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.
Zernig
G
G
Department of Pharmacology, University of Michigan, Ann Arbor 48109.
D000345:Affinity Labels; D001386:Azides / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000378:metabolism; D015220:Calcium Channels / Q000032:analysis*; D004095:Dihydropyridines / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008928:Mitochondria / Q000737:chemistry*; D014700:Verapamil / Q000031:analogs & derivatives / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; C051537:LU 49888; C038806:1,4-dihydropyridine; D014700:Verapamil