Regulation of the cardiac Na (�) -Ca 2� exchanger by Ca 2� . Mutational analysis of the Ca (2�) -binding domain

The sarcolemmal Na+-Ca 2+ exchanger is regulated by intracellular Ca ~+ at a high affinity Ca 2+ binding site separate from the Ca ~+ transport site. Previous data have suggested that the Ca 2+ regulatory site is located on the large intracellular loop of the Na +-Ca 2+ exchange protein, and we have identified a highaffinity 4SCa2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca ~+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside-out giant membrane patch technique. Ca ~+ regulation was measured as the stimulation of reverse Na+-Ca ~+ exchange (intracellular Na + exchanging for extracellular Ca ~+) by intracellular Ca ~+. Single-site mutations within two acidic clusters of the Ca 2+ binding domain lowered the apparent Ca ~+ affinity at the regulatory site from 0.4 to 1.1-1.8 ~.M. Mutations had parallel effects on the affinity of the exchanger loop for 45Ca~

Anomalous regulation of the Drosophila Na � - Ca 2� exchanger by Ca 2

ABSTRACT The Na+-Ca 2+ exchanger from Drosophila was expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique. This protein, termed Calx, shares 49 % amino acid identity to the canine cardiac Na+-Ca 2+ exchanger, NCX1. Calx exhibits properties similar to previously characterized Na+-Ca z+ exchangers including intracellular Na + affinities, current-voltage relationships, and sensitivity to the peptide inhibitor, XIP. However, the Drosophila Na+-Ca 2+ exchanger shows a completely opposite response to cytoplasmic Ca 2+. Previously cloned Na+-Ca 2+ exchangers (NCX1 and NCX2) are stimulated by cytoplasmic Ca 2+ in the micromolar range (0.1-10 jxM). This stimulation of exchange current is mediated by occupancy of a regulatory Ca 2+ binding site separate from the Ca 2+ transport site. In contrast, Calx is inhibited by cytoplasmic Ca 2+ over this same concentration range. The inhibition of exchange current is evident for both forward and reverse modes of transport. The characteristics of the inhibition are consistent with the binding of Ca 2+ at a regulatory site distinct from the transport site. These data provide a rational basis for subsequent structure-function studies targeting the intracellular Ca z+ regulatory mechanism. Key words: sodium-calcium exchange 9 calcium regulation 9 Drosophila melanogaste