Valerenic acid potentiates and inhibits GABA_A receptors: Molecular mechanism and subunit specificity

Abstract

Valerian is a commonly used herbal medicinal product for the treatment of anxiety and insomnia. Here we report the stimulation of chloride currents through GABA_A receptors (I_GABA) by valerenic acid (VA), a constituent of Valerian. To analyse the molecular basis of VA action, we expressed GABA_A receptors with 13 different subunit compositions in Xenopus oocytes and measured I_GABA using the two-microelectrode voltage-clamp technique. We report a subtype-dependent stimulation of I_GABA by VA. Only channels incorporating β₂ or β₃ subunits were stimulated by VA. Replacing β_2/3 by β₁ drastically reduced the sensitivity of the resulting GABA_A channels. The stimulatory effect of VA on α₁β₂ receptors was substantially reduced by the point mutation β_2N265S (known to inhibit loreclezole action). Mutating the corresponding residue of β₁ (β_1S290N) induced VA sensitivity in α₁β_1S290N comparable to α₁β₂ receptors. Modulation of I_GABA was not significantly dependent on incorporation of α₁, α₂, α₃ or α₅ subunits. VA displayed a significantly lower efficiency on channels incorporating α₄ subunits. I_GABA modulation by VA was not γ subunit dependent and not inhibited by flumazenil (1 μM). VA shifted the GABA concentration–effect curve towards lower GABA concentrations and elicited substantial currents through GABA_A channels at ≥30 μM. At higher concentrations (≥100 μM), VA and acetoxy-VA inhibit I_GABA. A possible open channel block mechanism is discussed. In summary, VA was identified as a subunit specific allosteric modulator of GABA_A receptors that is likely to interact with the loreclezole binding pocket.

Introduction

γ-Aminobutyric acid (GABA) mediates fast synaptic inhibition by interaction with the GABA type A (GABA_A) receptor. GABA_A receptors are assembled from individual subunits forming a pentameric structure. Nineteen isoforms of mammalian GABA_A receptor subunits have been cloned: α_1–6, β_1–3, γ_1–3, δ, ɛ, π, ρ_1–3 and θ (Barnard et al., 1998, Simon et al., 2004).

The subunit composition determines the GABA sensitivity and the pharmacological properties of the GABA_A receptor (Hevers and Lüddens, 1998, Sieghart, 1995, Boileau et al., 2002). Both binding sites for GABA and for benzodiazepines (BZDs) are assumed to be located at subunit interfaces (for review see Galzi and Changeux, 1995, Sigel and Buhr, 1997). Mutational studies suggest that the binding pocket for BZDs is located at the interface between α and γ subunits, whereas binding of GABA is believed to occur at the interfaces between α and β subunits (Sigel, 2002, Ernst et al., 2003, Ernst et al., 2005).

GABA_A channels are modulated by numerous structurally distinct substances including clinically important drugs as BZDs, barbiturates and various general anaesthetics (see Sieghart, 1995 for review), but also by several compounds of plant origin, including flavonoids, e.g. methyl-apigenin (Marder et al., 2003) or wogonin (from Scutellaria baicalensis, Hui et al., 2002, Huen et al., 2003), monoterpenes, e.g. borneol (Granger et al., 2005) and thymol (Garcia et al., 2006) or polyacetylenes, e.g. MS-1, MS-2 and MS-4 (from Cussonia zimmermannii, Baur et al., 2005). In the present study, we analysed the molecular mechanism of action of VA and the two structurally closely related compounds acetoxy-VA and hydroxy-VA from Valerian root on GABA_A channels. Previous in vitro studies on a neonatal rat brainstem preparation suggest that the Valerian effects may be mediated through VA action on GABA_A receptors (Yuan et al., 2004). The molecular mechanism of action of VA remained, however, unknown. In order to clarify the molecular basis of VA action on GABA_A receptors, we expressed 13 different subunit combinations in Xenopus oocytes and analysed the modulation of the corresponding receptors by VA.