KINETIC-PROPERTIES OF THE SODIUM-CALCIUM EXCHANGER IN RAT-BRAIN SYNAPTOSOMES

1. The kinetic properties of the internal Na+ (Na-i(+))-dependent Ca-45(2+) influx and external Na+ (Na-o(+))-dependent Ca-45(2+) efflux were determined in isolated rat brain nerve terminals (synaptosomes) under conditions in which the concentrations of internal Na+ ([Na+]i), external Na+ ([Na+](o)), external Ca2+ ([Ca2+](o)), and external K+ ([K+](o)) were varied. Both fluxes are manifestations of Na+-Ca2+ exchange. 2. Ca2+ uptake was augmented by raising [Na+](i) and/or lowering [Na+](o). The increase in Ca2+ uptake induced by removing external Na+ was, in most instances, quantitatively equal to the Na-i(+)-dependent Ca2+ uptake. 3. The Na-i(+)-dependent Ca2+ uptake (measured at 1 s) was activated with an apparent half-maximal [Ca2+](o) (K-Ca(o)) of about 0.23 mM. External Na+ inhibited the uptake in a noncompetitive manner: increasing [Na+](o) from 4.7 to 96 mM reduced the maximal Na-i(+)-dependent Ca2+ uptake but did not affect K-Ca(o). 4. The inhibition of Ca2+ uptake by Na-o(+) was proportional to ([Na+](o))(2), and had a Hill coefficient (n(H)) of similar to 2.0. The mean apparent half-maximal [Na+](o) for inhibition ((K) over bar(I(Na))) was about 60 mM, and was independent of [Ca2+](o) between 0.1 and 1.2 mM; this, too, is indicative of noncompetitive inhibition. 5. Low concentrations of alkali metal ions (M(+)) in the medium, including Na+, stimulated the Na-i(+)-dependent uptake. The external Na+ and K+ concentrations required for apparent half maximal activation (K-M(Na) and K-M(k), respectively) were 0.12 and 0.10 mM. Thus, the relationship between Ca2+ uptake and [Na+](o) was biphasic: uptake was stimulated by [Na+](o) less than or equal to 10 mM, and inhibited by higher [Na+](o). 6. The calculated maximal Na-i(+)-dependent Ca2+ uptake (J(max)) was about 1530 pmol (mg protein)(-1) s(-1) at 30 degrees C at saturating [Ca2+](o) and external M(+) concentration ([M(+)](o)), and with negligible inhibition by external Na+. 7. Internal Na+ activated the Ca2+ uptake with an apparent half-maximal concentration (K-Na(i)) of about 20 mM and a Hill coefficient, n(H), of similar to 3.0. 8. The J(max) for the Na-o(+)-dependent efflux of Ca2+ from Ca-45(2+)-loaded synaptosomes treated with carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) and caffeine (to release stored Ca2+ and raise the internal Ca2+ concentration ([Ca2+]i) was about 1800-2000 pmol (mg protein)(-1) s(-1) at 37 degrees C. 9. When the membrane potential (V-m) was reduced (depolarized) by increasing [K+](o), the Na-i(+)-dependent Ca2+ influx increased, and the Na-o(+)-dependent Ca2+ efflux declined. Both fluxes changed about 2-fold per 60 mV change in V-m. This voltage sensitivity corresponds to the movement of one elementary charge through about 60% of the membrane electric field. The symmetry suggests that the voltage-sensitive step is reversible. 10. The J(max) values for both Ca2+ influx and efflux correspond to a Na+-Ca2+ exchange-mediated flux of about 425-575 mu mol Ca2+ (1 cell water)(-1) s(-1) or a turnover of about one quarter of the total synaptosome Ca2+ in 1 s. We conclude that the Na+-Ca2+ exchanger may contribute to Ca2+ entry during nerve terminal depolarization; it is likely to be a major mechanism mediating Ca2+ extrusion during subsequent repolarization and recovery.