Abstract
Volatile anesthetics and alcohols enhance transmission mediated by γ-aminobutyric acid type A receptors (GABAARs) 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 GABAAR α1 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 α1(S270H,L277A)β2γ2S GABAAR 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 GABAARs containing the α1 subunit.
Footnotes
-
This study was supported by National Institutes of Health Grants T32-MH18273, GM47818, GM55719, AA10422, AA06399, and GM45129; Integrative Neuroscience Initiative on Alcoholism Consortium AA13520; and the Waggoner Center for Alcohol and Addiction Research.
-
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
-
doi:10.1124/jpet.106.104406.
-
ABBREVIATIONS: GABAAR, γ-aminobutyric acid type A receptor; HEK, human embryonic kidney; CRC, concentration-response curve; HA/HA, homozygous knockin mice; SL/HA, heterozygous knockin mice; SL/SL, control wild-type mice; RT-PCR, reverse transcription-polymerase chain reaction; ANOVA, analysis of variance.
- Received March 13, 2006.
- Accepted June 26, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|