title: Substitution of the Walker A lysine by arginine in the nucleotide-binding domains of sulphonylurea receptor SUR2B: effects on ligand binding and channel activity.
abstract: Sulphonylurea receptors (SURs) serve as regulatory subunits of ATP-sensitive K(+) channels. SURs are members of the ATP-binding cassette (ABC) protein superfamily and contain two conserved nucleotide-binding domains (NBDs) which bind and hydrolyse MgATP; in addition, they carry the binding sites for the sulphonylureas like glibenclamide (GBC) which close the channel and for the K(ATP) channel openers such as P1075. Here we have exchanged the conserved Lys in the Walker A motif by Arg in both NBDs of SUR2B, the regulatory subunit of the vascular K(ATP) channel. Then the effect of the mutation on the ATPase-dependent binding of GBC and P1075 to SUR2B and on the activity of the recombinant vascular (Kir6.1/SUR2B) channel was assessed. Surprisingly, in the absence of MgATP, the mutation weakened binding of P1075 and the extent of allosteric inhibition of GBC binding by P1075. The mutation abolished most, but not all, of the MgATP effects on the binding of GBC and P1075 and prevented nucleotide-induced activation of the channel which relies on SUR reaching the posthydrolytic (MgADP-bound) state; the mutant channel was, however, opened by P1075 at higher concentrations. The data provide evidence that mutant SUR2B binds MgATP but that the posthydrolytic state is insufficiently populated. This suggests that the mutation locks SUR2B in an MgATP-binding prehydrolytic-like state; binding of P1075 may induce a posthydrolytic-like conformation to open the channel.
authors.affiliation: Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, Eberhard-Karls-University Hospitals and Clinics, University of Tübingen, Wilhelmstr. 56, 72074, Tübingen, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Amann
authors.forename: Sophie
authors.initials: S
authors.lastname: Schell
authors.forename: Petra
authors.initials: P
authors.lastname: Kühner
authors.forename: Marcus
authors.initials: M
authors.lastname: Winkler
authors.forename: Mathias
authors.initials: M
authors.lastname: Schwanstecher
authors.forename: Ulrich
authors.initials: U
authors.lastname: Russ
authors.forename: Ulrich
authors.initials: U
authors.lastname: Quast
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D005905:Glyburide / Q000378:metabolism; D006146:Guanidines / Q000378:metabolism; D006801:Humans; D007004:Hypoglycemic Agents / Q000378:metabolism; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D051379:Mice; D009154:Mutation; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011725:Pyridines / Q000378:metabolism; D051381:Rats; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D064233:Sulfonylurea Receptors
chemical_list: D018528:ATP-Binding Cassette Transporters; C577511:Abcc9 protein, mouse; D006146:Guanidines; D007004:Hypoglycemic Agents; D008024:Ligands; D024661:Potassium Channels, Inwardly Rectifying; D011725:Pyridines; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; C073896:N-cyano-N'-(1,1-dimethylpropyl)-N''-(3-pyridinyl)guanidine; D000255:Adenosine Triphosphate; D001120:Arginine; D008239:Lysine; D005905:Glyburide