Roles of Two Ca2+-binding Domains in Regulation of the Cardiac Na+-Ca2+ Exchanger

We expressed full-length Na(+)-Ca(2+) exchangers (NCXs) with mutations in two Ca(2+)-binding domains (CBD1 and CBD2) to determine the roles of the CBDs in Ca(2+)-dependent regulation of NCX. CBD1 has four Ca(2+)-binding sites, and mutation of residues Asp(421) and Glu(451), which primarily coordinate Ca(2+) at sites 1 and 2, had little effect on regulation of NCX by Ca(2+). In contrast, mutations at residues Glu(385), Asp(446), Asp(447), and Asp(500), which coordinate Ca(2+) at sites 3 and 4 of CBD1, resulted in a drastic decrease in the apparent affinity of peak exchange current for regulatory Ca(2+). Another mutant, M7, with 7 key residues of CBD1 replaced, showed a further decrease in apparent Ca(2+) affinity but retained regulation, confirming a contribution of CBD2 to Ca(2+) regulation. Addition of the mutation K585E (located in CBD2) into the M7 background induced a marked increase in Ca(2+) affinity for both steady-state and peak currents. Also, we have shown previously that the CBD2 mutations E516L and E683V have no Ca(2+)-dependent regulation. We now demonstrate that introduction of a positive charge at these locations rescues Ca(2+)-dependent regulation. Finally, our data demonstrate that deletion of the unstructured loops between beta-strands F and G of both CBDs does not alter the regulation of the exchanger by Ca(2+), indicating that these segments are not important in regulation. Thus, CBD1 and CBD2 have distinct roles in Ca(2+)-dependent regulation of NCX. CBD1 determines the affinity of NCX for regulatory Ca(2+), although CBD2 is also necessary for Ca(2+)-dependent regulation.