Identification of the putative binding pocket of valerenic acid on GABAA receptors using docking studies and site-directed mutagenesis

BACKGROUND AND PURPOSE beta 2/3-subunit-selective modulation of GABA(A) receptors by valerenic acid (VA) is determined by the presence of transmembrane residue beta 2/3N265. Currently, it is not known whether beta 2/3N265 is part of VA's binding pocket or is involved in the transduction pathway of VA's action. The aim of this study was to clarify the localization of VA's binding pocket on GABA(A) receptors. EXPERIMENTAL APPROACH Docking and a structure-based three-dimensional pharmacophore were employed to identify candidate amino acid residues that are likely to interact with VA. Selected amino acid residues were mutated, and VA-induced modulation of the resulting GABA(A) receptors expressed in Xenopus oocytes was analysed. KEY RESULTS A binding pocket for VA at the beta(+)/alpha(-) interface encompassing amino acid beta 3N265 was predicted. Mutational analysis of suggested amino acid residues revealed a complete loss of VA's activity on beta 3M286W channels as well as significantly decreased efficacy and potency of VA on beta 3N265S and beta 3F289S receptors. In addition, reduced efficacy of VA-induced I-GABA enhancement was also observed for alpha 1M235W, beta 3R269A and beta 3M286A constructs. CONCLUSIONS AND IMPLICATIONS Our data suggest that amino acid residues beta 3N265, beta 3F289, beta 3M286, beta 3R269 in the beta 3 subunit, at or near the etomidate/propofol binding site(s), form part of a VA binding pocket. The identification of the binding pocket for VA is essential for elucidating its pharmacological effects and might also help to develop new selective GABA(A) receptor ligands.