γ-Amino Butyric Acid Type A Receptor Mutations at β2N265 Alter Etomidate Efficacy While Preserving Basal and Agonist-dependent Activity

Female Xenopus laevis  were housed in a veterinary-supervised environment and used in accordance with local and federal guidelines and with approval from the Massachusetts General Hospital subcommittee on research and animal care (Boston, Massachusetts). Frogs were anesthetized by immersion in ice-cold 0.2% tricaine (Sigma-Aldrich, St. Louis, MO) before mini-laparotomy for oocyte harvest.

R(+)-Etomidate was obtained from Bedford Laboratories (Bedford, OH). The clinical preparation in 35% propylene glycol was diluted directly into buffer. Previous studies have shown that propylene glycol at the dilutions used for these studies has no effect on GABA^Areceptor function.5Picrotoxin was purchased from Sigma-Aldrich and dissolved in electrophysiology buffer (2 mm) by prolonged gentle shaking. Alphaxalone was purchased from MP Biomedical (Solon, OH) and prepared as a stock solution in dimethylsulfoxide. Salts and buffers were purchased from Sigma-Aldrich.

Complementary DNA sequences for human GABA^Areceptor α1, β2, β1, and γ2L subunits were cloned into pCDNA3.1 vectors (Invitrogen, Carlsbad, CA). To create expression plasmids for β2(N265S), β2(N265M), and α1(L264T) mutants, oligonucleotide-directed mutagenesis was performed on the appropriate wild-type clone using QuickChange kits (Stratagene, La Jolla, CA). Clones from each mutagenesis reaction were sequenced through the entire subunit gene to confirm the presence of the mutation and absence of stray mutations.

Messenger RNA was synthesized in vitro  from linearized DNA templates and purified using commercial kits (Ambion Inc., Austin, TX). Subunit messenger RNAs were mixed at 1α:1β and at least two-fold excess γ to promote homogeneous receptor expression.21,22,Xenopus  oocytes were microinjected with 25–50 nl (15–25 ng) of messenger RNA mixture and incubated at 18°C in ND96 (in mm: 96 NaCl, 2 KCl, 0.8 MgCl², 1.8 CaCl², 5 HEPES, pH 7.5) supplemented with gentamicin (0.05 mg/ml) for 24–48 h before electrophysiology. HEK293 cells were cultured on glass cover slips, maintained as previously described,23and transfected with plasmids encoding GABA^Areceptor subunit mixtures (1α:1β:2γ) using lipofectamine (Invitrogen). A eukaryotic green fluorescent protein expression plasmid, pmaxGFP (Amaxa, Gaithersburg, MD), was mixed with the GABA^Areceptor subunit plasmids to aid in identification of transfected cells. Transfected cells were maintained in culture medium for 24–48 h before electrophysiology experiments.

GABA^Areceptor responses to GABA were assessed in Xenopus  oocytes using two-microelectrode voltage clamp electrophysiology, as previously described.24GABA pulses were from 5 to 20 s, depending on the concentration of GABA used and the time to steady-state peak current. Normalizing GABA responses were recorded at maximal GABA (1-10 mm). Picrotoxin-sensitive leak currents were measured by superfusion with 2 mm picrotoxin, followed by washout for at least 5 min before testing maximal GABA response. Alphaxalone (2 μm) was used as a gating enhancer in combination with 10 mm GABA to provide estimates of GABA efficacy. Oocyte currents were low-pass filtered at 1 kHz (Model OC-725B, Warner Instruments, Hamden, CT) and digitized at 1–2 kHz using commercial digitizer hardware (Digidata 1200; Molecular Devices, Sunnyvale, CA) and software (pClamp 7; Molecular Devices).

