title: Characterization of binding of the ATP-sensitive potassium channel ligand, [3H]glyburide, to neuronal and muscle preparations.
abstract: Binding of the hypoglycemic sulfonylurea, [3H]glyburide, to crude membrane fractions from brain, heart and smooth (intestinal) muscle was saturable, linear with protein concentration and reversible. Saturation analysis revealed high affinity sites (KH values, 7 x 10(-11) M, 5 x 10(-11) M and 6 x 10(-11) M), with Bmax-H values 209, 36 and 23 fmol/mg protein in the brain, heart and smooth muscle, respectively. High affinity [3H]glyburide binding was pharmacologically specific, insensitive to a variety of receptor-active ligands, but sensitive to a series of sulfonylureas, and good, essentially 1:1, correlations were obtained between binding affinities and literature-derived pharmacologic activities. The K+ channel activators, cromakalim, nicorandil, pinacidil and minoxidil were not effective as inhibitors of [3H]glyburide binding. However, diazoxide was a modestly effective inhibitor. Putative low affinity sites (KL values, 3 x 10(-7) M, 1 x 10(-7) M and 2 x 10(-9) M) with Bmax-L values 4956, 336 and 53 fmol/mg protein in brain, heart and smooth muscle, respectively, were identified. Their significance remains to be established. Except for ATP gamma S, the ability of nucleotide triphosphates to inhibit high affinity [3H] glyburide binding was dependent on the presence of Mg++. ADP, in the presence of Mg++, inhibited binding with an IC50 value of 6.3 x 10(-4) M. Nucleotide monophosphates did not inhibit [3H] glyburide binding in the presence or absence of Mg++, whereas in the presence of Mg++, nucleotide triphosphates were equally potent inhibitors of binding. The rank order potency for nucleotide diphosphate inhibition of binding, in the presence of Mg++, is ADP greater than GDP greater than IDP = UDP. In the absence of Mg++, [3H]glyburide binding shows a biphasic response to ADP, and the inhibition of binding by ADP was prevented by ATP. It is suggested that this biphasic response is the result of a second nucleotide binding site.
authors.affiliation: Department of Biochemical Pharmacology, School of Pharmacy, State University of New York, Buffalo.
authors.forename: M
authors.initials: M
authors.lastname: Gopalakrishnan
authors.forename: D E
authors.initials: DE
authors.lastname: Johnson
authors.forename: R A
authors.initials: RA
authors.lastname: Janis
authors.forename: D J
authors.initials: DJ
authors.lastname: Triggle
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D005905:Glyburide / Q000378:metabolism*; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008566:Membranes / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008954:Models, Biological; D009132:Muscles / Q000378:metabolism*; D009711:Nucleotides / Q000494:pharmacology; D015221:Potassium Channels / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D013696:Temperature
chemical_list: D008670:Metals; D009711:Nucleotides; D015221:Potassium Channels; D000255:Adenosine Triphosphate; D005905:Glyburide