γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system which plays a fundamental role in maintaining the optimal balance between activation and inhibition (Sigel and Steinmann, 2012). GABA acts through two types of specific receptors, namely the ionotropic GABAA receptor (GABAAR) (Sieghart, 2006) and the slower metabotropic GABAB receptor (GABABR) (Shaye et al., 2021). Activation of GABAAR causes the opening of the receptors’ pore and the induction of the chloride current (IGABA), which hyperpolarizes the membrane and inhibits the generation of nerve impulses. GABAARs involves dozen or more heteropentameric subtypes containing different subunits (α1–6, β1–3, γ1–3, ρ1–3, ε, π, δ or θ) with the predominant receptor being α1β2γ2 and with a subunit stoichiometry of 2:2:1 (Sieghart, 2006). GABAAR is regulated by a wide variety of pharmacological agents, many of which have found clinical use (Möhler, 2006). Such regulation is due to the presence of high-affinity specific binding sites on GABAAR for certain molecules, for example, benzodiazepines, barbiturates, neurosteroids, penicillin, picrotoxin, etc. Under both physiologic and clinical conditions, GABAARs can be exposed to multiple active substances, and the functional output of the receptor reflects the interplay among all active agents (Shin et al., 2019). In literature, the experiments with a combination of different drugs demonstrate the interrelation of the main neurotransmitter/drug recognition sites of the GABAARs (Olsen, 2018; Vale et al., 1997; Li et al., 2009). In our previous work, we used various combinations of two groups of drugs, the positive allosteric modulators of GABAAR, allopregnanolone (Allo) and zolpidem (Zolp), and the competitive antagonists, bicuculline and gabazine. The blocking properties of competitive antagonists have been shown to weaken in the presence of PAMs (Bukanova et al., 2022). In present study, we used combinations of PAMs with a noncompetitive GABAAR antagonist, picrotoxin (PTX).

PTX is isolated from plants of the moonseed family and it is known to be the active nervous system stimulant (Masiulis et al., 2019). PTX is a molecular pair of two highly similar isomers: the active picrotoxinin and the much less active picrotin (Olsen 2018). The mechanism of action of PTX is associated with its ability to block the open chloride pore of GABAAR (Gielen et al., 2015). It was shown that PTX can interact with both resting and GABA-bound receptors, but their affinity for the latter is about 10 times greater than that for the former (Dillon et al., 1995; Inoue and Akaike, 1988.) The authors conclude that PTX reaches the binding site more easily via the hydrophilic rather than the hydrophobic pathway. The picrotoxinin is shown to be sequestered in the channel pore between the M2 2′ and 9′ rings (Masiulis et al., 2019). A point mutation T272Y (T6′Y in the second membrane-spanning domain) to the γ2 subunit imparts resistance to PTX (Sun et al., 2019). The effect of PTX on IGABA is use-dependent and shows a slow onset and offset. It was found that the recovery time after the blockade far exceeds the time for the effect development (Dillon et al., 1995; Korshoej et al., 2010; Gielen et al., 2015; Bali and Akabas, 2007). This aftereffect indicates that the blockade continues after PTX elimination from the external solution. Through allosteric interaction between the PTX site and the orthosteric site, the presence of PTX in the pore can cause a decrease in the affinity of the orthosteric site (Masiulis et al., 2019). The blockade by PTX is weakened, and its reversibility is facilitated with an increase in the concentration of the agonist (GABA). Such studies have been conducted on recombinant GABA receptors expressed in Xenopus oocytes, and the results are inconsistent (Goutman and Calvo, 2004; Gielen et al., 2015; Bali and Akabas, 2007). In the present work, the dependence of PTX effect on GABA concentration was studied on native neurons, namely, Purkinje cells of the rat cerebellum.

The effect of Allo on the blocking properties of PTX has not previously been studied. Allo is an endogenous neurosteroid (NS) that at nanomolar concentrations strongly facilitates IGABA in electrophysiological experiments (Lambert et al., 1995; Zorumski et al., 2019; Solntseva et al., 2022; Sugasawa et al., 2020). The sites for binding NSs are located in transmembrane domain (TMD) and do not overlap with the extracellular GABA-binding site (Hosie et al., 2007; Chen et al., 2019). Three discrete NS-binding sites were estimated, namely, α-intrasubnnit site, β-intrasubunit site and β-α intersubunit site (Miller et al., 2017; Sugasawa et al., 2020). Allo was shown to bind to all three sites (Chen et al., 2019; Germann et al., 2021), but β-α intersubunit site is considered to be the most important (Legesse et al., 2023). The binding of Allo increases sensitivity to GABA and increases ion channel width, thereby enhancing receptor activity (Legesse et al., 2023).

Zolpidem (Zolp) is also known as a positive GABAA receptor modulator (Zhu et al., 2022). Zolp is a well-known tranquilizer which is widely used to treat a number of neurological diseases (Bomalaski et al., 2017; Wisden et al., 2019). The mechanism of action of Zolp is associated with its ability to activate benzodiazepine (BDZ) site on the GABAAR (Arbitla et al., 1985; Pritchett et al., 1989). In Cryo-EM experiments, in addition to the density for diazepam at the classical high affinitive BDZ site in α-γ interface of extracellular domain (ECD), three distinct densities were observed for diazepam in the TMD, two at β-α interfaces and a third at the γ-β interface that overlaps with one of the phenobarbital sites (Kim et al., 2020). Occupancy of four sites by diazepam results in global stabilization compared to the GABA alone complex, and promotes global rotation of the TMD halves of all subunits, most noticeably in the β2 subunits (Kim et al., 2020).

Our experiments have shown that PAMs of GABAAR significantly accelerate the reversibility of the PTX blocking effect on Purkinje cells of the rat cerebellum. Purkinje cells were chosen for experiments because they generate a large GABA-induced chloride current with a minor cell-to-cell variability in EC50 values which corresponds to the homogeneity of the subunit composition of the GABAA receptors in these cells, α1β2γ2 (Laurie et al., 1992). In addition, the advantages of working with receptors on native neurons compared to recombinant receptors are that the former have a natural physiological status, for example, phosphorylation level.