Transmembrane Segment Packing of the Na+/Ca2+ Exchanger Investigated with Chemical Cross-Linkers

The Na+/Ca2+ exchanger (NCX1) is a plasma membrane protein important in regulating Ca2+ in cardiac myocytes. The topological model is comprised of nine transmembrane segments (TMSs). To gain insights into the TMS packing arrangement of NCX1, we performed cysteine cross-linking experiments. Pairs of amino acids in different TMSs were mutated to cysteine on the backbone of a cysteineless NCX1. The mutated exchangers were expressed in an insect cell line and treated with cysteine-specific chemical cross-linkers followed by SDS-PAGE to determine the proximity of the introduced cysteines. Previously, we showed that TMSs 2, 3, 7, and 8 are near one another and that residues in TMSs 1 and 2 are close to TMS 6. In this report, we use the same approach to provide evidence for the arrangement of the remaining three TMSs (4, 5, and 9). We present a computer-generated two-dimensional model of transmembrane packing that minimizes the lengths of all cross-links.