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.Â 1. WT GAT1, but not GAT1 C74A, is sensitive to membrane-impermeant sulfhydryl reagents. a A representative GABA-evoked (500Â Î¼M) 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 ~50Â % reduction in the GABA-evoked current. The membrane potential (Vm) was â50Â mV throughout the experiment. Labeling was carried out at 1Â mM MTSET for 5Â min in NaCl buffer bathing the oocyte (at 21Â Â±Â 2Â Â°C, pH 7.4). b Similar to WT GAT1, GAT1 C74A mediates Na+-dependent and Clâ-facilitated GABA transport (see Fig.Â 6). Exposure of GAT1 C74A to MTSET (1Â mM for 5Â min) had no effect on the magnitude of the GABA-evoked (500Â Î¼M) 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 12Â mM for 10Â 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 1Â mM for 5Â min at â50Â mV) was normalized to that prior to sulfhydryl modification in the same cell. Reported values represent the meanÂ Â±Â 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.Â 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 Clâ of the NaCl buffer had been isosmotically replaced by another cation or anion, respectively. The experimental protocol was similar to that shown in Fig.Â 1 (5-min exposure to 1Â mM MTSET at â50Â mV, 21Â Â±Â 2Â Â°C, pH 7.4) with the exception that sulfhydryl modification was carried out in the indicated buffer. The GABA-evoked (500Â Î¼M in NaCl buffer) current after MTSET modification was normalized to that obtained in the same cell before exposure to MTSET. Reported values represent the meanÂ Â±Â SE from three or more oocytes
	Fig.Â 3. Effect of membrane potential, pH and temperature on MTSET labeling of WT GAT1. The GABA-evoked (500Â Î¼M at â50Â mV) current mediated by WT GAT1 was measured before and after MTSET labeling (1Â mM for 5Â 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 â50Â 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 21Â Â°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 meanÂ Â±Â SE from three or more oocytes
	Fig.Â 4. Effect of MTSET concentration and labeling duration on sulfhydryl modification of WT GAT1. a Sulfhydryl modification of WT GAT1 was carried out for 5Â min in NaCl buffer in the presence of the indicated concentration of MTSET. b Sulfhydryl modification of WT GAT1 was carried out at 1Â mM MTSET for 5Â min in the presence of the indicated concentration of valproate. [Na+]Â =Â 100Â mM. c Duration of labeling with 1 or 2.5Â mM MTSET was varied (up to 20Â 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 â50Â mV. In all experiments, the GABA-evoked (500Â Î¼M in NaCl buffer at â50Â mV) current after MTSET modification was normalized to that obtained in the same cell before exposure to MTSET. Reported values represent the meanÂ Â±Â SE from three or more oocytes
	Fig.Â 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 1Â mM MTSET for 5Â min (b). VmÂ =Â â50Â mV. c Cells expressing GAT1 were exposed to 500Â Î¼M GABA and 20 nM [3H]-GABA for 5â10Â min. After washout of GABA and isotope, cells were solubilized in 10Â % 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 2.1Â Â±Â 0.1 whether or not the cell was exposed to MTSET (nÂ =Â 8 and 8). The smooth line in (c) is a linear regression through all data points
	Fig.Â 6. Steady-state kinetic parameters of WT GAT1 before and after sulfhydryl modification with MTSET. Representative GABA (aâc), Na+ (dâf) and Clâ (gâi) steady-state kinetic curves are shown for WT GAT1 before (left panels) and after (middle panels) treatment with MTSET (1Â mM for 5Â min at â50Â mV) as well as for GAT1 C74A without MTSET treatment (right panels). The half-maximal concentration values (K0.5) for GABA, Na+ and Clâ were similar for WT GAT1 and GAT1 C74A. The Hill coefficient values for Na+ and Clâ 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. VmÂ =Â â50Â mV. For GABA kinetic experiments (aâc), [Na+]0 was 100Â mM and [Clâ] was 106Â mM. For sodium kinetic experiments (dâf), [Clâ] was 106Â mM and [GABA]0 was 5Â mM. For chloride kinetic experiments (gâi), [Na+]0 was 100Â mM and [GABA]0 was 5Â mM. The smooth lines represent fits of the experimental data with Eq.Â 1 (see âExperimental Proceduresâ Section). Reported values represent the meanÂ Â±Â SE from three or more oocytes
	Fig.Â 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 (500Â Î¼M at â50Â mV) current with SKF-89976A (a specific inhibitor of GAT1) following sulfhydryl modification with MTSET (1Â mM for 5Â min at â50Â 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 (1Â mM for 5Â min at â50Â 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 (bâg) represent fits of the experimental data with an equation for competitive inhibition at a single binding site (Eq.Â 4, see âExperimental Proceduresâ section). Reported Ki values represent the meanÂ Â±Â SE from three or more oocytes
	Fig.Â 8. Presteady-state charge movements of WT GAT1 before and after sulfhydryl modification with MTSET. aâc Representative current relaxations are shown in response to 400-ms voltage pulses from +80 to â130Â mV for WT GAT1 before (a) and after (b) treatment with MTSET (1Â mM for 5Â min) as well as for GAT1 C74A without MTSET treatment (c). Holding potential was â50Â mV. dâf 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 QâV relationship for WT GAT1 before (d) and after (e) labeling with MTSET as well as for GAT1 C74A (f). The smooth lines in (dâf) represent the fit of the data with a Boltzmann function (Eq.Â 3, see âExperimental Proceduresâ section). gâi Presteady-state currents monoexponentially decay to the zero level. The time constant plotted as a function of the test voltage yielded the ÏâV relationship. The smooth lines in (gâi) 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 QâV 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.Â 9. Sulfhydryl modification with MTSET has no effect on WT GAT1 turnover rate. The ratio of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ I_{\text{NaCl}}^{\text{GABA}} $$\end{document} to QNaCl is used as an index of the transporter turnover rate (Gonzales et al. 2007). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ I_{\text{NaCl}}^{\text{GABA}} $$\end{document} represents the maximum GABA-evoked current and was measured at 5Â mM GABA and â50Â mV (in NaCl buffer). QNaCl represents the total charge moved in response to ON voltage pulses and was measured in NaCl buffer according to the procedures described for the experiments of Fig.Â 8. Sulfhydryl modification with MTSET had no effect on the ratio of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ I_{\text{NaCl}}^{\text{GABA}} $$\end{document} to QNaCl (4.1Â Â±Â 0.2 vs. 4.0Â Â±Â 0.2Â sâ1, nÂ =Â 4 and 4). The ratio was also similar in GAT1 C74A (4.1Â Â±Â 0.2Â sâ1, nÂ =Â 7)

References [+] :

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