MEMBRANE TOPOLOGY OF THE RAT-BRAIN NA-CA2+ EXCHANGER()

To provide experimental evidence for the topology of the Na+-Ca2+ exch anger protein NCX1 in the membrane, indirect immunofluorescence studie s using site specific anti-peptide antibodies and Flag-epitope inserti on into chosen locations of the protein were carried out. Anti-peptide antibodies AbO-6 mid AbO-8 were raised against peptide segments prese nt in a large hydrophilic loop of about 500 amino acids, which separat es the hydrophobic amino terminal part of the protein from the hydroph obic carboxy terminal. AbO-10 was raised against the C-terminal tail o r the protein. All three antibodies hound to the exchanger protein exp ressed in transfected cells, in rat brain synaptic plasma membrane and in dog sarcolemmal preparations. The antibodies bound only to those N CX1 isoforms that contained the epitope against which they were raised . Detection of the exchanger protein in transfected cells in situ requ ired the addition of permeabilizing agents suggesting an intracellular location of the epitopes to which AbO-6. AbO-8 and AbO-10 bind. The F lag epitope was inserted into ten putative extramembraneous segments a long the exchanger protein. For topology studies, only the Flag-mutant s that retained Na+-Ca2+ exchange activity in whole HeLa cells, were u sed. Immunofluorescence studies indicated, that the N-terminnl of the protein is extracellular, the first hydrophilic loop separating transm embrane helices 1 and 2 as well as the C-terminal, are intracellular. (C) 1998 Elsevier Science B.V.