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045P Queen Elizabeth II Conference Centre London
Pharmacology 2014

 

 

Structural requirements for potent, efficiacious and β-subunit selective modulation of GABAA receptors by valerenic acid

D Luger, S Khom, T Linder, S Hering. University of Vienna, Department of Pharmacology and Toxicology, Vienna, Austria

GABAA receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and important targets for anxiolytics, sedatives, anticonvulsants and anaesthetics. The widely used benzodiazepines induce severe side effects due to unselective stimulation of many GABAA receptor subtypes. Valerenic acid (VA), a natural compound of common Valerian, has been identified as β2/3-subunit selective GABAA receptor modulator (1). A single asparagine residue in β2/3 (N265) – also relevant for the action of etomidate (4,5) – was found to determine VA’s modulatory effect in vitro and in vivo (1,2). The aim of this study is the identification of key amino acids of a potential VA binding pocket on GABAA receptors.

Homology modelling and docking into a GABAA receptor model suggests that seven amino acids at the α1/β2/3 interface are likely to interact with VA. Corresponding point mutations were introduced into GABAA receptors and constructs expressed in Xenopus laevis oocytes. Modulation of GABA-induced chloride currents (IGABA) was studied using 2-microelectrode voltage-clamp and a fast perfusion system (3) (Table 1).

Valerenic acid (VA)
Subtype Emax (%) EC50 ( μ M) n
α 1 β 3 γ 2S 692 ± 107 22.1 ± 5.7 7
α 1 β 1 γ 2S 111 ± 16 (***) 74.4 ± 19.3 8
α 1M235W β 3 γ 2S 193 ± 16 (***) 24.3 ± 7.7 6
α 1 β 3T262S γ 2S 562 ± 25 3.2 ± 0.5 8
α 1 β 3N265S γ 2S 108 ± 19 (***) 86.4 ± 32.9 6
α 1 β 3T266A γ 2S 621 ± 94 8.1 ± 3.0 11
α 1 β 3M286W γ 2S 76 ± 11 (***) 26.3 ± 5.4 8
α 1 β 3F289S γ 2S 191 ± 59 (***) 164.6 ± 87.6 (*) 7
α 1 β 1S266N γ 2S 582 ± 29 25.2 ± 2.2 8

Table 1: IGABA enhancement through wildtype and mutated GABAA receptors.

Stars indicate significant difference from corresponding VA values on α1β3γ2S.

(***): p<0.005; (*): p<0.05; One-way ANOVA with post hoc Dunnet test

Our data (Table 1) show that four amino acid residues (α1M235, β3N265, β3M286, β3F289) are likely to form part of a putative binding pocket for VA on GABAA receptors composed of α1-, β3- and γ2S subunits. Significantly reduced IGABA enhancement through α1M235Wβ3γ2S and α1β3M286Wγ2S receptors suggest a potential overlap with the etomidate binding site (4,5).

References:

(1) Khom et al. (2007). Neuropharmacology. 53: (1):178-187.

(2) Benke et al. (2009). Neuropharmacology. 56: (1):174-181.

(3) Baburin et al. (2006). Pflugers Arch. 453: (1):117-123.

(4) Li et al. (2006). J Neurosc. 26: (45):11599-11605.

(5) Olsen and Li (2013). Can J Anaesth. 58: (2):206-215.