Regulation of the cardiac Na+-Ca2+ exchanger by the endogenous XIP region

The cardiac sarcolemmal Na+-Ca2+ exchanger is modulated by intrinsic regulatory mechanisms. A large intracellular loop of the exchanger participates in the regulatory responses. We have proposed (Li, Z., D.A. Nicoll, A. Collins, D.W. Hilgemann, A.G. Filoteo, J.T. Penniston, J.N. Weiss, J.M. Tomich, and KD. Philipson. 1991. J. Biol. Chem. 266:1014-1020) that a segment of the large intracellular loop, the endogenous XIP region, has an autoregulatory role ill exchanger function. We now test this hypothesis by mutational analysis of the XIP region. Nine XIP-region mutants were expressed in Xenopus oocytes and all displayed altered regulatory properties. The major alteration was in a regulatory mechanism known as Na+-dependent inactivation. This inactivation is manifested as a partial decay in outward Na+-Ca2+ exchange current after application of Na+ to the intracellular surface of a giant excised patch. Two mutant phenotypes were observed. In group 1 mutants, inactivation was markedly accelerated; in group 2 mutants, inactivation was completely eliminated. All mutants had normal Naf affinities. Regulation of the exchanger by nontransported, intracellular Ca2+ was also modified by the XIP-region mutations. Binding of Ca2+ to the intracellular loop activates exchange activity and also decreases Na+-dependent inactivation. XIP-region mutants were all still regulated by Ca2+, However, the apparent affinity of the group 1 mutants for regulatory Ca2+ Mras decreased. The responses of all mutant exchangers to Ca2+ application or removal were markedly accelerated. Na+-dependent inactivation and regulation by Ca2+ are interrelated and are not completely independent processes. We conclude that the endogenous XIP region is primarily involved in movement of the exchanger into and out of the Na+-induced inactivated state, but that the XIP region is also involved in regulation by Ca2+.