Allosteric Interaction of Semotiadil Fumarate, a Novel Benzothiazine, with 1,4-Dihydropyridines, Phenylalkylamines, and 1,5-Benzothiazepines at the Ca2+-Channel Antagonist Binding Sites in Canine Skeletal Muscle Membranes

Abstract

We assessed the binding characteristics of a benzothiazine Ca(2+)-channel antagonist, semotiadil, in canine skeletal muscle membranes. Semotiadil inhibited binding of (+)-[3H]PN 200-110 (maximum inhibition 80%), and almost completely inhibited binding of both (-)-[3H]desmethoxyverapamil and D-cis-[3H]diltiazem to their specific binding sites with an IC50 value of 0.2-2 microM and a Hill slope of 0.6-0.9. Saturation isotherm and dissociation kinetic studies suggest that semotiadil acts as a noncompetitive inhibitor at the 1,4-dihydropyridine, phenylalkylamine, and benzothiazepine (BTZ) recognition sites in the L-type Ca2+ channel: (a) Scatchard analysis showed that semotiadil decreased maximum binding (Bmax) of the three classes of Ca2+ channel antagonists, while causing a slight increase in the equilibrium dissociation constant (Kd) in the case of (+)-[3H]PN 200-110 binding or no significant change in Kd values for binding of (-)-[3H]desmethoxyverapamil and D-cis-[3H]diltiazem to their specific binding sites; and (b) dissociation kinetics of the (+)-[3H]PN 200-110 and D-cis-[3H]diltiazem bindings were accelerated by semotiadil. These results suggest that semotiadil has a strong negative allosteric interaction with three classes of Ca2+ channel antagonists, including 1,4-dihydropyridines, phenylalkylamines, and BTZ at their specific binding sites.