title: Residues in the extracellular loop 4 are critical for maintaining the conformational equilibrium of the gamma-aminobutyric acid transporter-1.
abstract: We mutated residues Met345 and Thr349 in the rat gamma-aminobutyric acid transporter-1 (GAT-1) to histidines (M345H and T349H). These two residues are located four amino acids apart at the extracellular end of transmembrane segment 7 in a region of GAT-1 that we have previously suggested undergoes conformational changes critical for the transport process. The two single mutants and the double mutant (M345H/T349H) were expressed in Xenopus laevis oocytes, and their steady-state and presteady-state kinetics were examined and compared with wild type GAT-1 by using the two-electrode voltage clamp method. Oocytes expressing M345H showed a decrease in apparent GABA affinity, an increase in apparent affinity for Na+, a shift in the charge/voltage (Q/Vm) relationship to more positive membrane potentials, and an increased Li+-induced leak current. Oocytes expressing T349H showed an increase in apparent GABA affinity, a decrease in apparent Na+ affinity, a profound shift in the Q/Vm relationship to more negative potentials, and a decreased Li+-induced leak current. The data are consistent with a shift in the conformational equilibrium of the mutant transporters, with M345H stabilized in an outward-facing conformation and T349H in an inward-facing conformation. These data suggest that the extracellular end of transmembrane domain 7 not only undergoes conformational changes critical for the translocation process but also plays a role in regulating the conformational equilibrium between inward- and outward-facing conformations.
authors.affiliation: Department of Medical Physiology and the Department of Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark. nmacaulay@mfi.ku.dk
authors.forename: Nanna
authors.initials: N
authors.lastname: MacAulay
authors.forename: Anne-Kristine
authors.initials: AK
authors.lastname: Meinild
authors.forename: Thomas
authors.initials: T
authors.lastname: Zeuthen
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D004553:Electric Conductivity; D005260:Female; D050485:GABA Plasma Membrane Transport Proteins; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008094:Lithium / Q000494:pharmacology; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D026901:Membrane Transport Proteins; D008715:Methionine; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D027361:Organic Anion Transporters; D011487:Protein Conformation; D051381:Rats; D012964:Sodium / Q000378:metabolism; D013329:Structure-Activity Relationship; D013912:Threonine; D014162:Transfection; D014316:Tritium; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C493415:Slc6a1 protein, rat; D014316:Tritium; D013912:Threonine; D005680:gamma-Aminobutyric Acid; D008094:Lithium; D012964:Sodium; D008715:Methionine