Spontaneous activity, singly bound states and the impact of alpha 1 Phe64 mutation on GABA A R gating in the novel kinetic model based on the single-channel recordings

Distinct Modulation of Spontaneous and GABA-Evoked Gating by Flurazepam Shapes Cross-Talk Between Agonist-Free and Liganded GABAA Receptor Activity

“…Interestingly, for GABA-evoked currents, Gielen et al (2012) and Dixon et al (2015) suggested the impact of BDZs on the preactivation, but our recent report indicated that spontaneous openings do not require the preactivation step (Kisiel et al, 2018). To explore this problem, we compared the BDZ sensitivity of spontaneous activity mediated by WT receptors and by α 1 F64 mutants in which the preactivation step is impaired (Szczot et al, 2014; Kisiel et al, 2018). As expected, the WT receptor showed a low spontaneous activity revealed by 100 μM PTX ( Figures 1A,C ), but the mutations at the α 1 F64 residue strongly increased it ( Figures 1B,C ).…”

“…To further explore the modulation of spontaneous events by FLU, we conducted single-channel recordings as described by Kisiel et al (2018; see section “Materials and Methods”). Considering the similarity in the FLU effect on WT receptors and mutants, we limited these recordings to the WT and the CYS mutant ( Figures 2A,B ), which is most distinct from the native receptor (Kisiel et al, 2018).…”

“…Considering the similarity in the FLU effect on WT receptors and mutants, we limited these recordings to the WT and the CYS mutant ( Figures 2A,B ), which is most distinct from the native receptor (Kisiel et al, 2018). Open time distributions for WT and CYS receptors had two components with time constants that did not differ between these receptor types, but for CYS mutants the percentage of the slower component was larger ( Figures 2C,D,G ).…”

“…The saturating concentration of GABA for WT and mutated α 1 β 2 γ 2 GABA A R (at α 1 F64 residue) was previously determined by our group (Szczot et al, 2014) and was established as 100 mM [GABA] for leucine (LEU) and alanine (ALA) mutants, but this concentration was not fully saturating for the cysteine (CYS) mutant (Kisiel et al, 2018). In all experiments testing the impact of FLU, this compound was used at a concentration of 3 μM.…”

“…Spontaneous activity was described using the kinetic scheme proposed by Kisiel et al (2018), but to describe the modulation by FLU, an additional binding step for this modulator was added and the remaining rate constants were reassessed. To estimate the FLU binding/unbinding rates, ligand docking was used and the dissociation constant (K D ) was calculated using Eq.…”