Functional importance of the synaptic plasma membrane calcium pump and sodium-calcium exchanger

Two major Ca²⁺ transport mechanisms co-function in a preparation of synaptosomal plasma membrane vesicles: an (ATP + Mg²⁺)-dependent Ca²⁺ pump, and a reversible Na⁺-Ca²⁺ exchanger. An accurate comparative analysis of the kinetics of the two Ca²⁺ transporters under free Ca²⁺ conditions precisely buffered with EGTA, reveals that both mechanisms have high affinity for Ca²⁺. The ATP-dependent Ca²⁺ pump displays simple saturation kinetics with a K(m) for Ca²⁺ of 0.11 μM and a V(max) of 2.2 nmol/min/mg of protein. In contrast, the Na⁺-Ca²⁺ exchanger has a complex dependence on free Ca²⁺, the activity continuing to saturate over a wide range of free Ca²⁺ concentrations from 0.03 μM to 3 mM. The curvilinear Eadie-Hofstee analysis reveals a distinct high affinity component for the exchanger with a K(m) for Ca²⁺ of approximately 0.5 μM, and a lower affinity component not accurately resolvable into a discrete K(m) value. 2 mM amiloride blocks Na⁺-Ca²⁺ exchange-mediated Ca²⁺ uptake by 90% over a wide range of free Ca²⁺ (0.3-3000 μM), suggesting a similar noncompetitive inhibition of both low and high affinity Ca²⁺ sites. Ca²⁺ accumulated in vesicles via either the Ca²⁺ pump or Na⁺-Ca²⁺ exchanger is rapidly (in less than 1 min) released by 0.1% saponin (w/v), although a minor component (8-10%) of Ca²⁺ pump activity is resistant to saponin addition. The IC₅₀ for the effect of saponin is the same (0.01%, w/v) for both Ca²⁺ transport mechanisms. The ATP-dependent Ca²⁺ pump is shown to be highly sensitive to vanadate inhibition [K(i) = 0.5 μM]. The high saponin sensitivity of both Ca²⁺ transporters and the potent effect of vanadate on Ca²⁺ pumping, together with previous Na⁺ channel and Na⁺ pump flux studies in the same membrane vesicles, all strongly suggest that both of the high affinity Ca²⁺ transporters function in the plasma membrane where they are of major functional importance to the regulation of intrasynaptic free Ca²⁺ levels.