Excised outside-out membrane patches were voltage-clamped at –50 mV, and current recordings were performed at room temperature (21–23°C) as previously described.23Bath and superfusion solutions contained (in mm) 145 NaCl, 5 KCl, 10 HEPES, 2 CaCl², and 1 MgCl²at pH 7.4 (pH adjusted with N -methyl glucosamine). The intracellular (pipette) fluid contained (in mm) 140 KCl, 10 HEPES, 1 EGTA, and 2 MgCl²at pH 7.3 (pH adjusted with KOH). Currents were stimulated with brief (0.5–1.0 s) pulses of GABA delivered via  a multichannel superfusion pipette coupled to piezo-electric elements that switched superfusion solutions in under 1 ms. Currents were filtered at 5 kHz and digitized at 10 kHz for offline analysis.

Leak-correction and measurement of peak currents were performed offline using Clampfit 8.0 software (Molecular Devices). Peak GABA-activated or etomidate-activated oocyte currents were normalized to maximal GABA-activated currents (I^maxGABA) measured in the same cell. Concentration-response curves were assembled from pooled normalized data from multiple oocytes. Pooled data sets were fitted with logistic (Hill) functions using nonlinear least squares in Origin 6.1 (OriginLab, Northampton, MA) and Prism 5.02, (GraphPad Software, San Diego, CA):

where A is amplitude, EC⁵⁰is the half-maximal activating concentration, and nH is the Hill slope.

Etomidate potentiation of GABA responses was quantified as the ratio of the GABA EC⁵⁰values in the absence of anesthetic to that in the presence of 3.2 μm etomidate. GABA concentration-response curves shift leftward (i.e. , to a lower GABA EC⁵⁰) in the presence of etomidate; thus large EC⁵⁰ratios indicate strong modulation, whereas a ratio of 1.0 or less indicates lack of positive modulation.25 

Picrotoxin-sensitive leak currents (I^PTX) were normalized to I^maxGABA, providing estimates of basal open probability (P⁰). Incorporation of the α1(L264T) mutation was used to enhance basal gating for comparison of the effects of β2N265 mutations. Maximal GABA efficacy was assessed by first activating oocyte-expressed channels with 10 mm GABA. After full current activation and partial desensitization, superfusate was switched to 10 mm GABA plus 2 μm alphaxalone, a strong positive modulator of wild-type and both mutant receptors. Maximal GABA efficacy was calculated as the ratio of current immediately before the addition of alphaxalone (I^maxGABA) to the secondary current peak after the addition of alphaxalone (I^GABA+alphax).

Estimated open probability (P^openest), defined as the fraction of activatable receptors in the open state, was calculated by explicitly adding spontaneous current and renormalizing to the full range of open probability, assuming that picrotoxin-blocked leak represents no activation (P^open= 0) and that maximal GABA plus alphaxalone activates all nondesensitized channels (P^open= 1.0).26In practice, the elements used to calculate P^openestare all normalized to I^maxGABA:

Quantitative analysis based on Monod-Wyman-Changeux coagonism5was performed as follows: average P^openestvalues calculated from GABA concentration-responses (with and without etomidate) and etomidate direct activation data were pooled. With both [GABA] and [etomidate] specified as independent variables, these data were globally fitted to equation 3using nonlinear least squares:

This equation describes a two-state equilibrium allosteric mechanism with two classes of agonist sites (one for GABA and one for etomidate), each with two equivalent sites. L⁰in equation 3is a dimensionless basal equilibrium gating variable, approximately P⁰⁻¹. K^Gand K^Eare equilibrium dissociation constants for GABA and etomidate binding to inactive states, and c and d are dimensionless parameters representing the respective ratios of binding constants in active versus  inactive states. The agonist efficacies of GABA and etomidate are inversely related to c and d, respectively.

To analyze membrane patch macrocurrents for activation, desensitization, and deactivation kinetics, data windows were specified in each trace for different phases of the waveform. Activation windows were from 10% above the baseline trace to a point where desensitization had reduced the peak current by 3–5%. Desensitization windows were from the current peak to the end of GABA application. Deactivation windows were from the end of GABA application to the end of the sweep. Windowed data were fitted to multiple exponential functions using nonlinear least squares:

The number of components for each fit was determined by comparison of single-, double-, and triple-exponential fits using an F test to choose the best exponential fit model with a confidence value of P = 0.99 (Clampfit8.0; Molecular Devices).

