Pharmacological characterization of a novel cell line expressing human α4β3δ GABAA receptors

The pharmacology of the stable cell line expressing human α4β3δ GABAA receptor was investigated using whole-cell patch-clamp techniques. α4β3δ receptors exhibited increased sensitivity to GABA when compared to α4β3γ2 receptors, with EC50's of 0.50 (0.46, 0.53) μM and 2.6 (2.5, 2.6) μM respectively. Additionally, the GABA partial agonists piperidine-4-sulphonate (P4S) and 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP) displayed markedly higher efficacy at α4β3δ receptors, indeed THIP demonstrated greater efficacy than GABA at these receptors. The δ subunit conferred slow desensitization to GABA, with rate constants of 4.8±0.5 s for α4β3δ and 2.5±0.2 s for α4β3γ2. However, both P4S and THIP demonstrated similar levels of desensitization on both receptor subtypes suggesting this effect is agonist specific. α4β3δ and α4β3γ2 demonstrated equal sensitivity to inhibition by the cation zinc (2–3 μM IC50). However, α4β3δ receptors demonstrated greater sensitivity to inhibition by lanthanum. The IC50 for GABA antagonists SR-95531 and picrotoxin, was similar for α4β3δ and α4β3γ2. Likewise, inhibition was observed on both subtypes at high and low pH. α4β3δ receptors were insensitive to modulation by benzodiazepine ligands. In contrast Ro15-4513 and bretazenil potentiated GABA responses on α4β3γ2 cells, and the inverse agonist DMCM showed allosteric inhibition of α4β3γ2 receptors. The efficacy of neurosteroids at α4β3δ receptors was greatly enhanced over that observed at α4β3γ2 receptors. The greatest effect was observed using THDOC with 524±71.6% potentiation at α4β3δ and 297.9±49.7% at α4β3γ2 receptors. Inhibition by the steroid pregnenolone sulphate however, showed no subtype selectivity. The efficacy of both pentobarbitone and propofol was slightly augmented and etomidate greatly enhanced at α4β3δ receptors versus α4β3γ2 receptors. We show that the α4β3δ receptor has a distinct pharmacology and kinetic profile. With its restricted distribution within the brain and unique pharmacology this receptor may play an important role in the action of neurosteroids and anaesthetics. Keywords: Delta subunit, GABAA receptor, anaesthetic, neurosteroid, alpha 4 subunit, benzodiazepine, desensitization, inhibitory neurotransmission, allosteric modulation Introduction GABA (γ-Aminobutyric acid) is the major inhibitory neurotransmitter in the mammalian central nervous system. Its primary action is through the GABAA receptor, which is composed of a family of functionally diverse subunits that assemble into a pentameric structure (McKernan & Whiting, 1996). To date there are 17 different subunits identified (α1–6, β1–3, γ1–3, ρ1–2, δ, e, θ). These subunits have discrete locations within the brain, but the most abundant receptor subtypes have been found to express α, β and γ subunits (Barnard, 1998; Sieghart, 1995). The GABAA receptor can be modulated by a number of therapeutic agents, including benzodiazepines, barbiturates, anaesthetics, ethanol and neuroactive steroids. The extent of this modulation is subunit specific. Recombinant studies have shown the α and γ subunits are responsible for benzodiazepine and zinc sensitivity (Pritchett et al., 1989; Draguhn et al., 1990). The role of subunits for other modulators remains the subject of investigation. The α4 and δ subunits have a very restricted distribution within the brain, but primarily co-localize in the thalamus and hippocampus (Sperk et al., 1997; Sur et al., 1999; Pirker et al., 2000). The α4 subunit is most homologous to the α6 subunit, both are insensitive to diazepam but have high affinity for the benzodiazepines Ro15-4513 and bretazenil (Wisden et al., 1991). The role of the δ subunit is currently still relatively unclear as this has proved to be very difficult to express in transient recombinant systems. In this study we have created a dexamethasone-inducible, stable cell line in mouse L(-tk) cells, which expresses the human α4β3δ GABAA subtype and the pharmacology of this δ subunit containing receptor is investigated and compared to a similar cell line expressing human α4β3γ2 GABAA receptors allowing a direct comparison of the δ subunit with γ2 containing receptors, using the whole-cell patch-clamp technique.