Abstract
2’-Methoxy-6-methylflavone (2’MeO6MF) is an anxiolytic flavonoid which has been shown to display GABAA receptor (GABAAR) β2/3-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 α2β3γ2L GABAARs 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 α2β3γ2L GABAARs 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 (α2, β3, γ2L, α2β3, α2γ2L, β3γ2L and α2β3γ2L) in Xenopus oocytes using the two-electrode voltage clamp technique. We found that 2’MeO6MF activated non-α-containing β3γ2L 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 β3:γ2L 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 β3γ2L 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 α-lacking β3γ2L receptors raises questions about the structural requirements for their respective binding sites at GABAARs.
Highlights
Materials and MethodsGABA, flumazenil, zinc chloride (ZnCl2), DMSO and all buffer ingredients mentioned in this study were purchased from Sigma-Aldrich
In contrast to what was reported by Karim et al (2012) [30], we found that 300 μM 2’MeO6MF only activated α2β3γ2L receptors 8.2 ± 3.6% of the 3 mM GABA response (n = 5; Fig 1E and 1F)
We hypothesised that the variations in the relative efficacy of 2’MeO6MF measured at α2β3γ2L receptors expressed at different ratios may be due to the presence of mixed receptor populations
Summary
GABA, flumazenil, zinc chloride (ZnCl2), DMSO and all buffer ingredients mentioned in this study were purchased from Sigma-Aldrich Maximal GABA current responses (usually 3 mM, unless otherwise stated) were used as controls to allow for comparisons between different oocytes. Raw data from GABA concentration-response experiments were fitted to either a monophasic (1) or biphasic (2) Hill equation, and the fitted maximal values from the preferred model (Extra sum-of-squares F test) were used for normalisation of each dataset. Concentration-response curves of other agonists such as 2’MeO6MF and etomidate were constructed in a similar manner, except the data were normalised to the 3 mM GABA responses, unless otherwise stated. Statistical comparisons of results for different injection ratios of β3γ2L receptors were performed using ANOVA with Tukey’s posthoc test.
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