A single glycine residue at the entrance to the first membrane-spanning domain of the gamma-aminobutyric acid type A receptor beta(2) subunit affects allosteric sensitivity to GABA and anesthetics.
Site-directed mutagenesis of the gamma-aminobutyric acid type A (GABA(A)) receptor beta(2) subunit has demonstrated that conversion of a conserved glycine residue located at the entrance to the first transmembrane domain into the homologous rho(1) residue phenylalanine alters the modulating effects of four different i.v. anesthetics: pentobarbital, alphaxalone, etomidate, and propofol. Using the baculovirus expression system in Spodoptera frugiperda 9 cells, anesthetic-induced enhancement of [(3)H]muscimol and [(3)H]flunitrazepam binding in receptors containing the beta(2)(G219F) point mutation displayed a significantly reduced efficacy in modulation by all four i.v. anesthetics tested. Furthermore, GABA(A) receptors containing the alpha(1)(G223F) point mutation also significantly decreased the maximal effect of etomidate- and propofol-induced enhancement of ligand binding. Conversely, the homologous point mutation in rho(1) receptors (F261G) changed the i.v. anesthetic-insensitive receptor to confer anesthetic modulation of [(3)H]muscimol binding. Consistent with the binding, functional analysis of pentobarbital-enhanced GABA currents recorded with whole-cell patch clamp demonstrated the beta(2)(G219F) subunit mutation eliminated the potentiating effect of the anesthetic. Similarly, propofol-enhanced GABA currents were potentiated less in alpha(1)beta(2)(G219F)gamma(2) receptors than in alpha(1)beta(2)gamma(2) receptors. Although ligand binding displayed comparable K(D) values for muscimol among wild-type, alpha(1)beta(2)gamma(2), and mutant receptors, patch-clamp recordings showed that alpha(1)beta(2)(G219F)gamma(2) receptors had a significantly more potent response to GABA than did alpha(1)beta(2)gamma(2) or alpha(1)(G223F)beta(2)gamma(2). The alpha(1)beta(2)(G219F)gamma(2) receptors also were more sensitive to direct channel activation by pentobarbital and propofol in the absence of GABA. These results suggest that the first transmembrane glycine residue on the beta(2) subunit may be important for conformational or allosteric interactions of channel gating by both GABA and anesthetics.
Carlson
B X
BX
Department of Pharmacology, The Royal Danish School of Pharmacy, Copenhagen, Denmark.
Engblom
A C
AC
Kristiansen
U
U
Schousboe
A
A
Olsen
R W
RW
D000494:Allosteric Regulation / Q000187:drug effects; D000595:Amino Acid Sequence; D000777:Anesthetics / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D018118:Chloride Channels / Q000502:physiology; D004594:Electrophysiology; D005045:Etomidate / Q000494:pharmacology; D018757:GABA Modulators / Q000494:pharmacology; D005998:Glycine / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010424:Pentobarbital / Q000494:pharmacology; D011277:Pregnanediones / Q000494:pharmacology; D015742:Propofol / Q000494:pharmacology; D011487:Protein Conformation; D018079:Receptors, GABA / Q000378:metabolism; D011963:Receptors, GABA-A / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018080:Receptors, GABA-B; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000166:cytology; D005680:gamma-Aminobutyric Acid / Q000502:physiology*
D000777:Anesthetics; D018118:Chloride Channels; D018757:GABA Modulators; C106350:GABRR2 protein, human; D008024:Ligands; D011277:Pregnanediones; D018079:Receptors, GABA; D011963:Receptors, GABA-A; D018080:Receptors, GABA-B; D005680:gamma-Aminobutyric Acid; C006477:alphaxalone; D010424:Pentobarbital; D005998:Glycine; D015742:Propofol; D005045:Etomidate