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.

Fig. 1.

GABA responses and their modulation in α2 wild-type and mutant (SHLA) subunits coexpressed with β2/3γ2s subunits in Xenopus oocytes. A, GABA concentration response curves (n = 7–8). B, maximal GABA-induced current 3 days after injection (n = 4–13). C, isoflurane (0.3 mM) modulation of EC_5–10 GABA (n = 4). *, p < 0.05, t test.

Fig. 2.

Production of α2 knockin mice by gene targeting. A, partial DNA sequence of wild-type and mutant α2 genes. Note that the nucleotides highlighted in yellow in the knockin sequence denote the bases changed to alter the codons at amino acid positions 270 and 277. An additional nucleotide was altered to introduce an EcoRI restriction endonuclease recognition site that did not change the codon at position 274. B, gene-targeting strategy used to modify the α2 locus in embryonic stem cells. Exon 9 corresponds to nucleotides 859 to 1059 (note: nucleotide 1 corresponds to the start of translation) of the mouse α2 cDNA (Ensembl Transcript ID: ENSMUST00000000572). C, DNA sequence analysis of whole brain α2 reverse transcription-PCR products from SL/SL (top) or HA/HA (bottom) mice. This analysis demonstrates the presence of the introduced mutations in the α2 gene product in the brain of knockin mice. D, Western blot analysis of α2 GABA_A-R subunits (top) or β-actin (bottom) in cortex. Shown is a representative immunoblot. After normalization to β-actin, the amount of α2 did not differ with respect to genotype (n = 4/genotype).

Fig. 3.

Whole-cell patch clamp recordings from hippocampal dentate gyrus granule cells. A, the left traces illustrate the averaged mIPSCs (from 100 single events) recorded from a granule neuron from a SL/SL mouse in the absence or presence of 0.15 and 0.30 mM isoflurane. The right traces show the averaged mIPSCs from an α2 ΗΑ/ΗΑ animal. Under control conditions, the decay time constant of mIPSCs recorded from HA/HA mice was reduced compared with SL/SL (P < 0.01). B, histogram summarizing the effects of isoflurane on decay time constants. Isoflurane dose-dependently increased the decay time constants in both genotypes, but the increase in HA/HA mice was reduced compared with SL/SL mice in response to 0.30 mM isoflurane. **, p < 0.01.

Fig. 4.

Recovery from isoflurane anesthesia. A, recovery time after exposure to 1.5% atm isoflurane was significantly different between genotypes when studied on mice on a N₂ genetic background. B, recovery time after exposure to 1.5% atm halothane did not differ between mice on a N₂ genetic background. C, no differences between genotypes were observed on this same assay when mice of the N₄ genetic background were tested with 1.5% atm isoflurane. *, p < 0.05.

Fig. 5.

Concentration response curves for LORR and tail clamp/withdrawal assays in response to isoflurane (A; n = 11–13/genotype) and halothane (B; n = 10–12/genotype). Genotypes did not differ on either assay for either anesthetic.

Fig. 6.

Fear conditioning during training. A and B, HA/HA mice show facilitated acquisition of conditional fear during training to the three tone–1-mA shock pairings (A, p < 0.0001) and the six tone–1-mA shock pairings (B, p < 0.005) compared with SL/SL mice. T refers to tone, and PT refers to posttone. C, HA/HA mice displayed increased performance on the fear to context test compared with SL/SL mice after training with either the three or six tone–shock pairings. D, HA/HA mice displayed increased performance on the fear to tone test compared with SL/SL mice after the six, but not the three, tone–shock pairings. n = 8–14/genotype. *, p < 0.05; **, p < 0.001; ***, p < 0.005.

Fig. 7.

Fear conditioning with white noise. A, SL/SL and HA/HA mice were trained by using three white noise (0 Hz, 75–80 db)–shock (0.6 mA) pairings. HA/HA show facilitated acquisition of learning compared with SL/SL mice (p < 0.05). n = 8–10/genotype. B, There was no difference between genotypes during testing, however. C, Effect of isoflurane on fear to context. SL/SL and HA/HA mice were statistically similar at 0% isoflurane during the fear to context test. EC₅₀ values did not differ between genotypes for fear to context. D, SL/SL and HA/HA mice did not differ in fear to white noise at 0% isoflurane. EC₅₀ values did not differ between genotypes for fear to tone. n = 6–10/genotype per concentration.