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.

Staged Surgical Management of Open Navicular Fracture Secondary to a Gunshot Injury.

The management guidelines of gunshot wound (GSW) injuries to the lower extremities have primarily been described more recently in the literature. A navicular fracture with adjacent joint involvement is presented from a GSW with initial external fixation management to prevent loss of anatomical alignment and successful staged definitive treatment with internal fixation. Based on previous experiences with rearfoot joint involvement from GSW injuries, we were able to direct definitive treatment with arthrodesis of violated joints. After a 1-year follow-up, the patient has returned to normal activities without any limitations. This case report demonstrates a stepwise approach to management of an open navicular fracture secondary to a GSW.

Inhibitors of the gamma-aminobutyric acid transporter 1 (GAT1) do not reveal a channel mode of conduction.

We expressed the gamma-aminobutyric acid (GABA) transporter GAT1 (SLC6A1) in Xenopus laevis oocytes and performed GABA uptake experiments under voltage clamp at different membrane potentials as well as in the presence of the specific GAT1 inhibitors SKF-89976A and NO-711. In the absence of the inhibitors, GAT1 mediated the inward translocation of 2 net positive charges across the plasma membrane for every GABA molecule transported into the cell. This 2:1 charge flux/GABA flux ratio was the same over a wide range of membrane potentials from -110 mV to +10 mV. Moreover, when GABA-evoked (500 microM) currents were measured at -50 and -90 mV, neither SKF-89976A (5 and 25 microM) nor NO-711 (2 microM) altered the 2:1 charge flux/GABA flux ratio. The results are not consistent with previous hypotheses that (i) GABA evokes an uncoupled channel-mediated current in GAT1, and (ii) GAT1 inhibitors block the putative uncoupled current gated by GABA. Rather, the results suggest tight coupling of GAT1-mediated charge flux and GABA flux.