Physiological functions of the regulatory domains of the cardiac Na(+)/Ca(2+) exchanger NCX1

Physiologicalfunctions of the intracellular regulatory domains of theNa⁺/Ca²⁺ exchanger NCX1 were studied byexamining Ca²⁺ handling in CCL39 cells expressing alow-affinity Ca²⁺ regulatory site mutant (D447V/D498I), anexchanger inhibitory peptide (XIP) region mutant displaying noNa⁺ inactivation (XIP-4YW), or a mutant lacking most of thecentral cytoplasmic loop (246-672). We found that D447V/D498Iwas unable to efficiently extrude Ca²⁺ from the cytoplasm,particularly during a small rise in intracellular Ca²⁺concentration induced by the physiological agonist -thrombin orthapsigargin. The same mutant took up Ca²⁺ much lessefficiently than the wild-type NCX1 in Na⁺-free medium whentransfectants were not loaded with Na⁺, although itappeared to take up Ca²⁺ normally in transfectantspreloaded with Na⁺. XIP-4YW and, to a lesser extent,246-672, but not NCX1 and D447V/D498I, markedly accelerated theloss of viability of Na⁺-loaded transfectants. Furthermore,XIP-4YW was not activated by phorbol ester, whereas XIP-4YW andD447V/D498I were resistant to inhibition by ATP depletion. The resultssuggest that these regulatory domains play important roles in thephysiological and pathological Ca²⁺ handling by NCX1, aswell as in the regulation of NCX1 by protein kinase C or ATP depletion.