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To provide experimental evidence for the topology of the Na+-Ca2+ exchanger protein NCX1 in the membrane, indirect immunofluorescence studies using site specific anti-peptide antibodies and Flag-epitope insertion into chosen locations of the protein were carried out. Anti-peptide antibodies AbO-6 and AbO-8 were raised against peptide segments present in a large hydrophilic loop of about 500 amino acids, which separates the hydrophobic amino terminal part of the protein from the hydrophobic carboxy terminal. AbO-10 was raised against the C-terminal tail of the protein. All three antibodies bound to the exchanger protein expressed in transfected cells, in rat brain synaptic plasma membrane and in dog sarcolemmal preparations. The antibodies bound only to those NCX1 isoforms that contained the epitope against which they were raised. Detection of the exchanger protein in transfected cells in situ required the addition of permeabilizing agents suggesting an intracellular location of the epitopes to which AbO-6, AbO-8 and AbO-10 bind. The Flag epitope was inserted into ten putative extramembraneous segments along the exchanger protein. For topology studies, only the Flag-mutants that retained Na+-Ca2+ exchange activity in whole HeLa cells, were used. Immunofluorescence studies indicated, that the N-terminal of the protein is extracellular, the first hydrophilic loop separating transmembrane helices 1 and 2 as well as the C-terminal, are intracellular.

A ft~n~tjon~l rat heart Na"-Ca" exchanger gene has been obtained by splicing and ligating two partially overlapping clones isolated from a rat heart /ZZAP cDNA library. The deduced primary structure of the protein encoded by the open reading frame corresponds to 971 amino acids, that can be organized into 12 transmembrane helices. The cloned gene was functionally expressed in HeLa cells. Maximal expression was detected 18 h after transfection, after which transport activity rapidly declined. The electrogenic properties of the cloned transporter were demonstrated following reconstitution of the expressed exchanger protein into a tightly sealed phospholipid membrane.

The rat brain Na(+)-Ca2+ exchanger (RBE) gene, as well as other isoforms of this protein family, can be organized into 12 transmembrane alpha helices, the first of which was proposed by Durkin et al. (14) to constitute a cleavable signal peptide. We have prepared three amino-terminal mutants, in which 21, 26, and 31 amino acids beyond the initiating methionine were deleted. The deletions include the hydrophobic core of the putative signal peptide (N21), the entire putative signal peptide and parts of the putative signal peptidase cleavage site (N26), and the entire putative signal peptide and putative signal peptidase cleavage site (N31). All three mutant clones were transiently expressed in HeLa cells. The average Na+ gradient-dependent Ca2+ transport activity of the mutant exchangers was 108% (N21), 37.2% (N26), and 60.06% (N31) of the wild-type clone. Mutation of the putative cleavage site by an exchange of Ala-32 --> Asp, resulted in a decrease in Na(+)-Ca2+ exchange activity to 7.7%, relative to the wild-type exchanger. Functional reconstitution of the proteins that were expressed in the transfected cells, resulted in transport activities of: 60.1% (N21), 26.75% (N26), 85.36% (N31), and 31% (Ala-32 --> Asp) relative to the wild-type exchanger. Western blot analysis of the protein profile of RBE-1, N21, N26, N31 and Ala-32 --> Asp-transfected HeLa cells was carried out by using an antipeptide antibody directed against a pentadecapeptide segment derived from the large putative cytoplasmic loop of the cloned rat exchanger gene. In the total cell extract and in the plasma membrane-enriched fraction, in addition to a major protein band of about 125 kDa, which corresponds to the molecular mass of the mature fully processed Na(+)-Ca2+ exchanger, an additional protein of about 135 kDa is revealed in the profile of N21- and N26-transfected cells. This band is not detected in the protein profile of RBE-1, N31, or Ala-32 -->Asp. The amino-terminal truncated mutants of the cloned Na(+)-Ca2+ exchanger could be expressed and processed also in a reticulocyte lysate supplemented with dog microsomes. Our results suggest that the putative signal peptide of the cloned Na(+)-Ca2+ exchanger gene does not play a mandatory role in functional expression of the protein in HeLa cells.