title: 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 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.
authors.affiliation: Department of Physiology, University of California, Los Angeles, California 90095, USA. dnicoll@mednet.ucla.edu
authors.forename: Debora A
authors.initials: DA
authors.lastname: Nicoll
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Ren
authors.forename: Michela
authors.initials: M
authors.lastname: Ottolia
authors.forename: Martin
authors.initials: M
authors.lastname: Phillips
authors.forename: Alfredo R
authors.initials: AR
authors.lastname: Paredes
authors.forename: Jeff
authors.initials: J
authors.lastname: Abramson
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Philipson
mesh_terms: D003461:Crystallography; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship
chemical_list: D011993:Recombinant Fusion Proteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1