Characterization of D-[3H]cis-diltiazem binding to membrane fractions and specific binding of calcium channel blockers to isolated flagellar membranes of Chlamydomonas reinhardtii

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Abstract
Plasma membranes were separated from the intracellular membranes by using an aqueous two polymerphase system. D-[3H]cis-diltiazem was employed to characterize benzothiazepine-selective receptors in these different membrane fractions of Chlamydomonas reinhardtii. The separation revealed that one type of binding site with higher affinity (KD = 33 nm) can be attributed to the intracellular membrane fraction and a second type with lower affinity (KD = 313nm) to the plasma membrane fraction. The apparent dissociation constants determined from the association and dissociation rate constants in kinetic experiments are comparable to those determined by saturation experiments. The maximum numbers of binding sites of the intracellular membrane fraction and the plasma membrane fraction are Bmax = 6.4 pmol mg−1 protein and Bmax = 19 pmol mg−1 protein, respectively. D-[3H]cis-diltiazem binding is inhibited by (±)verapamil and calcium chloride in both fractions. Moreover, nifedipine stimulates D-[3H]cis-diltiazem diltiazem binding by the intracellular membrane fraction, but shows no effect on the plasma membrane fraction. Ligand displacement binding studies with isolated flagella revealed the occurrence of a d-cisdiltiazem binding site with about the same affinity to this drug (KD = 400 nm) as the one found inthe plasma membrane fraction. The maximum number of binding sites is 4.5 pmol mg−1 protein. The apparent dissociation constants for specific[3H]nimodipine and [3H]verapamil binding to the flagella were calculated to be 8 nm and 38 nm, respectively. The corresponding Bmax values are 345f mol mg−1 protein and 1.3 pmol mg−1 protein, respectively.