Inhaled Anesthetic Responses of Recombinant Receptors and Knockin Mice Harboring α2(S270H/L277A) GABA_AReceptor 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 ␣2 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 ␣2 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 wildtype ␣2 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 ␣2 subunit.

Loss of Ethanol Conditioned Taste Aversion and Motor Stimulation in Knockin Mice with Ethanol-Insensitive α2-Containing GABAAReceptors

“…Mice were originally produced on a mixed C57BL/6Jϫ129SvJ background and subsequently backcrossed to C57BL/6J for two to four generations as described previously (Werner et al, 2010). After weaning, mice were housed under specific pathogen-free conditions at the University of Pittsburgh and in a conventional facility at the University of Texas with ad libitum access to rodent chow and water with 12-h light/dark cycles (lights on at 7:00 AM).…”

“…All mice were genotyped by Southern blot (University of Pittsburgh) or polymerase chain reaction (University of Texas). Southern blot analysis of tail DNA was performed as described previously (Werner et al, 2010). Mouse genotypes were determined by polymerase chain reactions performed on DNA derived from tail biopsies.…”

“…For detailed materials and methods, see Borghese et al (2006) and Werner et al (2010). In brief, Xenopus laevis oocytes were manually isolated, treated with collagenase, and injected with cDNA encoding GABA A subunits (␣/ ␤/␥ 1:1:3 in ng/oocyte).…”

“…Indeed, knockin mice with Ser270 to histidine and Leu277 to alanine mutations in the ␣1 subunit of the GABA A -R, showed alterations in specific ethanol-induced behavioral effects . To extend these studies, we generated mice with Ser270 to His and Leu277 to Ala mutations in the ␣2 subunit of GABA A -Rs (Werner et al, 2010).…”

Characterization of Two Mutations, M287L and Q266I, in the α1 Glycine Receptor Subunit That Modify Sensitivity to Alcohols

“…Details of RNA extraction, cDNA preparation, and PCR are found in Werner et al (2011). Glycine ␣1 cDNA in the region of the mutations was selectively amplified by using an exon 7 primer (5Ј-gctcatcgtcatcctgtcctgg-3Ј) and an exon 9 primer (5Ј-gtggtgttgttgttgttggcac3Ј).…”