Characterization and photoaffinity labeling of receptor sites for the Ca2+ channel inhibitors d-cis-diltiazem, (+/-)-bepridil, desmethoxyverapamil, and (+)-PN 200-110 in skeletal muscle transverse tubule membranes.

J P Galizzi,M Borsotto,J Barhanin,M Fosset,M Lazdunski

doi:10.1016/s0021-9258(17)36105-7

Abstract

In order to further understand the molecular nature of the voltage-sensitive Ca2+ channel in skeletal muscle, we have performed classical radioligand binding studies and photoaffinity labeling with different types of tritiated inhibitors of the Ca2+ channel. The equilibrium dissociation constants (KD) for (-)-[3H]desmethoxyverapamil, d-cis-[3H]diltiazem, and (+/-)-[3H]bepridil at their receptor sites in skeletal muscle transverse tubule membranes are: 1.5 +/- 0.5, 50 +/- 5, and 20 +/- 5 nM, respectively. Maximum binding capacities in picomoles/milligram of protein were: 70 +/- 10 for (-)-[3H]desmethoxyverapamil, 50 +/- 15 for d-cis-[3H]diltiazem, and 75 +/- 15 for (+/-)-[3H]bepridil. The kinetics of association at 10 degrees C for the three types of tritiated compounds were relatively slow (3 X 10(5) M-1 S-1 for (-)-[3H]desmethoxyverapamil, 8 X 10(3) M-1 S-1 for d-cis-[3H]diltiazem, and 4.2 X 10(5) M-1 S-1 for (+/-)-[3H]bepridil). The dissociation of (-)-[3H]desmethoxyverapamil and d-cis-[3H]diltiazem from their receptor sites was also a slow process with half-lives of dissociation of 33 and 36 min, respectively. Competition studies using the three tritiated ligands suggest that they bind to the same receptor site which appears to be in a 1:1 stoichiometry with the dihydropyridine receptor. Photoaffinity labeling with high intensity ultraviolet light in the presence of (+/-)-[3H]bepridil or d-cis[3H]diltiazem resulted in the specific covalent incorporation of radioactivity into a polypeptide of Mr 170,000 +/- 10,000. A polypeptide of Mr 170,000 was also specifically labeled in photoaffinity labeling experiments using the high affinity dihydropyridine derivative (+)-[3H]PN 200-100.

Highlights

Of the voltage-sensitive Ca2+channel in skeletal mus- Verapamil-binding sites have been identified in several cle, we have performed classical radioligand binding tissues (8-12), andd-~is-[~H]diltiazembinding to skeletal studies and photoaffinity labeling with different typemsuscle microsomes has been described (13)
Preparation of Membranes from Rabbit White Skeletal Muscle-Ttubule membranes were prepared from rabbit white skeletal muscle according to Refs. 14 and 15 in the presence of 0.1 mM phenylmethylsulfonyl fluoride to miminize proteolytic degradation
Bepridil Binding to T-tubule Membranes-Fig. 1 (A-C) illustrates equilibrium binding of (-)-[3H]D888, d-cis-['H]dilti

Summary

The experiments reported in this paper were undertaken

The equilibrium dissociation constants (KO)for (-)- (i) to analyze in more detail the properties of the receptors [3H]desmethoxyverapamil, d-~is-[~H]diltiazem,and for (-)-[3H]D888, d-~is-[~H]diltiazem, an(d+)-[3H]bepridil;. Maximum binding capacities in picomoles/milligram of protein were: 70 2 10 for (-)-[3H]desmethoxyverapamil, f 15 for d~is-[~H]diltiazem, a7n5df15for (f)-[3H]bepridil. Tritiated compounds were relatively slow (3x 10' M-' Skeletal muscle T-tubule membranes, the richest source for s" for (-)-[3H]desmethoxyverapamil, 8 X lo M" s-' dihydropyridine receptors (14), were used in all experiments for d-~is-[~H]diltiazern, an4.d2 X 10' M" s" for (2)- described in this paper. Measurements of nonspecific binding were performed using 1PM (-)D888 with (-)-[3H]D888, 10 ,.IM d-cis-diltiazem with d-ci~-[~H]dilticifically labeled in photoaffinity labeling experiments azem, or 1 p~ (+)-bepridil with (+.)-[3H]bepridil.Specific binding using the high affinity dihydropyridine derivative(+)- was checked to be a linear function of increasing concentration of T-. Association and Dissociation Kinetics-Kinetic experiments were performed under conditions described for equilibrium binding assays.

RESULTS

Effects of Enantiomers of the Different Calcium Channel

KD Kr

DISCUSSION