What We Know about the Structure of NCX1 and How It Relates to Its Function

Abstract:  NCX1 is modeled to contain nine transmembrane segments (TMS) with a large intracellular loop between TMS 5–6 and two reentrant loops connecting TMS 2–3 and TMS 7–8. NCX1 also contains two regions of internal repeats. The α repeats are composed of TMS 2 and 3 and TMS 7 and 8 and are involved in ion binding and transport. The β repeats are in the large intracellular loop and are involved in binding of regulatory Ca2+. Our studies on the structure/function analysis of NCX1 have focused on the α‐ and β‐repeat regions and on how the TMS pack in the membrane. We have examined the α1 repeat by mutagenesis of residues modeled to be in the reentrant loop and TMS 3 and by determination of ion affinities of the mutants. Our results show that TMS 3 and not the reentrant loop is involved in Na+ binding. No mutants demonstrated altered affinity for transported Ca2+. We have synthesized a fusion protein composed of the β1 repeat. This fusion protein was expressed in Escherichia coli and purified. The fusion protein binds Ca2+ and shows conformational changes on binding. The crystal structure of the β1 repeat shows that it is composed of a seven‐stranded β‐sandwich with Ca2+‐binding sites located at one end of the sandwich. Four Ca2+ ions bind to the β1 repeat in a manner reminiscent of Ca2+ binding to C2 domains. Packing of TMS in the membrane has been studied by cross‐linking induced mobility shifts on SDS‐PAGE. Interactions between TMS 1, 2, 3, 6, 7, and 8 have been identified.