Results are reported as mean ± SD unless otherwise indicated. Nonlinear regression errors are those from fits in Origin 6.1 (OriginLab). Statistical comparison of fitted parameters was performed using Prism 5.02 (GraphPad Software). Single parameter group comparisons were performed using either a two-tailed Student t  test (with independent variances) or ANOVA with Tukey post hoc  multiple comparisons test in Microsft Excel (Microsoft Corporation, Redmond, WA) with an add-on statistical toolkit (StatistiXL; Nedlands, Australia). Statistical significance was inferred at P < 0.05.

GABA concentration-responses with and without etomidate were measured in Xenopus  oocytes and normalized to I^maxGABAwithout etomidate (fig. 1, table 1). A logistic fit to pooled wild-type α1β2γ2L receptor data revealed a GABA EC⁵⁰of 26 μm and a Hill slope of 1.2. Addition of 3.2 μm etomidate dramatically enhanced currents elicited with low GABA, causing a 14-fold decrease in the wild-type GABA EC⁵⁰to 1.9 μm and a small increase in the maximal GABA-activated current. Currents from α1β2(N265S)γ2L receptors expressed in oocytes were characterized by GABA EC⁵⁰= 27 μm, which was not significantly different from that of wild-type (P = 0.67). Addition of etomidate weakly enhanced currents elicited with low GABA and produced a 2.3-fold reduction in GABA EC⁵⁰to 12 μm. This shift was significantly smaller than that observed in wild-type (P < 0.0001). GABA-activated currents from α1β2(N265M)γ2L receptors were characterized by an EC⁵⁰value of 32 μm, significantly different from wild-type (P = 0.011). Etomidate did not enhance GABA-activated currents from this mutant, and GABA EC⁵⁰in the presence of 3.2 μm etomidate was 34 μm, not significantly different from GABA EC⁵⁰without etomidate (P = 0.64).

Etomidate, in the absence of GABA, directly activated chloride currents in oocytes expressing wild-type α1β2γ2L GABA^Areceptors (fig. 2, table 1). Maximal direct activation averaged 43% of the maximal GABA-activated current (I^maxGABA) and was observed at 0.3 mm etomidate. Half-maximal direct activation was at 36 μm etomidate. In α1β2(N265S)γ2L receptors, etomidate elicited small currents with maximum amplitude only 3% of that elicited by high GABA. Half-maximal direct activation in α1β2(N265S)γ2L receptors was at about 80 μm etomidate, but it was not significantly different from wild-type because of large uncertainty (P = 0.24). Etomidate at up to 1 mm did not elicit any currents in oocytes expressing α1β2(N265M)γ2L receptors.

Maximal GABA efficacy was estimated by first activating receptors with a saturating concentration of GABA (10 mm, approximately 380 × EC⁵⁰), then adding 2 μm alphaxalone (fig. 3). Alphaxalone more than doubled currents elicited with GABA concentrations below EC⁵⁰, showing that it is a strong positive modulator of all three receptors (not shown). In oocytes expressing wild-type α1β2γ2L receptors, alphaxalone enhanced maximal GABA currents on average by 9 ± 3.6% (n = 8). Alphaxalone enhanced maximal currents in receptors containing β2(N265S) and β2(N265M) mutations by 7 ± 3.2% (n = 10) and 19 ± 6.1% (n = 7), respectively. Assuming that 10 mm GABA plus alphaxalone activates all nondesensitized channels, we infer that GABA alone has an efficacy of 0.92 ± 0.030 in wild-type, 0.93 ± 0.030 in α1β2(N265S)γ2L, and 0.84 ± 0.043 in α1β2(N265M)γ2L. The results for the β2(N265M) mutant demonstrate significantly lower GABA efficacy (ANOVA P < 0.001) than either of the other two receptors. Wild-type and β2(N265S) mutant results were not significantly different (P = 0.74).

