title: Inhaled anesthetic responses of recombinant receptors and knockin mice harboring α2(S270H/L277A) GABA(A) receptor subunits that are resistant to isoflurane.
abstract: The mechanism by which the inhaled anesthetic isoflurane produces amnesia and immobility is not understood. Isoflurane modulates GABA(A) receptors (GABA(A)-Rs) in a manner that makes them plausible targets. We asked whether GABA(A)-R α2 subunits contribute to a site of anesthetic action in vivo. Previous studies demonstrated that Ser270 in the second transmembrane domain is involved in the modulation of GABA(A)-Rs by volatile anesthetics and alcohol, either as a binding site or a critical allosteric residue. We engineered GABA(A)-Rs with two mutations in the α2 subunit, changing Ser270 to His and Leu277 to Ala. Recombinant receptors with these mutations demonstrated normal affinity for GABA, but substantially reduced responses to isoflurane. We then produced mutant (knockin) mice in which this mutated subunit replaced the wild-type α2 subunit. The adult mutant mice were overtly normal, although there was evidence of enhanced neonatal mortality and fear conditioning. Electrophysiological recordings from dentate granule neurons in brain slices confirmed the decreased actions of isoflurane on mutant receptors contributing to inhibitory synaptic currents. The loss of righting reflex EC(50) for isoflurane did not differ between genotypes, but time to regain the righting reflex was increased in N(2) generation knockins. This effect was not observed at the N(4) generation. Isoflurane produced immobility (as measured by tail clamp) and amnesia (as measured by fear conditioning) in both wild-type and mutant mice, and potencies (EC(50)) did not differ between the strains for these actions of isoflurane. Thus, immobility or amnesia does not require isoflurane potentiation of the α2 subunit.
authors.affiliation: Department of Anesthesiology, University of Pittsburgh, 3501 Fifth Avenue, Pittsburgh, PA 15261, USA.
authors.forename: D F
authors.initials: DF
authors.lastname: Werner
authors.forename: A
authors.initials: A
authors.lastname: Swihart
authors.forename: V
authors.initials: V
authors.lastname: Rau
authors.forename: F
authors.initials: F
authors.lastname: Jia
authors.forename: C M
authors.initials: CM
authors.lastname: Borghese
authors.forename: M L
authors.initials: ML
authors.lastname: McCracken
authors.forename: S
authors.initials: S
authors.lastname: Iyer
authors.forename: M S
authors.initials: MS
authors.lastname: Fanselow
authors.forename: I
authors.initials: I
authors.lastname: Oh
authors.forename: J M
authors.initials: JM
authors.lastname: Sonner
authors.forename: E I
authors.initials: EI
authors.lastname: Eger
authors.forename: N L
authors.initials: NL
authors.lastname: Harrison
authors.forename: R A
authors.initials: RA
authors.lastname: Harris
authors.forename: G E
authors.initials: GE
authors.lastname: Homanics
mesh_terms: D018685:Anesthetics, Inhalation / Q000008:administration & dosage*; D000818:Animals; D003213:Conditioning, Psychological / Q000187:drug effects / Q000502:physiology; D004351:Drug Resistance / Q000187:drug effects / Q000502:physiology*; D005239:Fear / Q000187:drug effects / Q000502:physiology; D005260:Female; D055879:Gene Knock-In Techniques; D006801:Humans; D007530:Isoflurane / Q000008:administration & dosage*; D051379:Mice; D008810:Mice, Inbred C57BL; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000819:agonists / Q000235:genetics; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D018685:Anesthetics, Inhalation; C484841:GABRA2 protein, human; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid; D007530:Isoflurane
