Substrate-induced conformational changes of extracellular loop 1 in the glycine transporter GLYT2

The neurotransmitter glycine is removed from the synaptic cleft by two Na+- and Cl--dependent transporters, the glial (GLYT1) and neuronal (GLYT2) glyc ine transporters. GLYT2 lacks a conserved cysteine in the first hydrophilic loop (EL1) that is reactive to [2-(trimethylammonium)ethyl] methanethiosul fonate (MTSET) in related transporters. A chimeric GLYT2 (GLYT2a-EL1) that contains GLYT1 sequences in this region, including the relevant cysteine, w as sensitive to the reagent, and its sensitivity was decreased by co-substr ates. We combined cysteine-specific biotinylation to detect transporter-rea gent interactions with MTSET inactivation assays and temperature dependence analysis to study the mechanism by which Cl-, Na+, and glycine reduce meth anethiosulfonate reagent inhibition. We demonstrate a Na+ protective effect rather than an increased susceptibility to the reagent exerted by Li+, as reported for the serotonin transporter. The different inhibition, protectio n, and reactivation properties between GLYT2a-EL1 and serotonin transporter suggest that EL1 is a source of structural heterogeneity involved in the s pecific effect of lithium on serotonin transport. The protection by Na+ or Cl- on GLYT2a-EL1 was clearly dependent on temperature, suggesting that EL1 is not involved in ion binding but is subjected to ion-induced conformatio nal changes. Na+ and Cl- were required for glycine protection, indicating t he necessity of prior ion interaction with the transporter for the binding of glycine. We conclude that EL1 acts as a fluctuating hinge undergoing seq uential conformational changes during the transport cycle.