Radioligand binding and patch‐clamp techniques were used to study the actions of γ‐aminobutyric acid (GABA) and the general anaesthetics propofol (2,6‐diisopropylphenol), pentobarbitone and 5α‐pregnan‐3α‐ol‐20‐one on rat α1 and β3 GABA_A receptor subunits, expressed either alone or in combination. Membranes from HEK293 cells after transfection with α1 cDNA did not bind significant levels of [³⁵S]‐tert‐butyl bicyclophosphorothionate ([³⁵S]‐TBPS) (<0.03 pmol mg⁻¹ protein). GABA (100 μm) applied to whole‐cells transfected with α1 cDNA and clamped at −60 mV, also failed to activate discernible currents. The membranes of cells expressing β3 cDNAs bound [³⁵S]‐TBPS (∼1 pmol mg⁻¹ protein). However, the binding was not influenced by GABA (10 nm–100 μm). Neither GABA (100 μm) nor picrotoxin (10 μm) affected currents recorded from cells expressing β3 cDNA, suggesting that β3 subunits do not form functional GABA^A receptors or spontaneously active ion channels. GABA (10 nm–100 μm) modulated [³⁵S]‐TBPS binding to the membranes of cells transfected with both α1 and β3 cDNAs. GABA (0.1 μm–1 mm) also dose‐dependently activated inward currents with an EC₅₀ of 9 μm recorded from cells transfected with α1 and β3 cDNAs, clamped at −60 mV. Propofol (10 nm–100 μm), pentobarbitone (10 nm–100 μm) and 5α‐pregnan‐3α‐ol‐20‐one (1 nm–30 μm) modulated [³⁵S]‐TBPS binding to the membranes of cells expressing either α1β3 or β3 receptors. Propofol (100 μm), pentobarbitone (1 mm) and 5α‐pregnan‐3α‐ol‐20‐one (10 μm) also activated currents recorded from cells expressing α1β3 receptors. Propofol (1 μm–1 mm) and pentobarbitone (1 mm) both activated currents recorded from cells expressing β3 homomers. In contrast, application of 5α‐pregnan‐3α‐ol‐20‐one (10 μm) failed to activate detectable currents. Propofol (100 μm)‐activated currents recorded from cells expressing either α1β3 or β3 receptors reversed at the C1⁻ equilibrium potential and were inhibited to 34±13% and 39±10% of control, respectively, by picrotoxin (10 μm). 5α‐Pregnan‐3α‐ol‐20‐one (100 nm) enhanced propofol (100 μm)‐evoked currents mediated by α1β3 receptors to 1101±299% of control. In contrast, even at high concentration 5α‐pregnan‐3α‐ol‐20‐one (10 μm) caused only a modest facilitation (to 128±12% of control) of propofol (100 μm)‐evoked currents mediated by β3 homomers. Propofol (3–100 μm) activated α1β3 and β3 receptors in a concentration‐dependent manner. For both receptor combinations, higher concentrations of propofol (300 μm and 1 mm) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a ‘surge’ current on cessation of propofol (300 μm and 1 mm) application. Surge currents were also evident following pentobarbitone (1 mm) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5α‐pregnan‐3α‐ol‐20‐one (10 μm) to cells expressing α1β3 receptors. These observations demonstrate that rat β3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABA_A receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids. British Journal of Pharmacology (1997) 120, 899–909; doi:10.1038/sj.bjp.0700987

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