Basal gating of all three receptors was assessed using picrotoxin, a potent GABA^Areceptor inhibitor. Picrotoxin did not significantly reduce resting oocyte leak currents in cells expressing any of the three receptors (n ≥ 3; data not shown). Given that maximal GABA currents in oocytes were as high as 7 μA and our equipment can detect changes as small as 5 nA, we infer that the basal open probability of all three receptors is less than 0.1%. To more precisely quantify the effects of mutations on basal gating, we substituted wild-type α1 subunits with α1(L264T), containing a mutation that confers measurable basal gating activity (fig. 4). Picrotoxin (2 mm) produced an apparent outward current in α1(L264T)β2γ2L receptors that averaged 12 ± 4.5% (n = 7) of maximal GABA response. In oocytes expressing α1(L264T)β2(N265S)γ2L and α1(L264T)β2(N265M)γ2L receptors, picrotoxin produced apparent outward currents that averaged, respectively, 15 ± 7.0% (n = 11) and 7 ± 2.4% (n = 7) of maximal GABA currents. The α1(L264T)β2γ2L and α1(L264T)β2(N265S)γ2L results were not significantly different (ANOVA P = 0.15), whereas α1(L264T)β2(N265M)γ2L results differed significantly from the other two receptors (P = 0.0004 vs.  wt and P < 0.0001 vs.  N265M). These results indicate that the β2(N265S) mutation causes little or no change in basal gating, while the β2(N265M) mutation reduces basal gating probability approximately two-fold.

Global mechanism-based analyses of oocyte data were performed as previously described.26An initial fit with all parameters free to vary was used to determine the L⁰value for wild-type (25,000 ± 9,000). Our experimental results indicated that the β2(N265S) mutation does not alter basal gating; therefore, the L⁰value was constrained to equal the wild-type value in fitting α1β2(N265S)γ2L receptor data (table 2). Our data indicated that the β2(N265M) mutation reduced basal gating probability two-fold relative to wild-type channels; therefore, the L⁰value for fitting α1β2(N265M)γ2L data was fixed at twice that used for wild-type. Fits to calculated P^openestdata sets for both wild-type and α1β2(N265S)γ2L converged for all four remaining free parameters in equation 3(Methods). α1β2(N265M)γ2L receptors are totally insensitive to etomidate; therefore, fits to P^openestdata for this receptor did not converge on values for either K^Eor d. These parameters were removed from equation 3for subsequent fit to α1β2(N265M)γ2L data, resulting in well-determined parameters for both K^Gand c. Results of the nonlinear least squares fits are reported in table 2and displayed in figure 5.

GABA-activated currents in outside-out patches excised from transfected HEK293 cells were used to study macrocurrent kinetics of GABA^Areceptors (fig. 6, table 3). As previously reported,23α1β2γ2L wild-type currents elicited with high GABA concentrations display rapid activation, multiphasic desensitization, and biphasic deactivation after discontinuation of agonist. Currents elicited from patches expressing mutant receptors showed similar kinetics, which were analyzed in detail. For wild-type and both mutants, the best fit (F test; P = 0.99) number of exponential deactivation phases was two in all patches, and the best-fit number of desensitization phases was two in the majority (18 of 23) of patches. Thus, for comparison, deactivation and desensitization phases of all patches were reanalyzed by using double-exponential functions. Statistical comparison with ANOVA indicates that fitted time constants for activation, desensitization, and deactivation are indistinguishable for all three types of receptor channels (table 3). Fast deactivation rate analysis suggested that this phase in α1β2(N265M)γ2L may be faster than in wild-type receptors (59 s⁻¹vs.  42 s⁻¹), although this difference was not statistically significant (P = 0.15).

