γ-aminobutyric acid (GABA) is the main inhibitory transmitter of the brain, and the GABAA receptor is the main element of the rapid inhibition mechanism (Sigel and Steinmann, 2012). The GABAA receptor contains the water pore through which negatively charged chloride ions enter the cell (IGABA), hyperpolarize the membrane and inhibit the generation of nerve impulses. A large number of clinically important pharmacological agents are able to bind to the GABAA receptor and influence the conductivity of its chloride pore (Thompson, 2024; Möhler, 2006). Many of them have their own binding sites on the GABAA receptor that do not coincide with the orthosteric GABA site (Olsen, 2018). When several active ligands act simultaneously on the GABAA receptor, the total resulting response may depend on the allosteric interaction between the sites (Germann et al., 2019; Shin et al., 2019; Olsen, 2018; Vale et al., 1997; Li et al., 2009; Bukanova et al., 2024a, 2024b). In the present study, we investigated the interaction between benzodiazepine binding site and competitive antagonists binding sites in GABAA receptor.

Benzodiazepine drugs (BDZs) are CNS depressants that act as positive modulators of the GABAA receptor (Stephenson and Olsen, 1982; Goldschen-Ohm, 2022). Both extracellular and transmembrane BDZs sites are described (Walters et al., 2000; Zhu et al., 2022). The extracellular site is localized at the α/γ interface of the GABAA receptor and is homologous to the transmitter binding site in the β/α interface. Holding the α and γ subunits together, the BDZs make the extracellular domain more compact which helps GABA to rotate the extracellular domain counterclockwise and facilitates the opening of the activation gate in the pore of the GABAA receptor (Hanson and Czajkowski, 2011; Masiulis et al., 2019). 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, three distinct low affinity densities were observed for diazepam in the transmembrane domain, 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). In our experiments we used zolpidem (Zolp) as an agonist of BDZ-sites in GABAA receptor (Zhu et al., 2022). Zolp is a well-known tranquilizer which is widely used to treat a number of neurological disorders (Bomalaski et al., 2017; Wisden et al., 2019).

The group of competitive GABAA receptor antagonists we studied included three substances: bicuculline, gabazine and amiloride. Bicuculline (Bic) and gabazine (GBZ) are classic competitive inhibitors of GABA binding to the GABAA receptor (Akk et al., 2011; Johnston, 2013; Krall et al., 2015). They bind to the orthosteric site of the receptor (Zhang et al., 2008) and thereby directly compete with GABA for the same binding site by stabilizing the resting state of the receptor (Ueno et al., 1997; Steinbach and Akk, 2019). However, it appears that Bic and GBZ not simply block the binding of the agonist but can induce conformational changes in the GABAA receptor which can influence the state of other sites (Ueno et al., 1997; Chang and Weiss, 1999; Xu et al., 2016). Due to complex action of Bic and GBZ on GABAA receptor they are called also allosteric inhibitors or inverse agonists (Ueno et al., 1997).

Amiloride (Ami) is able to interact with many molecular targets and may cause a variety of physiological effects (Sun and Sever, 2020). The most sensitive targets for Ami are epithelial sodium channels in kidneys (Kd = 0.2 μM) (Sun and Sever, 2020), thus it is used for the treatment of hypertension, hypokalemia, edema, and congestive heart failure (Sun and Sever, 2020). One of the molecular targets for Ami is the GABAA receptor, which is blocked by this drug with the IC50 values within 19–454 μM (Inomata et al., 1988; Fisher, 2002; Liu et al., 2010). Although the binding site of Ami on the GABAA receptor has not yet been determined, it is believed that it inhibits IGABA by a competitive mechanism, i.e. by interacting with the orthosteric site, as evidenced by a decrease in its inhibitory effect with increasing GABA concentration (Liu et al., 2010). The ability of Ami to affect the operation of other sites of GABAA receptor has been poor studied (Bukanova et al., 2024b). In this work, we show that competitive antagonists of orthosteric site of the GABAA receptor, Bic, GBZ and Ami, can inhibit the potentiating effect of Zolp on the IGABA.