Inhaled anesthetic responses of recombinant receptors and knockin mice harboring α2(S270H/L277A) GABA(A) receptor subunits that are resistant to isoflurane.
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.
PubMed ID: 20807777
PMC ID: PMC3014300
Article link: J Pharmacol Exp Ther.
Grant support: AA06399 NIAAA NIH HHS , AA10422 NIAAA NIH HHS , AA16046 NIAAA NIH HHS , GM47818 NIGMS NIH HHS , P01 GM047818-150004 NIGMS NIH HHS , R01 AA006399-28 NIAAA NIH HHS , R01 AA006399-29 NIAAA NIH HHS , R37 AA010422-15 NIAAA NIH HHS , R37 AA010422-16 NIAAA NIH HHS , R01 AA006399-30 NIAAA NIH HHS , R01 AA006399 NIAAA NIH HHS , P01 GM047818 NIGMS NIH HHS , R37 AA010422 NIAAA NIH HHS