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 extracellu lar space by an electrogenic process where this neurotransmitter is co-tran sported with sodium and chloride ions. We report in this paper that tyrosin e at position 289 of GLYT2a is crucial for ion coupling, glycine affinity a nd 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 trans porters unable to catalyze glycine uptake. Measurements of glycine evoked s teady-state currents in transfected HEK-293 cells reveal EC50 values for gl ycine 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 d ramatic change corresponding to the less conservative substitution (Y289S), Accordingly, sodium selectivity is gradually lost in Y289F and Y289S mutan ts, and chloride dependence of glycine evoked currents is markedly decrease d in Y289F and Y289S mutants. In the absence of three-dimensional informati on from these transporters, these results provide experimental evidence sup porting the hypothesis of transmembrane domain III being part of a common p ermeation pathway for substrate and cotransported ions.