A final set of experiments addressed the efficacy and potency of etomidate direct activation in spontaneously active receptors containing the α1(L264T) mutation combined with γ2L and either β2 or β1 subunits (fig. 7). Oocyte-expressed α1β1γ2L receptors displayed no detectable (less than 0.1%; n = 5) picrotoxin-sensitive spontaneous activity and are characterized by GABA EC⁵⁰= 190 μm (fig. 7A, solid diamonds). Oocyte-expressed α1(L264T)β1γ2L receptors display 11 ± 2.1 (n = 5) spontaneous activity and GABA EC⁵⁰= 3.8 μm (fig. 7A, open diamonds). High GABA responses are not significantly enhanced by alphaxalone in either α1(L264T)β1γ2L or α1(L264T)β2γ2L (not shown), indicating that maximal GABA efficacy in both gating mutant channels is near 1.0. Etomidate potently (EC⁵⁰= 0.8 μm) activates α1(L264T)β2γ2L receptors, with efficacy that is similar to GABA in these channels (fig. 7B, solid triangles). By using a value of 8 for L⁰(calculated from I^ptx/I^GABAresults; fig. 4) along with K^E= 40 μm and d = 0.0076 from the wild-type α1β2γ2L global model fit (table 2), equation 3closely predicts etomidate direct activation in α1(L264T)β2γ2L receptors. In contrast, the etomidate EC⁵⁰for direct activation of α1(L264T)β1γ2L receptors is 43 μm, and maximal etomidate activation reaches about 60% of the maximal GABA response. The α1(L264T)β1γ2L etomidate direct activation data were used to calculate P^openest(equation 2, methods) and fitted with equation 3, resulting in L⁰= 9, K^E= 58 μm, and d = 0.26. Our estimated parameters for etomidate binding to closed receptors containing β1 versus β2 subunits differ by less than 50% (K^E= 58 μm with β1 vs.  40 μm with β2), whereas etomidate efficacy (inversely related to d) is 34 times larger in β2-containing receptors versus β1-containing receptors (d⁻¹= 3.85 with β1 vs.  132 with β2).

Our aim in this study was to better define the role of the β subunit residue at position 265 in GABA^Areceptor function in both the absence and presence of etomidate. Previous studies have reported diminished sensitivity to etomidate in heterologously expressed GABA^Areceptors containing either β2 or β3 subunits with N265S15or N265M mutations.13,16,17,27,28Unlike previously published reports, our experiments quantitatively assessed etomidate modulation of GABA activation as the ratio of GABA EC⁵⁰s in the absence and presence of a standard etomidate concentration, a robust measure of positive allosteric modulation.25,29This approach demonstrates that, in α1β2γ2L GABA^Areceptors, the β2(N265S) mutation reduces etomidate modulation by sixfold (2.3-fold shift vs.  14-fold for wild-type), whereas β2(N265M) eliminates this shift entirely. We further tested the impact of the β2N265 mutations on direct receptor activation by etomidate. We found that etomidate elicits more than 40% of maximal GABA current in wild-type, about 3% in α1β2(N265S)γ2L receptors, and no detectable current in α1β2(N265M)γ2L receptors. Thus, the effect of the β2N265 mutations on etomidate direct activation parallels that on modulation of GABA responses. Previous studies have also noted that both direct etomidate activation and modulation of GABA responses are reduced in receptors containing β1 versus β212or those containing β2/3(N265M) mutations.13 

We also evaluated whether β2N265 mutations alter GABA^Areceptor gating in the absence of etomidate, including experiments to measure spontaneous (0 GABA) gating activity, GABA EC⁵⁰, maximal GABA efficacy, and the macroscopic rates of transitions among major functional states. We found that none of these parameters was significantly altered by the β2(N265S) mutations, whereas the β2(N265M) mutation produced a 23% increase in GABA EC⁵⁰(table 1), and also significantly reduced maximal GABA efficacy from 0.92 to 0.84. Whereas we could detect no spontaneous gating in wild-type, α1β2(N265S)γ2L, or α1β2(N265M)γ2 channels, novel experiments exploiting the spontaneously gating α1(L264T)β2γ2L mutant channel background reveal that substituting β2(N265M) for β2 reduces basal gating activity by 50%, and substitution with β2(N265S) causes no significant change. Analysis of GABA-activated current kinetics recorded using submillisecond concentration jumps shows that activation, desensitization, and deactivation rates are not significantly altered by either β2(N265S) or β2(N265M), although rapid deactivation tended to be faster in α1β2(N265M)γ2L receptors than in wild-type (table 3).

