The Direct Actions of GABA, 2′-Methoxy-6-Methylflavone and General Anaesthetics at β3γ2L GABAA Receptors: Evidence for Receptors with Different Subunit Stoichiometries

2'-Methoxy-6-methylflavone (2'MeO6MF) is an anxiolytic flavonoid which has been shown to display GABA(A) receptor (GABA(A)R) beta 2/3-subunit selectivity, a pharmacological profile similar to that of the general anaesthetic etomidate. Electrophysiological studies suggest that the full agonist action of 2'MeO6MF at alpha 2 beta 3 gamma 2L GABA(A)Rs may mediate the flavonoid's in vivo effects. However, we found variations in the relative efficacy of 2'MeO6MF (2'MeO6MF-elicited current responses normalised to the maximal GABA response) at alpha 2 beta 3 gamma 2L GABA(A)Rs due to the presence of mixed receptor populations. To understand which receptor subpopulation(s) underlie the variations observed, we conducted a systematic investigation of 2'MeO6MF activity at all receptor combinations that could theoretically form (alpha 2, beta 3, gamma 2L, alpha 2 beta 3, alpha 2 gamma 2L, beta 3 gamma 2L and alpha 2 beta 3 gamma 2L) in Xenopus oocytes using the two-electrode voltage clamp technique. We found that 2'MeO6MF activated non-alpha-containing beta 3 gamma 2L receptors. In an attempt to establish the optimal conditions to express a uniform population of these receptors, we found that varying the relative amounts of beta 3:gamma 2L subunit mRNAs resulted in differences in the level of constitutive activity, the GABA concentration-response relationships, and the relative efficacy of 2'MeO6MF activation. Like 2'MeO6MF, general anaesthetics such as etomidate and propofol also showed distinct levels of relative efficacy across different injection ratios. Based on these results, we infer that beta 3 gamma 2L receptors may form with different subunit stoichiometries, resulting in the complex pharmacology observed across different injection ratios. Moreover, the discovery that GABA and etomidate have direct actions at the alpha-lacking beta 3 gamma 2L receptors raises questions about the structural requirements for their respective binding sites at GABA(A)Rs.