Fig. 1. Pharmacological inhibition of GAT1-mediated GABA-evoked current ($I_{\mathrm{NaCl}}^{\mathrm{GABA}}$) does not reveal a channel component conductive to Na⁺ ions.

(A–C) A representative GAT1-mediated GABA-evoked current $I_{\mathrm{NaCl}}^{\mathrm{GABA}}$ trace is shown (−50 mV), and the corresponding current-voltage relationships are shown for voltages ranging from −140 mV to +100 mV. [GABA] = 500 μM. When measured at an extracellular Na⁺ concentration of 100 mM (B) or 50 mM (C), $I_{\mathrm{NaCl}}^{\mathrm{GABA}}$ did not show any evidence of reversal. Therefore, under the zero-trans conditions of our experiments, $I_{\mathrm{NaCl}}^{\mathrm{GABA}}$ does not have an outward component even at membrane potentials more positive than the predicted Na⁺ equilibrium potential. When tested at 25 μM, SKF-89976A inhibited the inward current evoked by 500 μM GABA by ∼65% (B). (D–F) Application of NO-711 alone (25 μM) (D) or SKF-89976A alone (30 μM) (E) to a cell expressing GAT1 did not reveal a constitutive leak mode, however, both agents inhibited $I_{\mathrm{NaCl}}^{\mathrm{GABA}}$ in a concentration dependent manner. A representative trace is shown for SKF-89976A at 500 μM GABA and −50 mV (F). The current scale bar is the same for panels D–F. The time scale bar is 4 minutes for panels D and E, and 15 minutes for panel F. (G) SKF-89976A inhibition of $I_{\mathrm{NaCl}}^{\mathrm{GABA}}$ was carried out at 10 μM, 25 μM, and 500 μM GABA. V_m = −50 mV. The data were adequately fitted with Eq. 1 for competitive inhibition at a single site. (H) Replotting the data in a Dixon plot yielded a K_i value of 700 nM for SKF-89976A inhibition of GAT1-mediated $I_{\mathrm{NaCl}}^{\mathrm{GABA}}$. (I) NO-711 inhibition of $I_{\mathrm{NaCl}}^{\mathrm{GABA}}$ was carried out at 10 μM, 25 μM, and 500 μM GABA. V_m = −50 mV. The data were adequately fitted with Eq. 1 for competitive inhibition at a single site. (J) Replotting the data in a Dixon plot yielded a K_i value of 95 nM for NO-711 inhibition of GAT1-mediated $I_{\mathrm{NaCl}}^{\mathrm{GABA}}$. In panels H and J, each data point was normalized to the maximum GABA-evoked current (I_max) obtained in the same cell. I_max was obtained at a saturating GABA concentration (5 mM) and in the absence of inhibitor.