ION DEPENDENCE OF THE PARTIALLY PURIFIED MITOCHONDRIAL DIHYDROPYRIDINE CA2+ ANTAGONIST RECEPTOR

The mitochondrial inner membrane contains specific binding sites for d ihydropyridine (DHP) Ca2+ antagonists that are associated with an inne r mitochondrial membrane anion channel (IMAC) [Mol. Pharmacol. 38:362- 369 (1990)]. As in particulate preparations, binding of the DHP (+/-)- [H-3]nitrendipine ([H-3]NTR) to partially purified mitochondrial DHP r eceptors strongly depended on a variety of cations and inorganic as we ll as organic anions. Monovalent anions saturably stimulated [H-3]NTR binding with a potency rank order of 1- > Br- > Cl- > F-. The potency rank order for monovalent cations was Cs+ > Rb+ > Li+ > K+ > Na+. [H-3 ]NTR binding stimulation potency of the cations strikingly depended on their charge density, with EC50 values being 125 mM for K+, 5 mM for Ca2+, and 41-mu-M for La3+. This selectivity order clearly differed fr om one predicted on the basis of a simple surface charge-screening eff ect of the cations. In general, allosteric ion effects were due to cha nges in [H-3]NTR affinity for the partially purified mitochondrial DHP receptor. SCN- and NO3-, known permeators of the IMAC [J. Biol. Chem. 262: 15085-15093 (1987)], stimulated [H-3]NTR binding with EC50 value s of 26 mM and 96 mM, respectively. The IMAC permeators butylmalonate2 - and 1,2,3-benzenetricarboxylate3- were ineffective when given alone but dose-dependently inhibited 500 mM NaCl-stimulated [H-3]NTR binding , as did PO4(1.5-) and SO4(2-). Gluconate-, which was reported not to permeate the IMAC, qualitatively behaved as a partial agonist with res pect to Cl-. Glucuronate- was without effect on [H-3]NTR binding to th e partially purified mitochondrial DHP receptor. These results point t o the existence of rather large ion-binding domains. The cation-bindin g site was estimated to have a minimum diameter of 0.67 nm. The anion- binding domain could accommodate either spherical ligands with diamete rs of up to 0.6 nm or molecules with a flat backbone with dimensions o f approximately 0.9 nm x 0.7 nm x 0.3 nm.