Sodium-calcium exchange does not require allosteric calcium activation at high cytosolic sodium concentrations

The activity of the cardiac Na+-Ca2+ exchanger (NCX1.1) is allosterically regulated by Ca2+, which binds to two acidic regions in the cytosolically disposed central hydrophilic domain of the NCX protein. A mutation in one of the regulatory Ca2+ binding regions (D447V) increases the half-activation constant (K-h) for allosteric Ca2+ activation from similar to 0.3 to > 1.8 mu M. Chinese hamster ovary cells expressing the D447V exchanger showed little or no activity under physiological ionic conditions unless cytosolic [Ca2+] was elevated to > 1 mu M. However, when cytosolic [Na+] was increased to 20 mM or more (using ouabain-induced inhibition of the Na+, K+-ATPase or the ionophore gramicidin), cells expressing the D447V mutant rapidly accumulated Ca2+ or Ba2+ when the reverse (Ca2+ influx) mode of NCX activity was initiated, although initial cytosolic [Ca2+] was < 100 nM. Importantly, the time course of Ca2+ uptake did not display the lag phase that reflects allosteric Ca2+ activation of NCX activity in the wild-type NCX1.1; indeed, at elevated [Na+], the D447V mutant behaved similarly to the constitutively active deletion mutant Delta(241-680), which lacks the regulatory Ca2+ binding sites. In cells expressing wild-type NCX1.1, increasing concentrations of cytosolic Na+ led to a progressive shortening of the lag phase for Ca2+ uptake. The effects of elevated [Na+] developed rapidly and were fully reversible. The activity of the D447V mutant was markedly inhibited when phosphatidylinositol 4,5-bisphosphate (PIP2) levels were reduced. We conclude that when PIP2 levels are high, elevated cytosolic [Na+] induces a mode of exchange activity that does not require allosteric Ca2+ activation.