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.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1984|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology