A single glycine residue at the entrance to the first membrane-spanning domain of the γ-aminobutyric acid type a receptor β2 subunit affects allosteric sensitivity to GABA and anesthetics
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A single glycine residue at the entrance to the first membrane-spanning domain of the γ-aminobutyric acid type a receptor β2 subunit affects allosteric sensitivity to GABA and anesthetics. / Carlson, Berit X.; Engblom, A. Christine; Kristiansen, Uffe; Schousboe, Arne; Olsen, Richard W.
In: Molecular Pharmacology, Vol. 57, No. 3, 2000, p. 474-484.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - A single glycine residue at the entrance to the first membrane-spanning domain of the γ-aminobutyric acid type a receptor β2 subunit affects allosteric sensitivity to GABA and anesthetics
AU - Carlson, Berit X.
AU - Engblom, A. Christine
AU - Kristiansen, Uffe
AU - Schousboe, Arne
AU - Olsen, Richard W.
PY - 2000
Y1 - 2000
N2 - Site-directed mutagenesis of the γ-aminobutyric acid type A (GABA(A)) receptor β2 subunit has demonstrated that conversion of a conserved glycine residue located at the entrance to the first transmembrane domain into the homologous ρ1 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 [3H]muscimol and [3H]flunitrazepam binding in receptors containing the β2(G219F) point mutation displayed a significantly reduced efficacy in modulation by all four i.v. anesthetics tested. Furthermore, GABA(A) receptors containing the α1(G223F) point mutation also significantly decreased the maximal effect of etomidate- and propofol-induced enhancement of ligand binding. Conversely, the homologous point mutation in ρ1 receptors (F261G) changed the i.v. anesthetic- insensitive receptor to confer anesthetic modulation of [3H]muscimol binding. Consistent with the binding, functional analysis of pentobarbital- enhanced GABA currents recorded with whole-cell patch clamp demonstrated the β2(G219F) subunit mutation eliminated the potentiating effect of the anesthetic. Similarly propofol-enhanced GABA currents were potentiated less in α1β2(G219F)γ2 receptors than in α1β2γ2 receptors. Although ligand binding displayed comparable K(D) values for muscimol among wild-type, α1β2α2, and mutant receptors, patch-clamp recordings showed that α1β2(G219F)γ2 receptors had a significantly more potent response to GABA than did α1β2γ2 or α1(G223F)β2γ2. The α1β2(G219F)γ2 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 β2 subunit may be important for conformational or allosteric interactions of channel gating by both GABA and anesthetics.
AB - Site-directed mutagenesis of the γ-aminobutyric acid type A (GABA(A)) receptor β2 subunit has demonstrated that conversion of a conserved glycine residue located at the entrance to the first transmembrane domain into the homologous ρ1 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 [3H]muscimol and [3H]flunitrazepam binding in receptors containing the β2(G219F) point mutation displayed a significantly reduced efficacy in modulation by all four i.v. anesthetics tested. Furthermore, GABA(A) receptors containing the α1(G223F) point mutation also significantly decreased the maximal effect of etomidate- and propofol-induced enhancement of ligand binding. Conversely, the homologous point mutation in ρ1 receptors (F261G) changed the i.v. anesthetic- insensitive receptor to confer anesthetic modulation of [3H]muscimol binding. Consistent with the binding, functional analysis of pentobarbital- enhanced GABA currents recorded with whole-cell patch clamp demonstrated the β2(G219F) subunit mutation eliminated the potentiating effect of the anesthetic. Similarly propofol-enhanced GABA currents were potentiated less in α1β2(G219F)γ2 receptors than in α1β2γ2 receptors. Although ligand binding displayed comparable K(D) values for muscimol among wild-type, α1β2α2, and mutant receptors, patch-clamp recordings showed that α1β2(G219F)γ2 receptors had a significantly more potent response to GABA than did α1β2γ2 or α1(G223F)β2γ2. The α1β2(G219F)γ2 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 β2 subunit may be important for conformational or allosteric interactions of channel gating by both GABA and anesthetics.
UR - http://www.scopus.com/inward/record.url?scp=0034006474&partnerID=8YFLogxK
U2 - 10.1124/mol.57.3.474
DO - 10.1124/mol.57.3.474
M3 - Journal article
C2 - 10692487
AN - SCOPUS:0034006474
VL - 57
SP - 474
EP - 484
JO - Molecular Pharmacology
JF - Molecular Pharmacology
SN - 0026-895X
IS - 3
ER -
ID: 276333954