REGULATION OF THE CARDIAC NA-CA2+ EXCHANGER BY CA2+ - MUTATIONAL ANALYSIS OF THE CA2+-BINDING DOMAIN()

The sarcolemmal Na+-Ca2+ exchanger is regulated by intracellular Ca2at a high affinity Ca2+ binding site separate from the Ca2+ transport site. Previous data have suggested that the Ca2+ regulatory site is lo cated on the large intracellular loop of the Na+-Ca2+ exchange protein , and we have identified a high-affinity Ca-45(2+) binding domain on t his loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Jo urnal of Biological Chemistry. 269:22847-22852). We now use electrophy siological and mutational analyses to further define the Ca2+ regulato ry site. Wild-type and mutant exchangers were expressed in Xenopus ooc ytes, and the exchange current was measured using the inside-out giant membrane patch technique. Ca2+ regulation was measured as the stimula tion of reverse Na+-Ca2+ exchange (intracellular Na+ exchanging for ex tracellular Ca2+) by intracellular Ca2+. Single-site mutations within two acidic clusters of the Ca2+ binding domain lowered the apparent Ca 2+ affinity at the regulatory site from 0.4 to 1.1-1.8 mu M. Mutations had parallel effects on the affinity of the exchanger loop for Ca-45( 2+) binding (Levitsky et al., 1994) and for functional Ca2+ regulation . We conclude that we have identified the functionally important Ca2binding domain. All mutant exchangers with decreased apparent affiniti es at the regulatory Ca2+ binding site also have a complex pattern of altered kinetic properties. The outward current of the wild-type Na+-C a2+ exchanger declines with a half time (t(h)) of 10.8 +/- 3.2 s upon Ca2+ removal, whereas the exchange currents of several mutants decline with t(h) values of 0.7-4.3 s. Likewise, Ca2+ regulation mutants resp ond more rapidly to Ca2+ application. Study of Ca2+ regulation has pre viously been possible only with the exchanger operating in the reverse mode as the regulatory Ca2+ and the transported Ca2+ are then on oppo site sides of the membrane. The use of exchange mutants with low affin ity for Ca2+ at regulatory sites also allows demonstration of secondar y Ca2+ regulation with the exchanger in the forward or Ca2+ efflux mod e. In addition, we find that the affinity of wild-type and mutant Na+- Ca2+ exchangers for intracellular Na+ decreases at low regulatory Ca2. This suggests that Ca2+ regulation modifies transport properties and does not only control the fraction of exchangers in an active state.