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

Abstract

gamma-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 beta(2) subunit for modulation by the general anesthetic 2,6-diisopropylphenol (propofol). Mutation of methionine 286 to tryptophan (M286W) in the beta(2) 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 beta(2)(M286W) mutation resulted in a decrease in the 'cut-off' volume for propofol analog molecules to enhance GABA responses at GABA(A) alpha(1)beta(2)gamma(2s) receptors. This suggests that mutation of M286 in the GABA(A) beta(2) subunit alters the dimensions of a 'binding pocket' for propofol and related alkylphenol general anesthetics.

Fig. 1

Amino acid sequence alignment of TM2 and TM3 from human glycine α₁ (Grenningloh et al., 1987), GABA_A α₁ (Schofield et al., 1989), α2 (Hadingham et al., 1993a), β₁ (Schofield et al., 1989), rat or human GABA_A β₂ (Ymer et al., 1989; Hadingham et al., 1993b), and human GABA_A ρ₁ receptor subunits (Cutting et al., 1991). Residue positions in bold type within TM2 and TM3 of glycine α₁ (S267 and A288), GABA_A α₁ and α₂ (S270 and A291), GABA_A β₁ (S265 and M286), and GABA_A β₂ (N265 and M286) receptor subunits are critical for potentiation of agonist responses by alcohol, alkane, and ether anesthetics (Mihic et al., 1997; Ye et al., 1998; Krasowski et al., 1998a; Koltchine et al., 1999; Ueno et al. 1999, 2000; Yamakura et al., 1999; Krasowski and Harrison, 2000; Jenkins et al., 2001). GABA_A β₁(M286) is necessary for potentiation of GABA responses by propofol (Krasowski et al., 1998b). GABA_A β₂(M286) is the main residue position of interest for the current study. GABA_A α₁(S270), α₁(A291), and β₂(N265) are also considered in this paper.

Fig. 2

GABA_A α₁β₂γ_2s and α₁β₂(M286W)γ_2s receptors have similar sensitivity to GABA, but the α₁β₂(M286W)γ_2s receptor is insensitive to the modulatory effects of propofol. (A) and (B) Traces from individual HEK 293 cells expressing either GABA_A α₁β₂γ_2s or GABA_A α₁β₂(M286W)γ_2s receptors in response to application of 2, 5, 20, 500, and 1000 μM GABA. Time of application is indicated by the bars above the traces. (C) Submaximal (EC₂₀) GABA current responses at the wild-type GABA_A α₁β₂γ_2s receptor are enhanced by co-application of 0.2, 1, or 5 μM propofol. Note also the direct activation by 1 and 5 μM propofol, evident during the pre-application of propofol. (D) In contrast, propofol does not potentiate submaximal GABA currents at the GABA_A α₁β₂(M286W)γ_2s receptor, but does produce direct receptor activation at a concentration of 10 μM. Traces shown in (A)–(D) are recordings from individual HEK 293 cells transfected with cDNAs encoding the indicated receptor subunit combinations.

Fig. 3

Concentration–response curves for GABA at the wild-type GABA_A α₁β₂γ_2s receptor and the α₁β₂(M286X)γ_2s series of mutant receptors (see Table 1 for parameters derived by curve fitting to the pooled data).

Fig. 4

Concentration–response relationships for potentiation of GABA responses by propofol at the wild-type GABA_A α₁β₂γ_2s receptor and the α₁β₂(M286X)γ_2s series of mutant receptors. (A) Propofol potentiates GABA responses at GABA_A α₁β₂γ_2s, α₁β₂(M286A)γ_2s, α₁β₂(M286F)γ_2s, and α₁β₂(M286Y)γ_2s receptors but fails to enhance GABA responses at GABA_A α₁β₂(M286W)γ_2s receptors. (B) Propofol potentiates GABA responses at GABA_A α₁βγ_2s, α₁β₂(M286C)γ_2s, α₁β₂(M286E)γ_2s, α₁β₂(M286K)γ_2s, and α₁β₂(M286S)γ_2s receptors (see Table 1 for parameters derived by curve fitting to the pooled data).

Fig. 5

Concentration–response relationships for direct activation by propofol at the wild-type GABA_A α₁β₂γ_2s receptor and the α₁β₂(M286X)γ_2s series of mutant receptors (see Table 1 for parameters derived by curve fitting to the pooled data).

Fig. 6

The GABA_A β₂(M286W) mutation alters the cut-off for potentiation of GABA responses by propofol analogs at the GABA_A α₁β₂γ_2s receptor. (A) The ‘small’ propofol analogs 2,6-dimethylphenol and 2-isopropylphenol (molecular volumes=0.126 and 0.143 nm³, respectively) potentiate submaximal (EC₂₀) GABA responses at the GABA_A α₁β₂(M286W)γ_2s receptor. (B) In contrast, the larger propofol analogs 2,6-diethylphenol (0.16 nm³) and 2,6-diethylphenyl isothiocyanate (0.187 nm³) fail to enhance submaximal GABA responses at the GABA_A α₁β₂(M286W)γ_2s receptor. Traces shown in (A) and (B) are recordings from individual HEK 293 cells transfected with cDNAs encoding the GABA_A α₁, β₂(M286W), and γ_2s receptor subunits.

Fig. 7

Summary of the effects of six propofol analogs on the wild-type GABA_A α₁β₂γ_2s receptor and the GABA_A α₁β₂(M286A)γ₂s, α₁β₂(M286S)γ_2s, and α₁β₂(M286W)γ_2s receptors. The wild-type GABA_A α₁β₂γ_2s receptor supports potentiation of GABA responses by 2,6-dimethylphenol, 2-isopropylphenol, 2,6-diethylphenol, 2,6-diethylphenyl isothiocyanate, and propofol, but not the bulkier analog 2,6-di-tert-butylphenol. The mutation of methionine 286 to the smaller alanine or serine residues does not change this profile. On the other hand, the β₂(M286W) mutation results in a receptor now insensitive to 2,6- diethylphenol, 2,6-diethylphenyl isothiocyanate, and propofol, as well as 2,6-di-tert-butylphenol. Thus, the molecular volume cut-off for propofol potentiation of GABA responses is reduced from 0.193 nm³ in the GABA_A α₁β₂γ₂, α₁β₂(M286A)γ2s, and α₁β₂(M286S)γ_2s receptors (‘Wildtype cut-off’, single arrow) to less than 0.16 nm3 in the GABA_A α₁β₂(M286W)γ_2s receptor (‘Altered cut-off’, double arrow).