The glial and the neuronal glycine transporters differ in their reactivity to 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 characterized under voltage clamp their reactivity toward the membrane impermeant 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 current, and the glycine uptake, indicating a complete inactivation of the transporters following cysteine modification. In contrast, this compound had no detectable effect on the glycine uptake and the glycine-evoked current of GlyT2a. The sensitivities to MTSET of the two transporters can be permutated 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 GlyT2a-EL1) resulting in AFQ <--> CYR modification. Inactivation by MTSET was five times faster in GlyT2a-A223C than in GlyT2a-EL1 or GlyT1b, suggesting that the arginine in position +2 reduced the cysteine reactivity. Protection assays indicate that EL1 cysteines are less accessible in the presence of all co-transported substrates: Na(+), Cl(-), and glycine. Application of dithioerythritol reverses the inactivation by MTSET of the sensitive transporters. Together, these results indicate that EL1 conformation differs between GlyT1b and GlyT2a and is modified by substrate binding and translocation.
Roux
M J
MJ
Laboratoire de Neurobiologie, Unité Mixte de Recherche 8544, Centre National de la Recherche Scientifique, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris cedex 05, France.
Martinez-Maza
R
R
Le Goff
A
A
Lopez-Corcuera
B
B
Aragon
C
C
Supplisson
S
S
D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D002352:Carrier Proteins / Q000502:physiology*; D050487:Glycine Plasma Membrane Transport Proteins; D008698:Mesylates / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009457:Neuroglia / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D009928:Organ Specificity; D018408:Patch-Clamp Techniques; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014981:Xenopus
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D050487:Glycine Plasma Membrane Transport Proteins; D008698:Mesylates; D013439:Sulfhydryl Reagents; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate