MODULATION BY GENERAL-ANESTHETICS OF RAT GABA(A)-RECEPTORS COMPRISED OF ALPHA-1-BETA-3-SUBUNIT AND BETA-3-SUBUNIT EXPRESSED IN HUMAN EMBRYONIC KIDNEY-293 CELLS

1 Radioligand binding and patch-clamp techniques were used to study th e actions of gamma-aminobutyric acid (GABA) and the general anaestheti cs propofol (2,6-diisopropylphenol), pentobarbitone and 5 alpha-pregna n-3 alpha-ol-20-one on rat alpha 1 and beta 3 GABA(A) receptor subunit s, expressed either alone or in combination. 2 Membranes from HEK293 c ells after transfection with alpha 1 cDNA did not bind significant lev els of [S-35]-tert-butyl bicyclophosphorothionate ([S-35]-TBPS) (<0.03 pmol mg(-1) protein). GABA (100 mu M) applied to whole-cells transfec ted with al cDNA and clamped at -60 mV, also failed to activate discer nible currents. 3 The membranes of cells expressing beta 3 cDNAs bound [S-35]-TBPS (similar to 1 pmol mg(-1) protein). However, the binding was not influenced by GABA (10 nM-100 mu M). Neither GABA (100 mu M) n or picrotoxin (10 mu M) affected currents recorded from cells expressi ng beta 3 cDNA, suggesting that beta 3 subunits do not form functional GABA(A) receptors or spontaneously active ion channels.4 GABA (10 nM- 100 mu M) modulated [S-35]-TBPS binding to the membranes of cells tran sfected with both alpha 1 and beta 3 cDNAs. GABA (0.1 mu M-1 mM) also dose-dependently activated inward currents with an EC(50) of 9 mu M re corded from cells transfected with alpha 1 and beta 3 cDNAs, clamped a t -60 mV. 5 Propofol (10 nM-100 mu M), pentobarbitone (10 nM-100 mu M) and 5 alpha-pregnan-3 alpha-ol-20-one (1 nM-30 mu M) modulated [S-35] -TBPS binding to the membranes of cells expressing either alpha 1 beta 3 or beta 3 receptors. Propofol (100 mu M), pentobarbitone (1 mM) and 5 alpha-pregnan-3 alpha-ol-20-one (10 mu M) also activated currents r ecorded from cells expressing alpha 1 beta 3 receptors. 6 Propofol (1 mu M-1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing beta 3 homomers. In contrast, application of 5 a lpha-pregnan-3 alpha-ol-20-one (10 mu M) failed to activate detectable currents.7 Propofol (100 mu M)-activated currents recorded from cells expressing either alpha 1 beta 3 or beta 3 receptors reversed at the Cl- equilibrium potential and were inhibited to 34+/-13% and 39+/-10% of control, respectively, by picrotoxin (10 mu M). 5 alpha-Pregnan-3 a lpha-ol-20-one (100 nM) enhanced propofol (100 mu M)evoked currents me diated by alpha 1 beta 3 receptors to 1101+/-299% of control. In contr ast, even at high concentration 5 alpha-pregnan-3 alpha-ol-20-one (10 mu M) caused only a modest facilitation (to 128+/-12% of control) of p ropofol (100 mu M))-evoked currents mediated by beta 3 homomers. 8 Pro pofol (3-100 mu M) activated alpha 1 beta 3 and beta 3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 mu M and 1 mM) caused a decline in cu rrent amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a 'surge' current on cessation of propofo l (300 mu M and 1 mM) application. Surge currents were also evident fo llowing pentobarbitone (1 mM) application to cells expressing either r eceptor combination. By contrast, this phenomenon was not apparent fol lowing applications of 5 alpha-pregnan-3 alpha-ol-20-one (10 mu M) to cells expressing alpha 1 beta 3 receptors. 9 These observations demons trate that rat beta 3 subunits form homomeric receptors that are not s pontaneously active, are insensitive to GABA and can be activated by s ome general anaesthetics. Taken together, these data also suggest simi lar sites on GABA(A) receptors for propofol and barbiturates, and a se parate site for the anaesthetic steroids.