Omoto JJ et al. (2012), Functional consequences of sulfhydryl modificat...

XB-ART-45902

J Membr Biol 2012 Dec 01;24512:841-57. doi: 10.1007/s00232-012-9492-9.

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Functional consequences of sulfhydryl modification of the γ-aminobutyric acid transporter 1 at a single solvent-exposed cysteine residue.

Omoto JJ , Maestas MJ , Rahnama-Vaghef A , Choi YE , Salto G , Sanchez RV , Anderson CM , Eskandari S .

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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.

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Species referenced: Xenopus laevis
Genes referenced: slc6a1 tbx2

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	Fig. 1. WT GAT1, but not GAT1 C74A, is sensitive to membrane-impermeant sulfhydryl reagents. a A representative GABA-evoked (500 μM) current trace is shown for WT GAT1 before and after labeling with the membrane-impermeant sulfhydryl reagent MTSET. Labeling of GAT1 with MTSET led to ~50 % reduction in the GABA-evoked current. The membrane potential (Vm) was −50 mV throughout the experiment. Labeling was carried out at 1 mM MTSET for 5 min in NaCl buffer bathing the oocyte (at 21 ± 2 °C, pH 7.4). b Similar to WT GAT1, GAT1 C74A mediates Na+-dependent and Cl−-facilitated GABA transport (see Fig. 6). Exposure of GAT1 C74A to MTSET (1 mM for 5 min) had no effect on the magnitude of the GABA-evoked (500 μM) current. c Sulfhydryl modification of WT GAT1 with MTSET was completely reversed with DTT. Labeling with MTSET was carried out as in (a), and DTT was applied at 12 mM for 10 min in NaCl buffer. d Summary of data collected from four or more oocytes expressing WT GAT1 or GAT1 C74A. For each experimental condition, the GABA-evoked current obtained after labeling with MTSET, NEM or TMR6M (all at 1 mM for 5 min at −50 mV) was normalized to that prior to sulfhydryl modification in the same cell. Reported values represent the mean ± SE from four or more oocytes. Note that GAT1 C74A is insensitive to the membrane-impermeant MTSET but sensitive to the membrane-permeant NEM. GAT1 C74A was also sensitive to TMR6M
	Fig. 2. Effect of buffer composition on MTSET labeling of WT GAT1. WT GAT1 was labeled with MTSET in the indicated extracellular bathing solution, in which Na+ and/or Cl− of the NaCl buffer had been isosmotically replaced by another cation or anion, respectively. The experimental protocol was similar to that shown in Fig. 1 (5-min exposure to 1 mM MTSET at −50 mV, 21 ± 2 °C, pH 7.4) with the exception that sulfhydryl modification was carried out in the indicated buffer. The GABA-evoked (500 μM in NaCl buffer) current after MTSET modification was normalized to that obtained in the same cell before exposure to MTSET. Reported values represent the mean ± SE from three or more oocytes
	Fig. 3. Effect of membrane potential, pH and temperature on MTSET labeling of WT GAT1. The GABA-evoked (500 μM at −50 mV) current mediated by WT GAT1 was measured before and after MTSET labeling (1 mM for 5 min) at different membrane potential values (a), extracellular pH values (b) and experimental temperatures (c). In all experiments, labeling was carried out in NaCl buffer. Labeling was performed at the indicated membrane potential values for the experiments of a and at −50 mV for the experiments of (b) and (c). Labeling was performed at pH 7.4 for the experiments of (a) and (c) and at the indicated values for the experiments of (b). Labeling was performed at 21 °C for the experiments of (a) and (b) and at the indicated values for the experiments of (c). The GABA-evoked current after MTSET modification was normalized to that obtained in the same cell before exposure to MTSET. Reported values represent the mean ± SE from three or more oocytes
	Fig. 4. Effect of MTSET concentration and labeling duration on sulfhydryl modification of WT GAT1. a Sulfhydryl modification of WT GAT1 was carried out for 5 min in NaCl buffer in the presence of the indicated concentration of MTSET. b Sulfhydryl modification of WT GAT1 was carried out at 1 mM MTSET for 5 min in the presence of the indicated concentration of valproate. [Na+] = 100 mM. c Duration of labeling with 1 or 2.5 mM MTSET was varied (up to 20 min) in NaCl, LiCl or Na-valproate buffer for WT GAT1 or GAT1 C74A. Note that under all conditions GAT1 C74A was functionally insensitive to MTSET exposure. See text for the second-order rate constants for MTSET labeling of WT GAT1 under different conditions. In all experiments, labeling was carried out at −50 mV. In all experiments, the GABA-evoked (500 μM in NaCl buffer at −50 mV) current after MTSET modification was normalized to that obtained in the same cell before exposure to MTSET. Reported values represent the mean ± SE from three or more oocytes