references.citation: Mol Pharmacol. 2005 Jul;68(1):61-8
references.pmid: 15833735
references.citation: Anesthesiology. 2008 Jun;108(6):1016-24
references.pmid: 18497602
references.citation: J Pharmacol Exp Ther. 1994 Sep;270(3):987-91
references.pmid: 7932211
references.citation: Mol Pharmacol. 1997 Apr;51(4):588-96
references.pmid: 9106623
references.citation: Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8424-8
references.pmid: 8378314
references.citation: Neurobiol Learn Mem. 2010 Mar;93(3):383-7
references.pmid: 20018248
references.citation: Anesthesiology. 1993 Dec;79(6):1244-9
references.pmid: 8267200
references.citation: J Neurophysiol. 2005 Dec;94(6):4491-501
references.pmid: 16162835
references.citation: Nature. 1999 Oct 21;401(6755):796-800
references.pmid: 10548105
references.citation: J Neurosci. 1994 Sep;14(9):5302-24
references.pmid: 8083738
references.citation: Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8980-5
references.pmid: 12084936
references.citation: J Neurosci. 2003 Sep 17;23(24):8608-17
references.pmid: 13679430
references.citation: Learn Mem. 2005 May-Jun;12(3):327-33
references.pmid: 15897254
references.citation: Nat Genet. 2000 Jun;25(2):139-40
references.pmid: 10835623
references.citation: Br J Pharmacol. 2000 Feb;129(4):731-43
references.pmid: 10683198
references.citation: Neuroscience. 1997 Oct;80(4):987-1000
references.pmid: 9284055
references.citation: Anesth Analg. 2008 Mar;106(3):985-90, table of contents
references.pmid: 18292450
references.citation: Mol Pharmacol. 1994 Nov;46(5):851-7
references.pmid: 7969071
references.citation: Anesthesiology. 1987 Jan;66(1):55-9
references.pmid: 3492157
references.citation: Genes Brain Behav. 2005 Feb;4(1):10-9
references.pmid: 15660664
references.citation: Anesth Analg. 2008 Sep;107(3):832-48
references.pmid: 18713892
references.citation: Trends Neurosci. 1996 Apr;19(4):139-43
references.pmid: 8658597
references.citation: J Physiol. 1992 Apr;449:279-93
references.pmid: 1326046
references.citation: Anesthesiology. 2007 Jan;106(1):107-13
references.pmid: 17197852
references.citation: Nat Rev Neurosci. 2008 May;9(5):370-86
references.pmid: 18425091
references.citation: Nature. 1994 Feb 17;367(6464):607-14
references.pmid: 7509043
references.citation: J Comp Neurol. 1995 Aug 14;359(1):154-94
references.pmid: 8557845
references.citation: FASEB J. 2003 Feb;17(2):250-2
references.pmid: 12475885
references.citation: Front Behav Neurosci. 2009 Oct 20;3:37
references.pmid: 19876409
references.citation: Anesth Analg. 2009 Dec;109(6):1816-22
references.pmid: 19923508
references.citation: Nature. 1997 Sep 25;389(6649):385-9
references.pmid: 9311780
references.citation: Nat Neurosci. 2000 Jun;3(6):587-92
references.pmid: 10816315
references.citation: Anesthesiology. 1993 Apr;78(4):707-12
references.pmid: 8466071
references.citation: Psychon Bull Rev. 1994 Dec;1(4):429-38
references.pmid: 24203551
references.citation: J Neurosci. 1996 Jan;16(1):283-97
references.pmid: 8613794
references.citation: J Biol Chem. 2004 Oct 1;279(40):41422-35
references.pmid: 15258161
references.citation: J Pharmacol Exp Ther. 1972 Dec;183(3):577-607
references.pmid: 4636393
references.citation: J Neurosci. 2006 Oct 4;26(40):10315-26
references.pmid: 17021187
references.citation: J Pharmacol Exp Ther. 2006 Oct;319(1):208-18
references.pmid: 16807363
references.citation: J Pharmacol Exp Ther. 2006 Oct;319(1):219-27
references.pmid: 16785315
references.citation: Hear Res. 1999 Apr;130(1-2):94-107
references.pmid: 10320101