Characterization of binding of the ATP-sensitive potassium channel ligand, [³H]glyburide, to neuronal and muscle preparations

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 (K(H)) values, 7 x 10^-11 M, 5 x 10^-11 M and 6 x 10^-11 M), with B(max-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 (K(L) values, 3 x 10^-7 M, 1 x 10^-7 M and 2 x 10^-9 M) with B(max-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 ATPγS, 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 > GDP > 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.