Electrophysiological Recordings.

Whole-cell voltage-clamp recordings were performed at room temperature with an EPC-10 HEKA amplifier (HEKA Elektronik, Lambrecht, Germany). Cells were bathed in an external Ringer’s solution consisting of 160 mM NaCl, 4.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM HEPES, pH 7.4 and 308 mOsm. Recording electrodes were pulled from soda lime glass microhematocrit tubes (Kimble Chase, Rochester, NY) and fire-polished to resistances of 1.8–3 MΩ. Electrodes were filled with an internal solution consisting of 154 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 10 mM EGTA, pH 7.2 and 302 mOsm. Cells were voltage-clamped at −80 mV, and control currents were recorded under the local application of EC₉₀ GABA (100 μM) for 5 seconds to the patch-clamped cell using an 8-channel pinch valve–controlled gravity perfusion system (VC3-8xG system; ALA Scientific) positioned within 100 μm of the cell. GABA applications were followed by a 50-second wash with Ringer’s solution. Washes and TETS additions to the chamber were performed through a separate, syringe-driven perfusion system with a short perfusion line (length 236 mm, line volume 210 µl) and with a volume (2 ml) that exchanged the chamber volume five times. TETS was allowed to sit for 3 minutes on the cell before reapplication of EC₉₀ GABA directly onto the cell through the gravity perfusion system. Percentage of block was calculated using the area under the curve (AUC). One cell was used per concentration of the convulsant. Cells that became leaky during the experiment or that did not produce the same magnitude of response to EC₉₀ GABA twice before the experiment and after washout of TETS were excluded from the analysis. For screening of mutant channels, 50 μM TETS and EC₉₀ GABA were used to evaluate whether the mutation affected TETS potency. Percentage of current blocked (mean ± S.D. from n = 5–8 cells per mutant) was analyzed with one-way ANOVA followed by Dunnett’s test to compare the means with the wild-type control and to correct for multiple comparisons. *P < 0.05; **P < 0.01; and ***P < 0.001.

Concentration-response curves for selected mutants were constructed by testing varying concentrations of TETS or picrotoxinin for their ability to block currents elicited by 100 µM GABA (=EC₉₀ GABA). For analysis of current blockade, the area under the current curve (AUC_Max) was determined for the control (EC₉₀ GABA) and the AUC_Ex after exposure. [AUC_Ex]/[AUC_Max] × 100 = % Current Blocked. Data analysis and data fitting to the Hill equation to obtain EC₅₀ or IC₅₀ values was performed using Origin 9.1 (OriginLab Corporation, Northampton, MA) software. Individual data points are presented as mean ± S.D. from five to eight independent recordings. EC₅₀ and IC₅₀ values are presented with 95% confidence intervals. Concentration-response curves were compared using an extra sum-of-squares F test (GraphPad Prism8; GraphPad Software, La Jolla, CA).