	Fig. 5. Sulfhydryl modification of WT GAT1 with MTSET does not alter the ion/GABA transport coupling ratio. GABA-uptake experiments were performed under voltage clamp without MTSET treatment (a) or after labeling with 1 mM MTSET for 5 min (b). Vm = −50 mV. c Cells expressing GAT1 were exposed to 500 μM GABA and 20 nM [3H]-GABA for 5–10 min. After washout of GABA and isotope, cells were solubilized in 10 % SDS and intracellular GABA content was determined using a liquid scintillation counter. In the same cell, the net inward charge flux was obtained from the time integral of the GABA-evoked current trace. The ratio of charge flux to GABA flux (i.e., net positive charges per GABA, e/GABA) was 2.1 ± 0.1 whether or not the cell was exposed to MTSET (n = 8 and 8). The smooth line in (c) is a linear regression through all data points
	Fig. 6. Steady-state kinetic parameters of WT GAT1 before and after sulfhydryl modification with MTSET. Representative GABA (a–c), Na+ (d–f) and Cl− (g–i) steady-state kinetic curves are shown for WT GAT1 before (left panels) and after (middle panels) treatment with MTSET (1 mM for 5 min at −50 mV) as well as for GAT1 C74A without MTSET treatment (right panels). The half-maximal concentration values (K0.5) for GABA, Na+ and Cl− were similar for WT GAT1 and GAT1 C74A. The Hill coefficient values for Na+ and Cl− activation of the inward currents were also similar for WT GAT1 and GAT1 C74A. Moreover, MTSET exposure had no effect on WT GAT1 steady-state kinetic parameters. Vm = −50 mV. For GABA kinetic experiments (a–c), [Na+]0 was 100 mM and [Cl−] was 106 mM. For sodium kinetic experiments (d–f), [Cl−] was 106 mM and [GABA]0 was 5 mM. For chloride kinetic experiments (g–i), [Na+]0 was 100 mM and [GABA]0 was 5 mM. The smooth lines represent fits of the experimental data with Eq. 1 (see “Experimental Procedures” Section). Reported values represent the mean ± SE from three or more oocytes
	Fig. 7. Sulfhydryl modification with MTSET has no effect on WT GAT1 sensitivity to transport inhibitors. a A representative WT GAT1 current trace is shown for inhibition of the GABA-evoked (500 μM at −50 mV) current with SKF-89976A (a specific inhibitor of GAT1) following sulfhydryl modification with MTSET (1 mM for 5 min at −50 mV). Similar to that shown in this panel, inhibition kinetics experiments were performed with SKF-89976A or NO-711 for WT GAT1 without (b, e) or with (c, f) prior exposure to MTSET (1 mM for 5 min at −50 mV) as well as for GAT1 C74A without MTSET treatment (d, g). Sulfhydryl modification of WT GAT1 had no effect on the Ki values for SKF-89976A or NO-711. Moreover, the Ki values for SKF-89976A (d) and NO-711 (g) inhibition of GAT1 C74A GABA-evoked currents were not different from those of WT GAT1. The smooth lines in (b–g) represent fits of the experimental data with an equation for competitive inhibition at a single binding site (Eq. 4, see “Experimental Procedures” section). Reported Ki values represent the mean ± SE from three or more oocytes
	Fig. 8. Presteady-state charge movements of WT GAT1 before and after sulfhydryl modification with MTSET. a–c Representative current relaxations are shown in response to 400-ms voltage pulses from +80 to −130 mV for WT GAT1 before (a) and after (b) treatment with MTSET (1 mM for 5 min) as well as for GAT1 C74A without MTSET treatment (c). Holding potential was −50 mV. d–f For each evoked current trace, time integration of the ON presteady-state currents yielded the total charge moved (QON) and, when plotted as a function of the test voltage, the Q–V relationship for WT GAT1 before (d) and after (e) labeling with MTSET as well as for GAT1 C74A (f). The smooth lines in (d–f) represent the fit of the data with a Boltzmann function (Eq. 3, see “Experimental Procedures” section). g–i Presteady-state currents monoexponentially decay to the zero level. The time constant plotted as a function of the test voltage yielded the τ–V relationship. The smooth lines in (g–i) represent the fit of the data with a gaussian function (Sacher et al. 2002). While MTSET treatment led to a reduction in the total charge moved (compare d and e), it had no effect on the midpoint of the Q–V relationship (V0.5) and no effect on the relaxation time constants (compare g and h). The presteady-state parameters of GAT1 C74A were not different from those of WT GAT1. See text for additional details
	Fig. 9. Sulfhydryl modification with MTSET has no effect on WT GAT1 turnover rate. The ratio of to QNaCl (4.1 ± 0.2 vs. 4.0 ± 0.2 s−1, n = 4 and 4). The ratio was also similar in GAT1 C74A (4.1 ± 0.2 s−1, n = 7)

References [+] :

Anderson, GATMD: γ-aminobutyric acid transporter mutagenesis database. 2010, Pubmed