2. responses to isoflurane. We then produced mutant (knockin) mice in which this mutated subunit replaced the wild-type 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 EC50for isoflurane did not differ between genotypes, but time to regain the righting reflex was increased in N2generation knockins. This effect was not observed at the N4generation. Isoflurane produced immobility (as measured by tail clamp) and amnesia (as measured by fear conditioning) in both wild-type and mutant mice, and potencies (EC50) did not differ between the strains for these actions of isoflurane. Thus, immobility or amnesia does not require isoflurane potentiation of the 2 2 subunit. == Introduction == The advent of general anesthesia in the mid-19th century was 20(S)-Hydroxycholesterol one of the most important developments in the history of medicine. Despite more than 160 years of clinical use, the mechanisms of action of inhaled anesthetics remained largely a mystery until the end of the 20th century (Franks and Lieb, 1994). Although such anesthetics are some of the most widely used drugs in clinical practice, no presently available inhaled anesthetic is ideal. These drugs have a low PLA2B therapeutic index and exhibit adverse side effects. Knowledge of the mechanism of action might enable the design, synthesis, and testing of improved anesthetics and the prevention of adverse events such as intraoperative awareness and postoperative cognitive dysfunction. Accumulating evidence suggests that inhaled anesthetics act by modulating various ligand- and/or voltage-gated ion channels, rather 20(S)-Hydroxycholesterol than through nonspecific perturbation of membrane lipids as suggested previously by the Meyer-Overton hypothesis (Franks and Lieb, 1994;Franks, 2008). Among the plausible targets, GABAAreceptors (GABAA-Rs) have been strongly implicated in anesthetic action because of their leading role in mediating both synaptic and tonic inhibition in the central nervous system. The 20(S)-Hydroxycholesterol function of synaptic and extrasynaptic GABAA-Rs is enhanced by inhaled anesthetics at clinically relevant concentrations (Jones et al., 1992). In contrast, GABAA-Rs are insensitive to structurally related compounds that are not anesthetic (nonimmobilizers) (Mihic et al., 1994). Potentiation of GABAA-R function in vitro parallels in vivo anesthetic potency for the inhaled agents (Zimmerman et al., 1994). These receptors are now accepted as the primary targets and mediators of the central nervous system depressant actions of the intravenous anesthetics propofol and etomidate (Jurd et al., 2003;Reynolds et al., 2003). Pentameric GABAA-Rs are encoded by 19 different GABAA-R subunit genes (Simon et al., 2004). Most native GABAA-Rs are composed of two , two , and a or subunit (McKernan and Whiting, 1996). Our understanding of the roles of various receptor isoforms and specific subunits in the behavioral effects of anesthetics is limited. One experimental strategy for defining the contribution of individual receptor subunits to drug action is to create and characterize point mutated gene knockin mice that harbor mutations that render individual subunits insensitive to the drug of interest but otherwise do not affect receptor function. This knockin 20(S)-Hydroxycholesterol strategy clarified the role of individual GABAA-R subunits in whole animal responses to benzodiazepines (Rudolph et al., 1999;McKernan et al., 2000) and intravenous anesthetics (Jurd et al., 2003;Reynolds et al., 2003). We used this same genetic strategy to dissect inhaled anesthetic action. We initially identified a key amino acid (serine at position 270; Ser270) in GABAA-R subunits that, when mutated to histidine (His), abolished sensitivity to isoflurane (Mihic et al., 1997). However, knockin mice with this single S270H mutation in the 1 subunit displayed behavioral impairments because of an increased sensitivity to GABA (Homanics et al., 2005). This increased GABA sensitivity could be corrected by a second mutation (Leu277 to alanine; L277A), which when combined with S270H, resulted in mutant receptors with isoflurane insensitivity and near normal GABA responses (Borghese et al., 2006). GABAA-R 1 subunit knockin mice with these two mutations showed reduced GABAergic cellular responses to isoflurane and reduced sensitivity to isoflurane-induced loss of righting reflex (LORR), but showed normal.