Physiological functions of the regulatory domains of the cardiac Na+/Ca2+exchanger NCX1

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Abstract
Physiological functions of the intracellular regulatory domains of the Na+/Ca2+exchanger NCX1 were studied by examining Ca2+handling in CCL39 cells expressing a low-affinity Ca2+regulatory site mutant (D447V/D498I), an exchanger inhibitory peptide (XIP) region mutant displaying no Na+inactivation (XIP-4YW), or a mutant lacking most of the central cytoplasmic loop (Δ246–672). We found that D447V/D498I was unable to efficiently extrude Ca2+from the cytoplasm, particularly during a small rise in intracellular Ca2+concentration induced by the physiological agonist α-thrombin or thapsigargin. The same mutant took up Ca2+much less efficiently than the wild-type NCX1 in Na+-free medium when transfectants were not loaded with Na+, although it appeared to take up Ca2+normally in transfectants preloaded with Na+. XIP-4YW and, to a lesser extent, Δ246–672, but not NCX1 and D447V/D498I, markedly accelerated the loss of viability of Na+-loaded transfectants. Furthermore, XIP-4YW was not activated by phorbol ester, whereas XIP-4YW and D447V/D498I were resistant to inhibition by ATP depletion. The results suggest that these regulatory domains play important roles in the physiological and pathological Ca2+handling by NCX1, as well as in the regulation of NCX1 by protein kinase C or ATP depletion.