Protons modulate gating of recombinant α1β2γ2 GABAA receptor by affecting desensitization and opening transitions

Electrophysiology of ionotropic GABA receptors

“…All GABA A receptor subtypes seem to be insensitive to intracellular pH [275] (reviewed in [4]), but certain subtypes are sensitive to extracellular pH because of their two extracellular proton-binding sites [170,275]. Conductance is negatively correlated with extracellular pH for α1β1, α1β2γ2 and α1β1δ [170,276], and positively for ρ subtypes [277,278], while α1β1γ2S is pH-insensitive [170] and the curves of α1β1γ2Sδ, α4β3γ2, α4β3δ and α1β2γ2S conductance as a function of extracellular pH are bell-shaped [73,170]. Extracellular protons slow the deactivation of the α1β3δ subtype and increase its opening probability in the absence of GABA without affecting its desensitization, resulting in an increase of currents at low pH [181].…”

GABAA Receptor β2E155 Residue Located at the Agonist-Binding Site Is Involved in the Receptor Gating

“…Expression of a pure fraction of α 1 β 2 E155Cγ 2 mutants is problematic because of a possibility to express either α 1 β 2 -or α 1 γ 2 -type receptors (Tretter et al, 1997;Brodzki et al, 2016). Typically, to reduce the expression of α 1 β 2 receptors, the γ 2 subunit is overexpressed (Boileau et al, 2005;Jatczak-Śliwa et al, 2018;Kisiel et al, 2018Kisiel et al, , 2019 and therefore, plasmids encoding α 1 , β 2 , and γ 2L subunits were added to a transfection solution at a 1:1:3 ratio. However, in the case of the α 1 β 2 E155Cγ 2 mutants, we observed a marked transfection-to-transfection variability in the time course and amplitude of recorded currents, which could be essentially avoided by the additional overexpression of the β 2 subunit.…”

“…For agonist concentration higher than 10 mM, it was necessary to reduce NaCl/KCl concentration to 87 mM to maintain the osmolarity at a constant level. In this case, the external solution was supplemented with glucose and the internal solution with 50 mM K-gluconate (Wagner et al, 2004;Szczot et al, 2014;Kisiel et al, 2019). All experiments were performed at room temperature (20-23 • C).…”

“…Considering that macroscopic recordings (rapid agonist application) are performed in dynamic conditions, whereas single-channel experiments are made in steady-state conditions, it is of interest to be able to compare the kinetic features of the receptors in these two situations (Kisiel et al, 2019). To assess the maximum open probability (P openMax ) in the dynamic conditions, we have used NSVA (Sigworth, 1980) for currents elicited by rapid agonist application.…”

“…Single-channel kinetic analysis was performed using SCAN and EKDIST software (DCProgs), which was given to our group by David Colquhoun (UCL, London). As described in detail in our recent study (Kisiel et al, 2018(Kisiel et al, , 2019, single-channel traces (stored in the form of *.abf-Axon Binary File) selected for analysis contained ∼10,000 events. Filtering of the trace was performed in two stages.…”