A number of previous studies have reported that β2(N265M) or β3(N265M) mutations increase GABA EC^{5016,17,28,30}. Nishikawa et al.  30studied a series β2N265 mutations in α1β2γ2L receptors in HEK293 cells and found that GABA EC⁵⁰approximately doubled with β2(N265M) and increased about 50% with β2(N265S). One previously published study by Miko et al.  31investigated spontaneous gating in homomeric β1 GABA^Areceptors and the effects of β1(S265) mutations. In that study, mutant β3(N265S) subunits did not induce spontaneous gating in homomeric or heteromeric channels. No previous studies have assessed the influence of β2N265 or β3N265 mutations on macrocurrent kinetics in heterologously expressed channels. However, because GABA reuptake from synapses occurs in less than a millisecond,32GABAergic inhibitory postsynaptic current decay is largely dependent on channel deactivation. Reynolds et al.  2reported that GABAergic miniature inhibitory postsynaptic current decays were similar in cerebellar Purkinje neurons from both wild-type and β2(N265S) knock-in mice. Drexler et al.  33found that inhibitory postsynaptic current decays in neocortical neurons of wild-type and β3(N265M) knock-in mice were not significantly different. The subunit compositions of channels mediating these neuronal inhibitory postsynaptic currents is uncertain, but inhibitory postsynaptic current prolongation by etomidate was dramatically reduced in currents recorded from knock-in versus  wild-type cells, demonstrating that GABA^Areceptors containing mutant β subunits were present. These results therefore indicate that in vivo  neuronal GABA^Achannel kinetics are minimally altered by N265S and N265M mutations.

Allosteric gating models have proven useful for interpretation of how mutations alter the function of ligand-gated ion channels.34,35We have used allosteric coagonist models to interpret both etomidate actions on GABA^Areceptors5and the effects of mutations on etomidate sensitivity,26and we have applied this approach here to analyze the impact of the β2N265 mutants we studied (fig. 5, table 2). Importantly, allosteric coagonist models appear to fit our wild-type and mutant data well, reinforcing the hypothesis that both etomidate modulation of GABA activation and direct etomidate activation of GABA^Areceptors are manifestations of interactions at a single class of etomidate sites observed under different experimental conditions.5After correcting L⁰values according to our basal gating results, GABA binding (K^G) and efficacy (c⁻¹) parameters in all three fitted models (table 2) are all quite similar and not statistically different, suggesting that these mutations have little or no impact on GABA interactions. The total lack of etomidate effects in α1β2(N265M)γ2L receptors makes it impossible to fit etomidate binding and efficacy parameters in the allosteric model. However, comparison of coagonist model parameters for wild-type versus α1β2(N265S)γ2L receptors indicates that the β2(N265S) mutation reduces etomidate efficacy (d⁻¹) five-fold from wild-type, whereas etomidate binding (1/K^E) affinity is reduced about two-fold.

Direct activation experiments using the gating mutant α1(L264T) expressed together with β1 versus β2 also addressed whether etomidate binding versus  efficacy is affected by changing β subunit subtype. Combining the gating mutant L⁰with fitted values for etomidate binding (K^E) and efficacy (d) from the α1β2γ2L model accurately predicts etomidate direct activation of α1(L264T)β2γ2L channels. This result suggests that the α1(L264T) mutation does not affect etomidate binding or efficacy, but simply sensitizes receptors to etomidate gating by increasing their tendency to open. Most importantly, α1(L264T)β1γ2L and α1(L264T)β2γ2L channels display similar spontaneous activity, but those with β1 subunits display less etomidate direct activation and much lower apparent affinity (etomidate EC⁵⁰) than those with β2 subunits. Allosteric mechanism analysis indicates that these changes are largely due to etomidate efficacy (d⁻¹) that is 34-fold lower in α1(L264T)β1γ2L versus α1(L264T)β2γ2L.

