Photoaffinity labeling of the partially purified mitochondrial phenylalkylamine calcium antagonist receptor.

Mitochondria contain specific Ca2+ antagonist binding sites that are associated with an inner mitochondrial membrane anion channel. These mitochondrial Ca2+ antagonist receptors can be solubilized with digitonin and partially purified [as assessed by postreversible (+/-)-[3H]nitrendipine binding] using ion exchange chromatography and sucrose density gradient centrifugation. In the present study, reversible binding of the phenylakylamine Ca2+ antagonist [3H]ludopamil, an optically pure photoaffinity analog of verapamil, to the partially purified mitochondrial Ca2+ antagonist receptor complex (Kd, 9 +/- 4 microM; Bmax, 1.2 +/- 0.5 nmol/mg of protein) depended on NaNO3 and was inhibited by the 1,4-dihydropyridine niludipine and by ATP. Accordingly, the unlabeled racemic analog of [3H]ludopamil, (+/-)-LU 47781, dose-dependently inhibited the binding of the 1,4-dihydropyridine (+/-)-[3H]nitrendipine to the purified mitochondrial receptors (IC50, 2.1 +/- 0.1 microM). After UV irradiation, [3H]ludopamil specifically incorporated into two polypeptides of 12.7 +/- 0.1 kDa and 11.7 +/- 0.1 kDa, with the pharmacological profile of [3H]ludopamil photoincorporation stimulation and protection being identical to that of reversible binding.