title: Direct visualization of glutamate transporter elevator mechanism by high-speed AFM.
abstract: Glutamate transporters are essential for recovery of the neurotransmitter glutamate from the synaptic cleft. Crystal structures in the outward- and inward-facing conformations of a glutamate transporter homolog from archaebacteriumPyrococcus horikoshii, sodium/aspartate symporter GltPh, suggested the molecular basis of the transporter cycle. However, dynamic studies of the transport mechanism have been sparse and indirect. Here we present high-speed atomic force microscopy (HS-AFM) observations of membrane-reconstituted GltPhat work. HS-AFM movies provide unprecedented real-space and real-time visualization of the transport dynamics. Our results show transport mediated by large amplitude 1.85-nm "elevator" movements of the transport domains consistent with previous crystallographic and spectroscopic studies. Elevator dynamics occur in the absence and presence of sodium ions and aspartate, but stall in sodium alone, providing a direct visualization of the ion and substrate symport mechanism. We show unambiguously that individual protomers within the trimeric transporter function fully independently.
authors.affiliation: U1006 INSERM, Aix-Marseille Université, Parc Scientifique et Technologique de Luminy, 13009 Marseille, France.
authors.forename: Yi
authors.initials: Y
authors.lastname: Ruan
authors.affiliation: U1006 INSERM, Aix-Marseille Université, Parc Scientifique et Technologique de Luminy, 13009 Marseille, France.|Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065.|Department of Anesthesiology, Weill Cornell Medicine, New York, NY 10065.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyagi
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Wang
authors.affiliation: BioEM Lab, Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, CH-4058 Basel, Switzerland.
authors.forename: Mohamed
authors.identifier: 0000-0002-8733-055X
authors.initials: M
authors.lastname: Chami
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065; sis2019@med.cornell.edu olb2003@med.cornell.edu.|Howard Hughes Medical Institute, Weill Cornell Medicine, New York, NY 10065.
authors.forename: Olga
authors.initials: O
authors.lastname: Boudker
authors.affiliation: U1006 INSERM, Aix-Marseille Université, Parc Scientifique et Technologique de Luminy, 13009 Marseille, France; sis2019@med.cornell.edu olb2003@med.cornell.edu.|Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065.|Department of Anesthesiology, Weill Cornell Medicine, New York, NY 10065.
authors.forename: Simon
authors.initials: S
authors.lastname: Scheuring
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000737:chemistry / Q000378:metabolism*; D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D001224:Aspartic Acid; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D018360:Crystallography, X-Ray; D018698:Glutamic Acid / Q000378:metabolism; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D043902:Pyrococcus horikoshii / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism
chemical_list: D027322:Amino Acid Transport System X-AG; D019843:Archaeal Proteins; D021122:Protein Subunits; D001224:Aspartic Acid; D018698:Glutamic Acid; D012964:Sodium
keywords: GltPh; HS-AFM; dynamics; elevator mechanism; transporter