title: Electrophysiological characterization of the archaeal transporter NCX_Mj using solid supported membrane technology.
abstract: Sodium-calcium exchangers (NCXs) are membrane transporters that play an important role in Ca(2+) homeostasis and Ca(2+) signaling. The recent crystal structure of NCX_Mj, a member of the NCX family from the archaebacterium Methanococcus jannaschii, provided insight into the atomistic details of sodium-calcium exchange. Here, we extend these findings by providing detailed functional data on purified NCX_Mj using solid supported membrane (SSM)-based electrophysiology, a powerful but unexploited tool for functional studies of electrogenic transporter proteins. We show that NCX_Mj is highly selective for Na(+), whereas Ca(2+) can be replaced by Mg(2+) and Sr(2+) and that NCX_Mj can be inhibited by divalent ions, particularly Cd(2+) By directly comparing the apparent affinities of Na(+) and Ca(2+) for NCX_Mj with those for human NCX1, we show excellent agreement, indicating a strong functional similarity between NCX_Mj and its eukaryotic isoforms. We also provide detailed instructions to facilitate the adaption of this method to other electrogenic transporter proteins. Our findings demonstrate that NCX_Mj can serve as a model for the NCX family and highlight several possible applications for SSM-based electrophysiology.
authors.affiliation: Nanion Technologies, 80636 Munich, Germany Center for Integrated Protein Science (CIPS-M) and Center for Drug Research, Department of Pharmacology, Ludwig Maximilians University and DZHK (German Center for Cardiovascular Research), partner site Munich Heart Alliance, 81377 Munich, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Barthmes
authors.affiliation: Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390 School of Life Science and Technology, Shanghai Tech University, Shanghai 201210, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Liao
authors.affiliation: Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390.
authors.forename: Youxing
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Nanion Technologies, 80636 Munich, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Brüggemann
authors.affiliation: Center for Integrated Protein Science (CIPS-M) and Center for Drug Research, Department of Pharmacology, Ludwig Maximilians University and DZHK (German Center for Cardiovascular Research), partner site Munich Heart Alliance, 81377 Munich, Germany christian.wahl@cup.uni-muenchen.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Wahl-Schott
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D002104:Cadmium / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D017136:Ion Transport; D008274:Magnesium / Q000378:metabolism; D063747:Methanocaldococcus / Q000737:chemistry; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000378:metabolism*; D013324:Strontium / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D019831:Sodium-Calcium Exchanger; D053835:Unilamellar Liposomes; C110374:sodium-calcium exchanger 1; D002104:Cadmium; D012964:Sodium; D008274:Magnesium; D002118:Calcium; D013324:Strontium