Abstract
Two specific amino acid residues in transmembrane segments (TM) 2 and 3 are critical for the enhancement of glycine receptor (GlyR) function by volatile anesthetics. To determine which physicochemical characteristics of these sites determine their roles in anesthetic actions, an extensive series of single amino acid mutations at amino acid residue 288 (Ala-288) in TM3 of the alpha1 GlyR subunit was tested for modulation by volatile anesthetics. The mutations changed the apparent affinities of receptors for glycine; replacements with larger volumes and less hydropathy exhibited higher affinities for glycine. Potentiation by anesthetics was reduced by specific mutations at Ala-288. The molecular volume of the substituents was negatively correlated with the extent of potentiation by isoflurane, enflurane, and 1-chloro-1,2,2-trifluorocyclobutane, whereas there was no correlation between anesthetic enhancement and polarity, hydropathy, or hydrophilicity of substituents. In contrast to anesthetics, no correlation was found between the effects of the nonanesthetics 1,2-dichlorohexafluorocyclobutane or 2, 3-dichlorooctafluorobutane and any physicochemical property of the substituent. These results suggest that the molecular volume and hydropathy of the amino acid at position 288 in TM3 regulate glycine and anesthetic sensitivity of the GlyR and that this residue might represent one determinant of an anesthetic binding site.
Highlights
Two specific amino acid residues in transmembrane segments (TM) 2 and 3 are critical for the enhancement of glycine receptor (GlyR) function by volatile anesthetics
Affinities of the Mutant GlyRs for Glycine—The homomeric GlyRs composed of the ␣1 subunit mutated at Ala-288 displayed glycine-activated currents after expression in Xenopus oocytes, except for those substituted with proline, arginine, aspartate, or tryptophan
We found that a series of single amino acid replacements at Ala-288 in the TM3 of the ␣1 GlyR subunit reduced the extent of potentiation of the resulting GlyRs by volatile anesthetics
Summary
Site-directed Mutagenesis—Site-directed mutagenesis of the human ␣1 GlyR subunit cDNA [20] in the modified pBK-CMV vector (pBKCMV N/B-200) [21] was performed by using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). Degenerate complementary oligonucleotides were used for the simultaneous creation of multiple mutants.. All mutations were verified by partial sequencing of both plasmid strands. Oocyte Expression—Preparation of xenopus laevis oocytes and microinjection of the cDNA was performed as described previously [22, 23]. Isolated oocytes were placed in modified Barth’s saline (MBS) containing (in mM): NaCl 88, KCl 1, HEPES 10, MgSO4 0.82, NaHCO3 2.4, CaCl2 0.91, and Ca(NO3)2 0.33 adjusted to pH 7.5.
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