Identification of the high affinity Ca(2+)-binding domain of the cardiac Na(+)-Ca2+ exchanger.
The cardiac sarcolemmal Na(+)-Ca2+ exchanger transports Na+ and Ca2+ but is also regulated by Ca2+ at a high affinity binding site. A large intracellular, hydrophilic loop of the exchanger has been suggested to contain the Ca(2+)-binding regulatory domain (Matsuoka S., Nicoll, D. A., Reilly, R. F., Hilgemann, D. W., and Philipson, K. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3870-3874). To localize the Ca(2+)-binding site(s), we expressed portions of the exchanger loop as fusion proteins and measured binding by 45Ca2+ overlay. The high affinity binding domain is located near the center of the loop and binds Ca2+ in a cooperative manner. K0.5 ranges from 0.3 to 3 microM in the presence of 0.2-5 mM Mg2+. The binding region (amino acids 371-508) has two highly acidic sequences, each characterized by 3 consecutive aspartic acid residues. Ca2+ affinity markedly decreases when these aspartates are mutated. The Ca(2+)-binding region does not contain an EF-hand motif. The mobilities during SDS-polyacrylamide gel electrophoresis of fusion proteins with high Ca2+ affinity differ depending on the presence or absence of Ca2+ in the gel loading buffer. The high affinity Ca(2+)-binding domain is probably responsible for the secondary Ca2+ regulation of the Na(+)-Ca2+ exchanger.
Levitsky
D O
DO
Department of Physiology, UCLA School of Medicine 90024-1760.
Nicoll
D A
DA
Philipson
K D
KD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D019831:Sodium-Calcium Exchanger; D001616:beta-Galactosidase / Q000096:biosynthesis
D002352:Carrier Proteins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019831:Sodium-Calcium Exchanger; D001616:beta-Galactosidase; D002118:Calcium