Allosteric Activation of Sodium–Calcium Exchange Activity by Calcium : Persistence at Low Calcium Concentrations

The activity of the cardiac Na⁺/Ca²⁺ exchanger is stimulated allosterically by Ca²⁺, but estimates of the half-maximal activating concentration have varied over a wide range. In Chinese hamster ovary cells expressing the cardiac Na⁺/Ca²⁺ exchanger, the time course of exchange-mediated Ca²⁺ influx showed a pronounced lag period followed by an acceleration of Ca²⁺ uptake. Lag periods were absent in cells expressing an exchanger mutant that was not dependent on regulatory Ca²⁺ activation. We assumed that the rate of Ca²⁺ uptake during the acceleration phase reflected the degree of allosteric activation of the exchanger and determined the value of cytosolic Ca²⁺ ([Ca²⁺]_i) at which the rate of Ca²⁺ influx was half-maximal (K_h). After correcting for the effects of mitochondrial Ca²⁺ uptake and fura-2 buffering, K_h values of ∼300 nM were obtained. After an increase in [Ca²⁺]_i, the activated state of the exchanger persisted following a subsequent reduction in [Ca²⁺]_i to values <100 nM. Thus, within 30 s after termination of a transient increase in [Ca²⁺]_i, exchange-mediated Ca²⁺ entry began without a lag period and displayed a linear rate of Ca²⁺ uptake in most cells; a sigmoidal time course of Ca²⁺ uptake returned 60–90 s after the transient increase in [Ca²⁺]_i was terminated. Relaxation of the activated state was accelerated by the activity of the endoplasmic reticulum Ca²⁺ pump, suggesting that local Ca²⁺ gradients contribute to maintaining exchanger activation after the return of global [Ca²⁺]_i to low values.