ATP-dependent calcium transport in rat parotid basolateral membrane vesicles is modulated by membrane potential

Abstract

The ATP-dependent Ca2+ transport activity (T. Takuma, B.L. Kuyatt and B.J. Baum, Biochem. J. 227:239-245, 1985) exhibited by inverted basolateral membrane vesicles isolated from rat parotid gland was further characterized. The activity was dependent on Mg2+. Phosphate (5 mM), but not oxalate (5 mM), increased maximum Ca2+ accumulation by 50%. Half-maximal Ca2+ transport was achieved at approximately 70 nM Ca2+ in EGTA-buffered medium while maximal activity required greater than 1 microM Ca2+ (Vmax = 54 nmol/mg protein/min). Optimal rates of Ca2+ transport were obtained in the presence of KCl, while in a KCl-free medium (mannitol or sucrose) approximately 40% of the total activity was achieved, which could not be stimulated by FCCP. The initial rate of Ca2+ transport could be significantly altered by preimposed membrane potentials generated by K+ gradients in the presence of valinomycin. Compared to the transport rate in the absence of membrane potential, a negative (interior) potential stimulated uptake by approximately 30%, while a positive (interior) potential inhibited uptake. Initial rates of Ca2+ uptake could also be altered by imposing pH gradients, in the absence of KCl. When compared to the initial rate of Ca2+ transport in the absence of a pH gradient, pHi = 7.5/pHo = 7.5; the activity was approximately 60% higher in the presence of an outwardly directed pH gradient, pHi = 7.5/pHo = 8.5; while it was approximately 80% lower when an inwardly directed pH gradient was imposed, pHi = 7.5/pHo = 6.2. The data show that the ATP-dependent Ca2+ transport in BLMV can be modulated by the membrane potential, suggesting therefore that there is a transfer of charge into the vesicle during Ca2+ uptake, which could be compensated by other ion movements.