title: Ca2+-dependent structural rearrangements within Na+-Ca2+ exchanger dimers.
abstract: Cytoplasmic Ca(2+) is known to regulate Na(+)-Ca(2+) exchanger (NCX) activity by binding to two adjacent Ca(2+)-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca(2+)-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane. Addition of Ca(2+) decreases the distance between the cytoplasmic loops of NCX pairs. The Ca(2+)-dependent movements detected between paired NCXs were abolished by mutating the Ca(2+) coordination sites in CBD1 (D421A, E451A, and D500V), whereas disruption of the primary Ca(2+) coordination site in CBD2 (E516L) had no effect. Thus, the Ca(2+)-induced conformational changes of NCX dimers arise from the movement of CBD1. FRET studies of CBD1, CBD2, and CBD1-CBD2 peptides displayed Ca(2+)-dependent movements with different apparent affinities. CBD1-CBD2 showed a Ca(2+)-dependent phenotype mirroring full-length NCX but distinct from both CBD1 and CBD2.
authors.affiliation: Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1751, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: John
authors.forename: Bernard
authors.initials: B
authors.lastname: Ribalet
authors.forename: James N
authors.initials: JN
authors.lastname: Weiss
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Philipson
authors.forename: Michela
authors.initials: M
authors.lastname: Ottolia
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials; D009154:Mutation; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D011487:Protein Conformation / Q000187:drug effects; D055503:Protein Multimerization; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D008164:Luminescent Proteins; D019831:Sodium-Calcium Exchanger; D002118:Calcium