Nucleotide Regulation and Characteristics of Potassium Channel Opener Binding to Skeletal Muscle Membranes

Abstract

[³H]P1075 binding to membrane preparations of rabbit skeletal muscle were observed in the presence of nucleotide triphosphates or diphosphates but not AMP, cAMP, adenosine, tripolyphosphate, or pyrophosphate. Nonhydrolyzable or poorly hydrolyzable ATP analogs inhibited MgATP-supported binding. The EC₅₀ value for MgATP-supported binding (0.4 mm) was decreased ∼10-fold in the presence of an ATP-regenerating system, and significant metabolism by membrane nucleotidases was confirmed by high performance liquid chromatographic analysis. [³H]P1075 bound to skeletal muscle with aK_d value of 37 ± 3 nm and a B_max value of 280 ± 14 fmol/mg of protein. [³H]P1075 binding to subcellular fractions was highest in membranes enriched in T tubules. Specific binding was reversible, trypsin-sensitive, maximal at pH 8, and stereoselective for the (3S,4R)-enantiomer of cromakalim. Potassium channel openers exhibited a rank order of potency of P1075 > pinacidil > levcromakalim = BMS-180448 > nicorandil > diazoxide = BRL 38226. Fluorescein analogs (ethyleosin, phloxine B, and rose bengal) were relatively potent inhibitors of binding (K_i = 200–300 nm). The potassium channel openers cromakalim and BMS-180448 were competitive inhibitors of [³H]P1075 binding. In contrast, rose bengal and the ATP-regulated potassium channel antagonist glyburide increased the rate of [³H]P1075 dissociation in a manner consistent with noncompetitive interaction.