Helix packing of the cardiac Na+-Ca2+ exchanger: proximity of transmembrane segments 1, 2, and 6

The cardiac Na+-Ca2+ exchanger (NCX1) is a membrane protein that extrudes Ca2+ from cells using the energy of the Na+ gradient and is a key protein in regulating intracellular Ca2+ and contractility. Based on the current topological model, NCX1 consists of nine transmembrane segments (TMSs). The N-terminal five TMSs are separated from the C-terminal four TMSs by a large intracellular loop. Cysteine 768 is modeled to be in TMS 6 close to the intracellular surface. In this study, the proximity of TMS 6 to TMSs 1 and 2 was examined. Insect High Five cells were transfected with cDNAs encoding mutant NCX1 proteins. Each mutant contained cysteine 768 and an introduced cysteine in TMS 1 or 2. Cross-linking between cysteines was determined after reaction with thiol-specific cross-linkers containing spacer arms of 6.5-12 A. The data indicate that residues in TMSs 1 and 2 are close to cysteine 768 in TMS 6. Cysteine 768 cross-linked with residues at both ends of TMSs 1 and 2 and is likely located toward the middle of TMS 6. Based on these results, we present an expanded helix-packing model for NCX1.