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.
Department of Physiology and the Cardiovascular Research Laboratories, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1760, USA. mottolia@mednet.ucla.edu
Michela
M
Ottolia
Debora A
DA
Nicoll
Kenneth D
KD
Philipson
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D018360:Crystallography, X-Ray / Q000379:methods; D003593:Cytoplasm / Q000378:metabolism; D005786:Gene Expression Regulation; D007477:Ions; D016296:Mutagenesis; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D019831:Sodium-Calcium Exchanger / Q000737:chemistry*; D014982:Xenopus laevis
D007477:Ions; D019831:Sodium-Calcium Exchanger; D002118:Calcium