Characterization and solubilization of the nitrendipine binding protein from canine small intestinal circular smooth muscle

The binding of nitrendipine, a calcium channel blocking agent, to the microsomes prepared from canine small intestinal circular smooth muscle was characterized. The binding of this 1,4-dihydropyridine to the membrane was reversible, saturable and of high affinity with a dissociation constant of 0.28 nM and a maximal binding of 91 fmol/mg microsomal protein. The binding occurred with an association rate constant of 0.094 nM-1 min-1 and dissociated at the rate of 0.0498 min-1. These rate constants gave a value of 0.52 nM for the dissociation constant of the binding. The binding was inhibited in a competitive fashion by other dihydropyridines (nifedipine, nimodipine, nisoldipine, PN200-110) with inhibition constants in the range of 0.1 to 1.0 nM. The binding was also inhibited by verapamil and D-600 but only at much higher concentrations. Diltiazem increased the nitrendipine binding to the membranes. The nitrendipine binding protein was solubilized using the detergent 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS). The solubilized material bound nitrendipine with a dissociation constant of 0.51 nM giving maximal binding of 70 fmol/mg protein. The binding of the solubilized material resembled the membrane bound material in inhibition by the dihydropyridines, verapamil and D-600 and in the increase by diltiazem. Thus we have solubilized the nitrendipine binding protein without changing its binding properties substantially.