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Ann N Y Acad Sci. 2007 Mar;1099:1-6. Epub 2007 Feb 15.

What we know about the structure of NCX1 and how it relates to its function.

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1
Department of Physiology, University of California, Los Angeles, California 90095, USA. dnicoll@mednet.ucla.edu

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 alpha repeats are composed of TMS 2 and 3 and TMS 7 and 8 and are involved in ion binding and transport. The beta 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 alpha- and beta-repeat regions and on how the TMS pack in the membrane. We have examined the alpha1 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 beta1 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 beta1 repeat shows that it is composed of a seven-stranded beta-sandwich with Ca2+-binding sites located at one end of the sandwich. Four Ca2+ ions bind to the beta1 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.

PMID:
17303833
DOI:
10.1196/annals.1387.014
[Indexed for MEDLINE]
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