ABSTRACT

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.

Two functional lsoforms of the rat brain Nat-Ca'+ exchanger were isolated from a 1ZAP hippocampus cDNA library. The open reading frame of clone RBE-1 codes for a protein 935 amino acids long, and that of clone RBE-2 codes for a protein 958 amino acids long. Expression in HeLa cells of Na' gradient dependent Ca" transport activity was determined following transfection of the cells with either RBE-1 or RBE-2. Both clones expressed proteins that exchange Na' with Ca" m an electrogemc manner and none of them exhibited a dependency of the antiport on K', since they transported Ca'+ in an Na+ gradient dependent manner in external choline chloride as well.

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 C terminus of the rat brain Na exchanger and from C10 indicated that resistance to the digestion was acquired after 1 and 5 h of chase, respectively. C29 did not acquire detectable resistance to endoglycosidase H digestion even after 10 h of chase. Taken together, these results suggest that the "cellular quality control machinery" can tolerate the structural change introduced by truncation of the C terminus up to Ser-893 albeit with reduced rate of ER3 Golgi transfer and reduced surface expression of the truncated protein. Further truncation of C-terminal amino acids leads to retention of the truncated protein in the ER, no transfer to the Golgi, and no surface expression.