Mapping of the cardiac sodium-calcium exchanger with monoclonal antibodies.
We used a panel of monoclonal antibodies raised against the canine cardiac Na(+)-Ca2+ exchanger expressed in Sf9 insect cells to analyze the immunoreactive domains and the topological organization of this membrane protein. Antibodies, which reacted strongly on Western blots of the recombinant protein, were used to screen an expression sublibrary composed of exchanger cDNA fragments. Positive clones thus indicated the expression of antibody binding sites. Linear epitopes, 16-155 amino acids in length, could be identified for four antibodies. One antibody recognized two neighboring, but nonoverlapping, sequences. All epitopes were localized to the large hydrophilic region of the exchanger connecting the putative transmembrane segments 5 and 6. The immunodominant region of the protein is a highly charged domain in the carboxy-terminal half of the hydrophilic region. Binding studies with the 3H-labeled high-affinity antibody R3F1 establish that the immunodominant region is located on the intracellular surface of the membrane. The same antibody was used to directly determine the membrane concentration of the exchanger in different cell types. Newborn rat heart cells contain approximately 6 x 10(5) exchanger molecules per cell. Exchanger densities in different cells seem to correlate with the Na(+)-dependent Ca2+ transport activity in the corresponding membrane vesicles.
Porzig
H
H
Department of Pharmacology, University of Bern, Switzerland.
Li
Z
Z
Nicoll
D A
DA
Philipson
K D
KD
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes; D015723:Gene Library; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D011233:Precipitin Tests; D019831:Sodium-Calcium Exchanger; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D018076:DNA, Complementary; D000939:Epitopes; D019831:Sodium-Calcium Exchanger