The analysis of β2N265 mutants contrasts with our recent study of tryptophan mutations at α1M236 and β2M286,26and it is most consistent with the conclusion that β2N265 is not a contact point for etomidate binding to GABA^Areceptors. Instead this residue may act as a transduction element between the etomidate sites and the ion channel. Other evidence also supports the conclusion that β2N265 does not contribute to etomidate binding. Recent structural models of the GABA^Areceptor transmembrane domains,11,18,19based on disulfide crosslinking and photolabeling data, depict βN265 located outside the intersubunit cleft where both etomidate and propofol bind. Furthermore, propofol does not protect cysteine substitutions at β2N265 from covalent modification, whereas it does protect cysteine modification at β2M286,36a site where there is abundant evidence for contact with etomidate.11,26An alternative interpretation of our findings is based on the concept of efficacy in allosteric gating models. Agonist (or coagonist) efficacy is the ratio of equilibrium binding dissociation constants in the two canonical states: active (open channel) versus  inactive (closed channel). Thus, reduced etomidate efficacy in β2N265 mutants implies reduced etomidate affinity for the transient open GABA^Areceptor state. This introduces the possibility that, although β2N265 does not contribute to etomidate binding to inactive GABA^Areceptors, it might make contact with etomidate during channel opening. This interpretation also points out the importance of defining anesthetic binding determinants in more than one receptor state, a task that may be achieved using time-resolved photolabeling.37,38 

Mutations that alter GABA^Areceptor anesthetic sensitivity in vitro  represent potential tools for knock-in animal studies linking subunits to the various actions of anesthetics.39In this regard, the β2(N265S) mutation is remarkable for reducing etomidate sensitivity without affecting basal or GABA-activated receptor activity. Indeed, Reynolds et al.  2reported that β2(N265S) knock-in mice have normal receptor expression, normal baseline and sleeping electroencephalographic activity, and normal baseline behavior. In comparison with β2(N265S), the β2(N265M) mutation produces two molecular effects: a small negative gating effect in the absence of etomidate combined with abolition of etomidate sensitivity. However, whereas the viability and general behavior of β3(N265M) knock-in mice is apparently normal, no data have been published on their awake or sleeping electroencephalographic patterns or susceptibility to seizures. One behavioral study40reported that baseline freezing in response to a learned fear context was significantly lower in β3(N265M) knock-ins versus  wild-type mice.

GABA^Areceptor mutations with negative gating effects in vitro , such as γ2(K289M), are associated with increased GABA EC⁵⁰and with epilepsy in vivo ,41whereas gain-of-function mutations such as α1(S270H) reduce GABA EC⁵⁰and have been associated with grossly abnormal anatomic and behavioral phenotypes in knock-in animals.42Allosteric models illustrate that in vitro  GABA EC⁵⁰is a function of baseline channel open probability (L⁰), GABA-binding affinity (K^G), and efficacy (c). A single mutation could simultaneously alter both L⁰and GABA efficacy, resulting in a near-normal GABA EC⁵⁰, but significantly abnormal channel activity. Therefore, GABA EC⁵⁰alone may be a misleading predictor of channel activity and in vivo  phenotype. Assessment of basal gating changes and maximal GABA efficacy, together with GABA EC⁵⁰, will provide a stronger basis for this prediction.

The authors thank Aiping Liu, M.S., Senior Technician, Department of Anesthesia & Critical Care, Massachusetts General Hospital, Boston, Massachusetts for technical assistance. They also thank Uwe Rudolph, M.D., Director of the Laboratory for Genetic Pharmacology, McLean Hospital, Belmont, Massachusetts, for his insights into knock-in animal phenotypes.