Methionine 286 in transmembrane domain 3 of the GABAA receptor β subunit controls a binding cavity for propofol and other alkylphenol general anesthetics

γ-Aminobutyric acid type A (GABA_A) receptors are an important target for general anesthetics in the central nervous system. Site-directed mutagenesis techniques have identified amino acid residues that are important for the positive modulation of GABA_A receptors by general anesthetics. In the present study, we investigate the role of an amino acid residue in transmembrane (TM) domain 3 of the GABA_A receptor β₂ subunit for modulation by the general anesthetic 2,6-diisopropylphenol (propofol). Mutation of methionine 286 to tryptophan (M286W) in the β₂ subunit abolished potentiation of GABA responses by propofol but did not affect direct receptor activation by propofol in the absence of GABA. In contrast, substitution of methionine 286 by alanine, cysteine, glutamate, lysine, phenylalanine, serine, or tyrosine was permissive for potentiation of GABA responses and direct activation by propofol. Using propofol analogs of varying molecular size, we show that the β₂(M286W) mutation resulted in a decrease in the ‘cut-off’ volume for propofol analog molecules to enhance GABA responses at GABA_A α₁β₂γ_2s receptors. This suggests that mutation of M286 in the GABA_A β₂ subunit alters the dimensions of a ‘binding pocket’ for propofol and related alkylphenol general anesthetics.