Abstract
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.
Highlights
Naϩ/ClϪ-coupled transporters control the extracellular and intracellular concentrations of neurotransmitters like bioamines, ␥-aminobutyric acid, and glycine
Similar applications of MTSET affected neither IT (Fig. 1c) nor the transient currents nor the outward rectifying leak conductance (Fig. 1d) recorded in GlyT2aϩ oocytes. To test whether these differences could result from the presence (GlyT1b) or the absence (GlyT2a) of a cysteine in the first extracellular loop (EL1), as suggested by results obtained on GAT-1 [8] and SERT [9], we applied
The topological organization of the Naϩ/ClϪ neurotransmitter transporter family has received good experimental support in favor of the original model of 12 putative transmembrane segments proposed for GAT-1 [13]
Summary
Aliquots of dithioerythritol (300 mM, Sigma), MTSET, and MTSES (100 mM, Toronto Research Chemical Co., Toronto, Canada) were prepared in ice-cold water and kept at Ϫ20 °C They were thawed and diluted in the appropriate solution just prior to perfusion (Ͻ10 s). Charge Movement—Transient currents specific to GlyT1b or GlyT2a were evoked in the absence of glycine by stepping the voltage at various potentials. These currents are Naϩ-dependent and capacitative in nature because their time integrals estimated following the onset and return from the voltage step are equal in amplitude but opposite in sign. After complete wash-out and after the current returned to the base-line level, oocytes were transferred in 5 ml of ice-cold Ringer solution, washed twice, lysed in vials containing 500 l of SDS (2%), and measured for radioactivity. The charge coupling was determined as the ratio between the glycine-evoked current integral and the glycine uptake as described by Roux and Supplisson [5]
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