A new topological model of the cardiac sarcolemmal Na+-Ca2+ exchanger

The current topological model of the Na+-Ca2+ exchanger consists of II tran smembrane segments with extracellular loops a, c, e, g, i, and k and cytopl asmic loops b, d, f, h, and j, Cytoplasmic loop f, which plays a role in re gulating the exchanger, is large and separates the first five from the last six transmembrane segments. We have tested this topological model by mutat ing residues near putative transmembrane segments to cysteine and then exam ining the effects of intracellular and extracellular applications of sulfhy dryl-modifying reagents on exchanger activity. To aid in our topological st udies, we also constructed a cysteineless Na+-Ca2+ exchanger. This mutant i s fully functional in Na+ gradient-dependent Ca-45(2+) uptake measurements and displays wild-type regulatory properties. It is concluded that the 15 e ndogenous cysteine residues are not essential for either activity or regula tion of the exchanger. Our data support the current model by placing loops c and e at the extracellular surface and loops d, j, and l at the intracell ular surface. However, the data also support placing Ser-788 of loop h at t he extracellular surface and Gly-837 of loop i at the intracellular surface , To account for these data, we propose a revision of the model that places transmembrane segment 6 in cytoplasmic loop f. Additionally, we propose th at putative transmembrane segment 9 does not span the membrane, but may for m a "P-loop"-like structure.