An Isoflurane- and Alcohol-Insensitive Mutant GABA_A Receptor α₁ Subunit with Near-Normal Apparent Affinity for GABA: Characterization in Heterologous Systems and Production of Knockin Mice

Abstract

Volatile anesthetics and alcohols enhance transmission mediated by ␥-aminobutyric acid type A receptors (GABA A Rs) in the central nervous system, an effect that may underlie some of the behavioral actions of these agents. Substituting a critical serine residue within the GABA A R ␣ 1 subunit at position 270 with the larger residue histidine eliminated receptor modulation by isoflurane, but it also affected receptor gating (increased GABA sensitivity). To correct the shift in GABA sensitivity of this mutant, we mutated a second residue, leucine at position 277 to alanine. The double mutant ␣ 1 (S270H,L277A)␤ 2 ␥ 2S GABA A R was expressed in Xenopus laevis oocytes and human embryonic kidney (HEK)293 cells, and it had near-normal GABA sensitivity. However, rapid application of a brief GABA pulse to receptors expressed in HEK293 cells revealed that the deactivation was faster in double mutant than in wild-type receptors. In all heterologous systems, the enhancing effect of isoflurane and ethanol was greatly decreased in the double mutant receptor. Homozygous knockin mice harboring the double mutation were viable and presented no overt abnormality, except hyperactivity. This knockin mouse line should be useful in determining which behavioral actions of volatile anesthetics and ethanol are mediated by the GABA A Rs containing the ␣ 1 subunit.

Knockin Mice with Ethanol-Insensitive α1-Containing γ-Aminobutyric Acid Type A Receptors Display Selective Alterations in Behavioral Responses to Ethanol

“…We chose mutations that render the receptor resistant to ethanol (and also to isoflurane) but provide a near normal response to GABA and normal modulation by flunitrazepam (Borghese et al, 2006b). This allowed us to test the importance of ␣1-containing GABA A -Rs to specific cellular and behavioral responses to ethanol.…”

“…All mice were genotyped by Southern blot or polymerase chain reaction analysis of tail DNA as described previously (Borghese et al, 2006b). All mice were of a mixed C57BL/6Jx129SvJ background of the F 3 to F 6 generations.…”

“…However, incorporating a second mutation, leucine 277 to alanine (L277A), resulted in receptors that displayed normal GABA sensitivity but no modulation by ethanol and a reduced potentiation by etomidate and pentobarbital. Importantly, these receptors showed normal modulation by flunitrazepam, and mice harboring this mutation appeared normal (Borghese et al, 2006b).…”

Regulation of GABAAReceptor Subunit Expression by Pharmacological Agents

“…␣1 subunit (Borghese et al, 2006). Recombinant receptors containing these mutations are insensitive to ethanol (Ueno et al, 2000;Borghese et al, 2006).…”

“…Recombinant receptors containing these mutations are insensitive to ethanol (Ueno et al, 2000;Borghese et al, 2006). CE treatment did not affect ␣1 polypeptide level in ␣1 knockin mice, although it reduced the level in wild-type mice (Werner et al, 2009).…”