The glial and the neuronal glycine transporters differ in their reactivityto sulfhydryl reagents

The neuronal (GlyT2) and glial (GlyT1) glycine transporters, two members of the Na+/Cl--dependent neurotransmitter transporter superfamily, differ by many aspects, such as substrate specificity and Na+ coupling. We have chara cterized under voltage clamp their reactivity toward the membrane impermean t sulfhydryl reagent [2-(trimethylammonium) -ethyl] -methanethiosulfonate ( MTSET). In Xenopus oocytes expressing GlyT1b, application of MTSET reduced to the same extent the Na+-dependent charge movement, the glycine-evoked cu rrent, and the glycine uptake, indicating a complete inactivation of the tr ansporters following cysteine modification. In contrast, this compound had no detectable effect on the glycine uptake and the glycine-evoked current o f GlyT2a, The sensitivities to MTSET of the two transporters can be permuta ted by suppressing a cysteine (C62A) in the first extracellular loop (EL1) of GlyT1b and introducing one at the equivalent position in GlyT2a, either by point mutation (A223C) or by swapping the EL1 sequence (GlyT1b-EL1 and G lyT2a-EL1) resulting in AFQ <----> CYR modification. Inactivation by MTSET was five times faster in GlyT2a-A223C than in GlyT2a-EL1 or GlyT1b, suggest ing that the arginine in position +2 reduced the cysteine reactivity, Prote ction assays indicate that EL1 cysteines are less accessible in the presenc e of all co-transported substrates: Na+, Cl-, and glycine, Application of d ithioerythritol reverses the inactivation by MTSET of the sensitive transpo rters. Together, these results indicate that EL1 conformation differs betwe en GlyT1b and GlyT2a and is modified by substrate binding and translocation .