrock1,
- Ferruh Artunc1,
- Cornelia Löffler-Walz1,
- Yoshiyuki Horio2,
- Yoshihisa Kurachi2 and
- Ulrich Quast1
- 1Department of Pharmacology, University of Tuebingen, Tuebingen, Germany (U.R., A.H., F.A., C.L.-W., U.Q.); and 2Department of Pharmacology II, Faculty of Medicine, Osaka University, Osaka, Japan (Y.H., Y.K.)
Abstract
ATP-sensitive K+ channels are closed by the hypoglycemic sulfonylureas like glibenclamide (GBC) and activated by a class of vasorelaxant compounds, the K+ 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+ 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 (KD) of GBC binding to SUR2B and to the SUR2B/Kir6.1 complex were determined to be 32 and 6 nM, respectively; the KD value of the opener P1075 (N-cyano-N′-(1,1-dimethylpropyl)-N′′-3-pyridylguanidine) (≈5 nM) was, however, not affected by cotransfection. In whole cell voltage-clamp experiments, GBC inhibited the SUR2B/Kir6.1 channel with IC50 ≈ 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+ channel in vascular tissue; and 4) theKD value of GBC binding to the octameric channel is 7 times lower than the IC50 value for channel inhibition. The latter finding suggests that occupation of all four GBC sites per channel is required for channel closure.
Footnotes
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Send reprint requests to: Dr. Ulrich Quast, Dept. of Pharmacology, University of Tübingen, Wilhemstr. 56, D-72074 Tübingen, Germany. E-mail: ulrich.quast{at}uni-tuebingen.de
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This study was supported by the Deutsche Forschungsgemeinschaft (Qu 100/2–2, U.Q. and U.R.) and by a grant from the Federal Ministry of Education, Science, Research and Technology, and the Interdisciplinary Center for Clinical Research (IZKF) Tübingen Fö.(01KS 9602, F.A.).
- Abbreviations:
- BTOT
- total binding
- GBC
- glibenclamide
- GFP
- green fluorescent protein
- HEK cells
- human embryonic kidney cells
- KATP channel
- ATP-sensitive K+ channel
- KD
- equilibrium dissociation constant of the radioligand
- P1075
- N-cyano-N′-(1,1-dimethylpropyl)-N′′-3-pyridylguanidine)
- PSS
- physiological salt solution
- SUR
- sulfonylurea receptor
- KNDP
- nucleoside diphosphate-dependent K+channel
- IK, NDP
- nucleoside diphosphate-activated K+ current
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- Received March 24, 1999.
- Accepted August 9, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
