Functional consequences of sulfhydryl modification of the γ-aminobutyric acid transporter 1 at a single solvent-exposed cysteine residue.
The aims of this study were to optimize the experimental conditions for labeling extracellularly oriented, solvent-exposed cysteine residues of γ-aminobutyric acid transporter 1 (GAT1) with the membrane-impermeant sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) and to characterize the functional and pharmacological consequences of labeling on transporter steady-state and presteady-state kinetic properties. We expressed human GAT1 in Xenopus laevis oocytes and used radiotracer and electrophysiological methods to assay transporter function before and after sulfhydryl modification with MTSET. In the presence of NaCl, transporter exposure to MTSET (1-2.5 mM for 5-20 min) led to partial inhibition of GAT1-mediated transport, and this loss of function was completely reversed by the reducing reagent dithiothreitol. MTSET treatment had no functional effect on the mutant GAT1 C74A, whereas the membrane-permeant reagents N-ethylmaleimide and tetramethylrhodamine-6-maleimide inhibited GABA transport mediated by GAT1 C74A. Ion replacement experiments indicated that MTSET labeling of GAT1 could be driven to completion when valproate replaced chloride in the labeling buffer, suggesting that valproate induces a GAT1 conformation that significantly increases C74 accessibility to the extracellular fluid. Following partial inhibition by MTSET, there was a proportional reduction in both the presteady-state and steady-state macroscopic signals, and the functional and pharmacological properties of the remaining signals were indistinguishable from those of unlabeled GAT1. Therefore, covalent modification of GAT1 at C74 results in completely nonfunctional as well as electrically silent transporters.
Biological Sciences Department, California State Polytechnic University, Pomona, 3801 West Temple Avenue, Pomona, CA 91768-4032, USA.
Jaison J
JJ
Omoto
Matthew J
MJ
Maestas
Ali
A
Rahnama-Vaghef
Ye E
YE
Choi
Gerardo
G
Salto
Rachel V
RV
Sanchez
Cynthia M
CM
Anderson
Sepehr
S
Eskandari
D000818:Animals; D003545:Cysteine / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000737:chemistry; D005260:Female; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008698:Mesylates / Q000737:chemistry*; D009154:Mutation; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D018408:Patch-Clamp Techniques; D012235:Rhodamines / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000737:chemistry*; D014162:Transfection; D014635:Valproic Acid / Q000737:chemistry / Q000494:pharmacology; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D050485:GABA Plasma Membrane Transport Proteins; D008698:Mesylates; D012235:Rhodamines; C493413:SLC6A1 protein, human; D013439:Sulfhydryl Reagents; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D012965:Sodium Chloride; D005680:gamma-Aminobutyric Acid; D014635:Valproic Acid; C005358:tetramethylrhodamine; D003545:Cysteine; D005033:Ethylmaleimide; D004229:Dithiothreitol