title: The cardiac Na+-Ca2+ exchanger has two cytoplasmic ion permeation pathways.
abstract: The Na(+)-Ca(2+) exchanger (NCX) is a ubiquitously expressed plasma membrane protein. It plays a fundamental role in Ca(2+) homeostasis by moving Ca(2+) out of the cell using the electrochemical gradient of Na(+) as the driving force. Recent structural studies of a homologous archaebacterial exchanger, NCX_Mj, revealed its outward configuration with two potential ion permeation pathways exposed to the extracellular environment. Based on the symmetry of NCX_Mj structure, an atomic model of an inward-facing conformation was generated showing similar pathways but directed to the cytoplasm. The presence of these water-filled cavities has yet to be confirmed experimentally, and it is unknown if the mammalian exchanger adopts the same structure. In this study, we mutated multiple residues within transmembrane segments 2 and 7 of NCX1.1 (cardiac isoform) to cysteines, allowing us to investigate their sensitivity to membrane-impermeable sulfhydryl reagents as exchanger current block. By trapping NCX1.1 in the inward-facing configuration, we have mapped two differently sized cytoplasmic aqueous cavities, the access of which is modified during exchange. This data reveals movements of the protein associated with ion transport. Electrophysiological characterization shows that the conserved residues within transmembrane segments 2 and 7, coordinating Na(+) and Ca(2+) ions in NCX_Mj, play a fundamental role in NCX1.1. Our results suggest a similar architecture between the mammalian and archaebacterial exchangers.
authors.affiliation: Department of Medicine (Cardiology) and Cardiovascular Research Laboratory, David Geffen School of Medicine at the University of California, Los Angeles, CA 90095, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: John
authors.forename: Jun
authors.initials: J
authors.lastname: Liao
authors.forename: Youxing
authors.initials: Y
authors.lastname: Jiang
authors.forename: Michela
authors.initials: M
authors.lastname: Ottolia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000235:genetics; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D017136:Ion Transport / Q000187:drug effects; D007477:Ions; D008698:Mesylates / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009206:Myocardium / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000378:metabolism*; D014981:Xenopus
chemical_list: D007477:Ions; D008698:Mesylates; D050505:Mutant Proteins; D019831:Sodium-Calcium Exchanger; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D003545:Cysteine