Transmembrane domain III plays an important role in ion binding and permeation in the glycine transporter GLYT2.
The neuronal glycine transporter GLYT2 takes up glycine from the extracellular space by an electrogenic process where this neurotransmitter is co-transported with sodium and chloride ions. We report in this paper that tyrosine at position 289 of GLYT2a is crucial for ion coupling, glycine affinity and sodium selectivity, stressing the essential role played by this residue of transmembrane domain III in the mechanism of transport. Substitution to tryptophan (Y289W), phenylalanine (Y289F), or serine (Y289S), renders transporters unable to catalyze glycine uptake. Measurements of glycine evoked steady-state currents in transfected HEK-293 cells reveal EC(50) values for glycine 17-fold (Y289F) and 45-fold (Y289S) higher than that of the wild type transporter. Sodium dependence is severely altered in tyrosine 289 mutants, both at the level of apparent affinity and cooperativity, with the more dramatic change corresponding to the less conservative substitution (Y289S). Accordingly, sodium selectivity is gradually lost in Y289F and Y289S mutants, and chloride dependence of glycine evoked currents is markedly decreased in Y289F and Y289S mutants. In the absence of three-dimensional information from these transporters, these results provide experimental evidence supporting the hypothesis of transmembrane domain III being part of a common permeation pathway for substrate and co-transported ions.
Ponce
J
J
Centro de Biologia Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autónoma, Consejo Superior de Investigaciones Cientificas, 28049 Madrid, Spain.
Biton
B
B
Benavides
J
J
Avenet
P
P
Aragon
C
C
D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D050487:Glycine Plasma Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014443:Tyrosine
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D050487:Glycine Plasma Membrane Transport Proteins; D014443:Tyrosine; D005998:Glycine