Regulation of the Cardiac Na+-Ca2+ Exchanger by the Endogenous XIP Region

Abstract

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 K.D. Philipson. 1991. J. Biol. Chem. 266:1014–1020) that a segment of the large intracellular loop, the endogenous XIP region, has an autoregulatory role in 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 Na+ 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+ was 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+.

Phospholemman regulates cardiac Na+/Ca2+exchanger by interacting with the exchanger's proximal linker domain

“…The ␣-repeats in TM segments 2, 3, and 7 of NCX1 are important in ion transport activity (15,24), while the large intracellular loop contains the regulatory domains of the exchanger (17,18,20). Specifically, the exchange inhibitory peptide (XIP) region (residues 219 -238) (18) associated with regulation of NCX1 activity by Na ϩ , Ca 2ϩ , and phosphatidylinositol 4,5-bisphosphate (13,21), the proximal linker domain (residues 259 -370), the two consecutive Ca 2ϩ -binding domains 1 (residues 371-508) (17) and 2 (residues 501-650) (14), and the interaction site for endogenous XIP (residues 562-679) (20) reside within the intracellular loop. Using glutathione S-transferase (GST) pull-down assays and split NCX1 exchangers consisting of NH 2 -or COOH-terminal domains with varying lengths of intracellular loop (26), we previously demonstrated that PLM interacts and associates with a region encompassing residues 218 -358 of the intracellular loop of NCX1 (38).…”

Inhibitory Interaction of the Plasma Membrane Na+/Ca2+ Exchangers with the 14-3-3 Proteins

“…Although this loop is not involved in Na ϩ and Ca 2ϩ translocation, it is responsible for the regulation of activity. Several factors are responsible for the regulation (3), among them the two transported ions, Na ϩ and Ca 2ϩ (13,14), the intracellular pH (15), metabolic components (e.g. ATP, phosphatidylinositol 4,5-bisphosphate (16), protein kinase A, and protein kinase C (17)), redox agents, hydroxyl radicals, H 2 , Cu 2ϩ , and OH (18).…”

Topology of a Functionally Important Region of the Cardiac Na+/Ca2+ Exchanger

“…Through mutagenesis studies, it has been determined that the endogenous XIP 1 region in loop f (8) is involved in Na ϩ -dependent inactivation (9) and that another portion of loop f is involved in the binding of regulatory Ca 2ϩ ( Fig. 1) (3, 10).…”