Whole-cell patch-clamp recordings

Whole-cell patch-clamp remains the gold standard for recording the activation of ligand-gated ion channels, and for the testing of disease variants in epilepsy, and many other neurological and psychiatric disorders (Yuan et al., 2015). No other single technique can generate data that establish the presence of functional receptors on the cell surface and simultaneously define how a variant alters the activation of the receptor on a millisecond timescale. When combined with the recombinant expression of a homogeneous population of variant receptors, the data generated provide the most accurate assessment of variant impact on the amplitude and time course of disease-associated changes in membrane excitability.

Whole-cell patch-clamp recording was performed on HEK293T cells expressing α_xβ_xγ_2s GABA_A receptors and GFP, similar to methods previously described (Williams et al., 2010). Patch pipettes were fabricated from thin-walled borosilicate glass (TW150F-4, World Precision Instruments, Inc.) using a horizontal puller (P-97, Sutter Instruments, Inc.) to give a resistance of 2–8 MΩ when filled with intracellular solution (120 mM KCl, 2 mM MgCl₂, 10 mM EGTA, and 10 mM HEPES, adjusted to pH 7.2 with NaOH, 315 mOsm). Extracellular solution contained 161 mM NaCl, 3 mM KCl, 1 mM MgCl₂, 1.5 mM CaCl₂, 10 mM HEPES, and 6 mM d-glucose, adjusted to pH 7.4 with NaOH (320–330 mOsm). A rapid solution changer (RSC-160, BioLogics Science Instruments) connected to a 10-channel infusion pump (KD Scientific Inc.) was used to deliver GABA and picrotoxin solutions. The rapid solution changer was controlled by protocols written in pClamp 9 (Molecular Devices, LLC). Whole-cell currents were recorded at −60 mV, filtered at 100 Hz, and sampled at 200 Hz with a MultiClamp 700B amplifier and DigiData 1322A digitizer (Molecular Devices, LLC).

GABA concentration-response assays were performed by exposing each whole-cell patch to increasing concentrations of GABA (0.3, 1, 3, 10, 30, 100, 300 and 1000 μM) for 2 s, with an 8-s washout between concentrations. Recordings were baseline corrected and analysed in MATLAB (MathWorks, Inc.). Peak currents (I) were measured from GABA exposures and fitted using least-of-squares non-linear regression analysis based on the Hill equation: I = I_max × [A]^nH / (EC₅₀^nH + [A]^nH), where I is current peak amplitude, I_max is maximum current amplitude, EC₅₀ is the GABA concentration producing the half-maximal response, A is agonist concentration, and nH is the Hill coefficient. GABA concentration-response assays were individually fitted to the Hill equation for each whole-cell recording. The maximum peak current, EC₅₀, and Hill coefficient were estimated based on averaged values for each mutant receptor and are shown as mean ± standard error of the mean (SEM). The EC₅₀, also known as apparent-affinity, is a compound measure of the binding affinity and gating efficacy of GABA for the receptor (Colquhoun, 1998).

The degree of baseline leak current for cells expressing α₂- and α₂(T292K)-containing receptors was calculated using whole-cell recordings from GABA concentration-response assays. The first 41 points (0.2 s) of whole-cell baseline current in extracellular solution was averaged for each patch to give a measurement of baseline leak. This was performed for all eight concentrations in the GABA concentration assay, and the values averaged for each cell.

Desensitization was measured for α₅- and α₅(V294L)-containing receptors from the whole-cell recordings of GABA concentration-response assays. Whole-cell analysis of desensitization was performed using previously described methods (Moody et al., 2017). Briefly, desensitization was measured from 2-s GABA exposures as follows: (I_peak − I_end) / I_peak × 100, where I_peak was the amplitude of the total peak current response and I_end was the amplitude of the peak current response at the end of the agonist exposure (at 2 s). For each cell, desensitization was measured for the eight GABA concentration responses. The log-linear concentration-desensitization relationship was fitted by linear regression. The slope of this function describes the extent of desensitization as GABA concentration increased.