Expression of recombinant GABAA receptors and two-electrode voltage-clamp recordings from Xenopus oocytes

Oocytes from Xenopus laevis (purchased from Xenopus 1, Dexter, MI) were used as the expression system for recombinant GABA_A receptors. Oocyte harvest and animal handling were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol for use of frogs was approved by the Animal Studies Committee of Washington University in St. Louis (Approval No. 20170071).

Electrophysiological recordings were conducted on rat GABA_A receptors consisting of β2-α1-γ2L and β2-α1 concatemeric constructs, which have been shown to assemble as βαγβα (counterclockwise, top view)⁴⁰. The generation and functional characterization of the concatemeric receptors have been reported previously⁴¹. The cDNAs in the pcDNA3 expression vector were linearized by digestion with Xba I (NEB Labs, Ipswich, MA). The cRNAs were produced using mMessage mMachine (Ambion, Austin, TX). Oocytes were injected with a total of 18 ng of cRNA in nuclease-free water (final volume 32 nl) and incubated in ND96 with supplements (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, and the supplements 2.5 mM Na pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamycin; adjusted to pH 7.4 with NaOH) at 16 °C. Electrophysiological recordings were conducted during the next 2–3 days.

Recordings were conducted using standard two-electrode voltage clamp. The oocytes were clamped at −60 mV. The chamber (RC-1Z, Warner Instruments, Hamden, CT) was perfused with bath (ND96) or bath containing the drugs at approximately 7 ml/min. The current responses were amplified with an Axoclamp 900 A amplifier (Molecular Devices, Sunnyvale, CA), filtered at 10 Hz, and digitized with a Digidata 1320 digitizer (Molecular Devices) at a 50 Hz sampling rate. The traces were subsequently analyzed with Clampfit (Molecular Devices) to determine the peak amplitude of the current response.

Receptor activity was analyzed in the co-agonist concerted transition model framework^18,42. Raw current amplitudes were converted to units of open probability (P_o)^43,44. To that end, the current responses from each cell were compared to the reference response to 3 mM GABA + 50 μM propofol, that was considered to generate a response with a P_o indistinguishable from 1^21,45. For example, exposure to saturating GABA produced a peak response that was 86 ± 8% (n = 9 cells) of the response to 3 mM GABA + 50 μM propofol. Accordingly, the maximal P_o for GABA was estimated at 0.86 ± 0.08. This is similar to previous estimates of maximal P_o for GABA in the ternary αβγ receptor^21,46,47. The level of activity in the absence of any GABAergic drugs (P_o = 0.00011²²) was excluded in these calculations.

The binding and gating parameters were determined from fitting the pooled estimated P_o data to the following equation^18,48:

where K is the closed receptor equilibrium dissociation constant for the drug (GABA, propofol, alfaxalone, or diazepam), c is a measure of receptor gating efficacy expressed as the ratio of the equilibrium dissociation constant of the open receptor to that of the closed receptor, and N is the number of binding sites for the agonist (constrained to 2 for GABA⁴⁹, 3 for propofol²¹, 2 for alfaxalone⁵⁰, and 1 for diazepam⁵¹). Stabilization energy provided by a drug was calculated as NRT × ln(c), where RT is the multiplication product of the gas constant and thermodynamic temperature, and other terms are as described above.

The parameter L reflects unliganded or background activity from the receptor. In the absence of any GABAergic agonists, it was constrained to 9000^21,22. In experiments where a low concentration of one GABAergic drug, i.e., a background drug, was coapplied with a range of concentrations of a second GABAergic agonist, L was calculated as (1 − P_o,background)/P_o,background, where P_o,background is the open probability of the response to the background drug. In a standard potentiation experiment, where several concentrations of a potentiator (propofol, alfaxalone or diazepam) were coapplied with a fixed low concentration of GABA, the response to GABA alone comprised the background response. Since exposure to 1 μM alfaxalone or 1 μM diazepam did not produce robust current responses, the P_o,background for alfaxalone and diazepam were calculated using Equation (¹) and the K and c values estimated for each drug in the presence of low GABA. Curve-fitting was carried out using Origin v. 7.5 (OriginLab Corp., Northampton, MA).