Sodium-calcium exchange does not require allosteric calcium activation at high cytosolic sodium concentrations.
The activity of the cardiac Na(+)-Ca(2+) exchanger (NCX1.1) is allosterically regulated by Ca(2+), which binds to two acidic regions in the cytosolically disposed central hydrophilic domain of the NCX protein. A mutation in one of the regulatory Ca(2+) binding regions (D447V) increases the half-activation constant (K(h)) for allosteric Ca(2+) activation from approximately 0.3 to > 1.8 microm. Chinese hamster ovary cells expressing the D447V exchanger showed little or no activity under physiological ionic conditions unless cytosolic [Ca(2+)] was elevated to > 1 microm. 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 Ca(2+) or Ba(2+) when the reverse (Ca(2+) influx) mode of NCX activity was initiated, although initial cytosolic [Ca(2+)] was < 100 nm. Importantly, the time course of Ca(2+) uptake did not display the lag phase that reflects allosteric Ca(2+) 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 Ca(2+) binding sites. In cells expressing wild-type NCX1.1, increasing concentrations of cytosolic Na(+) led to a progressive shortening of the lag phase for Ca(2+) 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 Ca(2+) activation.
Urbanczyk
Jason
J
Department of Pharmacology & Physiology, UMDNJ - NJ Medical School, 185 South Orange Avenue, PO Box 1709, Newark, NJ 07101-1709, USA.
Chernysh
Olga
O
Condrescu
Madalina
M
Reeves
John P
JP
D000494:Allosteric Regulation; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism*; D015640:Ion Channel Gating; D009154:Mutation; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D019269:Phosphatidylinositol 4,5-Diphosphate; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium