Point Mutation in the First Transmembrane Region of the β2 Subunit of the γ-Aminobutyric Acid Type A Receptor Alters Desensitization Kinetics of γ-Aminobutyric Acid- and Anesthetic-induced Channel Gating

A conserved glycine residue in the first transmembrane (TM1) domain of the β2 subunit has been identified to be involved with desensitization induced by γ-aminobutyric acid (GABA) and anesthetics. Recombinant GABAA receptors expressed in Sf9 cells were recorded using semi-fast agonist application. Upon direct activation by GABA or anesthetics, the main effect of the TM1 point mutation on the β2 subunit (G219F) was to slow the time constant (τ) of desensitization. At GABA concentrations eliciting maximum currents, the corresponding median τ values were 0.87 s (25–75% interval (0.76; 1.04 s)), 0.93 s (0.76; 1.23 s), and 1.36 s (1.17; 1.57 s) for α1β2γ2, α1(G223F)β2γ2, and α1β2(G219F)γ2, respectively. The τ value for the β2-mutant receptor was significantly longer than α1β2γ2 (p < 0.01) and α1(G223F)β2γ2 (p < 0.05). For pentobarbital-induced currents (500 μm), the corresponding median τ values were 1.36 s (0.81; 1.41 s), 1.47 s (1.31; 2.38 s), and 2.82 s (2.21; 5.56 s) for α1β2γ2, α1(G223F)β2γ2, and α1β2(G219F)γ2, respectively. The τ value for the β2-mutant receptor was significantly longer than that for α1β2γ2 (p < 0.01). The present findings suggest that this TM1 glycine residue is critical for the rate at which desensitization occurs and that both GABA and intravenous anesthetics implement an analogous pathway for generating desensitization.

Most volatile and intravenous anesthetics enhance the activity of ␥-aminobutyric acid type A (GABA A ) 1 receptors and directly activate this ligand-gated chloride ion channel in the absence of its endogenous ligand, GABA (1,2). Specifically, anesthetics are known to prolong GABA-induced Cl Ϫ channel opening (1,2), and, depending on the type of anesthetic, this potentiation of GABA-gated currents appears to alter deactivation and/or desensitization. For example, halothane, a volatile anesthetic, has been shown to slow the dissociation of GABA from its receptor, i.e. slows deactivation (3). Propofol, an intravenous anesthetic, slows both deactivation and the exit rate from desensitization (4), and neurosteroids, some of which have anesthetic qualities (5), also decrease the recovery rate from desensitization (6). From these studies, it is apparent that desensitization of GABA A receptors is altered in the presence of intravenous anesthetics.
Investigations using chimeras and site-directed mutagenesis have identified key amino acids in the second transmembrane domain (TM2) of both the ␣ and ␤ subunits, which are involved in the conformational state of desensitization (7,8). Furthermore, for GABA A receptors, structural determinants of anesthetic action have been primarily located to both the TM2 and TM3 regions of GABA A receptors (9 -12). These same regions have been described to be an integral part of the channel gating domain of the GABA A receptor (13,14). These data suggest that the desensitization machinery appears to lie within the channel gating region and that this domain is also allosterically sensitive to anesthetics.
Another GABA A receptor domain to be allosterically sensitive to intravenous anesthetics is the TM1 region. As shown below in Fig. 1, the N-terminal of TM1 is highly conserved among GABA A receptor subunits, including the 1 subunit, which comprises the homomeric receptors that are insensitive to most anesthetics (1). However, the TM1 glycine residue, which is conserved across GABA A receptor subunits, is replaced by a phenylalanine in the 1 subunit (see Fig. 1). In the previous study by Carlson et al. (2000), the mutation of the TM1 glycine of the ␤2 subunit to the homologous residue, phenylalanine, in the 1 subunit, i.e. ␤2(G219F) was shown to affect receptor gating induced by both GABA and anesthetics (15). This finding was consistent with the suggestion that the TM1 region may work together with TM2 for channel gating (16). Because this TM1 glycine on the ␤ subunit is perhaps linked with the channel gating region of TM2, the present study tests the hypothesis that glycine 219 on the ␤2 subunit affects the conformational events of desensitization induced by GABA and/or anesthetics. Kinetic analyses were performed on whole-cell patch clamp recordings from wild type GABA A receptors, ␣1␤2␥2, and mutant GABA A receptors, ␣1(G223F)␤2␥2 and ␣1␤2(G219F)␥2, which were recombinantly expressed in Sf9 cells. It was determined that a TM1 glycine on the ␤2 subunit is involved with desensitization of GABA-, pentobarbital-, and propofol-induced currents. These findings suggest that the structural determinants for regulating desensitization resulting from direct activation of the GABA A receptor chloride ionophore are similar for GABA and anesthetics.

Site-directed Mutagenesis and Generation of Recombinant
Baculoviruses-Point mutations were introduced into the cDNAs of rat ␣1 and ␤2 GABA A receptor subunits with an in vitro mutagenesis system (Altered Sites II, Promega). The coding region of ␣1 (and ␤2 subunit performed separately) was subcloned into pAlter, and both mutations, ␣1(G223F) and ␤2(G219F), were incorporated using a mutagenic oligonucleotide. Following verification of mutagenesis by DNA sequencing, the point-mutated GABA A receptor subunits were subcloned into the baculovirus transfer vectors for generation of recombinant baculovirus according to BAC-BAC expression system (Invitrogen) or BaculoGold transfection kit (BD PharMingen). All procedures were performed according to the manufacturer's suggestion and as previously described (15).
Cell Culture and Baculovirus Infection-Sf9 insect cells (Spodoptera frugiperda) were grown as a shaking culture (140 rpm) in serum-free medium (Sf900 II medium, Invitrogen) at 27°C. Sf9 cells were infected with multiple combinations of recombinant Autographa californica nuclear polyhedrosis viruses encoding for the following rat GABA A receptor subunits: ␣1, ␤2, ␥2, ␣1(G223F), and ␤2(G219F). The determination of virus titer and the amount of recombinant baculovirus added for each infection was performed according to the protocol from the Invitrogen instruction manual, Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques.
Drug Applications-Stock solutions of GABA (Sigma) and pentobarbital sodium (DAK, Denmark) were dissolved to a concentration at least 100ϫ greater than that required for perfusion and premixed by diluting solutions in ABSS. Propofol (Tocris) was dissolved in Me 2 SO and diluted in ABSS; the content of Me 2 SO in the perfusion solutions was at most 0.1%, which had no effect of its own on membrane current. The solutions were applied from a multibarreled perfusion pipette (18) ϳ100 m from the cell. Agonists were applied for 5 s every 1 min. In modulation experiments, pentobarbital or propofol was applied together with GABA as a premixed solution and in some experiments also for 10 s immediately before the combined application. Between drug applications the cell was perfused from one of the barrels with ABSS. The extracellular solution exchange rate was determined in separate experiments. Initially a stable (desensitized) current was established by application of 2 mM GABA in normal ABSS. Then the extracellular Cl Ϫ concentration was lowered by switching to application of the same GABA concentration dissolved in modified ABSS, where 90 mM of the NaCl was substituted by an equimolar concentration of sodium gluconate. The time constant and 10 -90% relaxation time for the resulting current relaxation were measured.
Quantification of Responses-Responses were quantified by measuring the peak current during application of agonist (e.g. GABA and/or anesthetics) and the current remaining after 5 s of application (end current). Rise time was estimated as the time needed for the current to increase from 10 -90% of the peak response. The time constant for desensitization ( desens ) was estimated from the current decay from the peak to the end of the 5-s application, whereas the time constant for deactivation ( deact ) was estimated using the current decay from the termination of agonist application to baseline. Time constants were fitted by mono-and biexponential functions, using PulseFit (HEKA) software. The quality of the fit was evaluated by the root mean square value. In general, the fit was not significantly improved by using two exponentials.
Statistics-Current data (peak currents, end currents) were normally distributed. They were described using mean and standard error (S.E.) and compared with analysis of variance followed where relevant by a Tukey multiple comparison procedure. Time data (rise times, time constants) were often not normally distributed and therefore described using median, 25 and 75% quartiles. Comparisons were made using the Kruskal-Wallis one-way analysis of variance followed where relevant by Dunn's multiple comparison procedure. Probabilities (p) Ͻ 0.05 were considered statistically significant.

Comparison of Kinetics of Wild Type and TM1 Glycine-mutated GABA A Receptors at Maximum GABA Currents-The
GABA concentration-response relationships for the wild type (␣1␤2␥2) GABA A receptor and the mutated ␣1(G223F)␤2␥2 and ␣1␤2(G219F)␥2 receptors have been characterized in our previous study (15), and the vital data are summarized in Table  I. Briefly, mutation of the ␣1 subunit did not significantly affect the concentration-response relation for GABA-induced peak currents. The corresponding mutation in the ␤2 subunit, on the other hand, significantly decreased the EC 50 of the receptor for GABA. The Hill coefficients determined for the three subunit combinations were not significantly different.
To estimate possible differences in the kinetics of the different GABA receptors, the lowest concentrations of GABA giving rise to maximum peak responses (saturating concentrations) were investigated (i.e. 2 mM for the wild type and the ␣1(G223F)␤2␥2 receptors, and 0.2 mM for the ␣1␤2(G219F)␥2 receptor (Fig. 2). The rate of current onset was described using the 10 -90% rise time. As shown in Fig. 3A, the rise time did not significantly differ between the wild type and the ␣1(G223F) and ␤2(G219F) mutated receptors. Increasing the GABA concentration from 0.2 to 2 mM for the ␤2-mutant receptor did not further decrease the rise time. None of the 10 -90% rise times for saturating GABA concentrations were significantly different from the 10 -90% exchange time for extracellular solution (median 112 ms, 25-75% interval (79; 141 ms)) determined in nine cells expressing the ␣1␤2␥2 receptor. The time constant for solution exchange was 52 ms (37; 67 ms).
Differences in the extent of desensitization were estimated from the proportion of the peak current remaining at the end of the 5-s application. No significant differences in the extent of current fade between the wild type and the mutant receptors There are five amino acids that are conserved in all the subunits listed, including 1 (asterisks) and seven amino acids that are conserved within subunit families (dots). The sources for all sequences were Gen-Bank TM and Cutting et al. (25). were detected using the saturating GABA concentrations mentioned above (Fig. 3B).
The desensitization kinetics were described using the time course of current fade, which for all three receptor types was described by one exponential component (Figs. 2 and 3C). Although the corresponding time constants ( desens ) for the wild type GABA A receptor and the ␣1(G223F) mutated receptor were similar, the desens for the ␤2(G219F) mutated receptor was significantly longer than the desens of the wild type (p Ͻ 0.01) and the ␣1-mutant (p Ͻ 0.05) receptors. Increasing the GABA concentration to 2 mM on the ␤2-mutant decreased desens (p Ͻ 0.05) and the end current (p Ͻ 0.05) significantly. Deactivation time course of the GABA-elicited currents were also adequately described by one exponential component ( Fig.  3D), with similar time constants ( deact ) for the wild type and mutant receptors at saturating concentrations. When 2 mM GABA concentration was applied to the ␤2-mutant receptor, the deact increased, and it was significantly longer than that for the ␣1(G223F) mutant receptor (p Ͻ 0.01).
Mutation of TM1 Glycine on ␣1 and ␤2 Subunit Diminishes Propofol-induced Enhancement of GABA Currents-The modulating effect of propofol on currents induced by GABA at EC 20 in the wild type and mutant receptors is summarized in Table  II, which shows the effect of the highest propofol concentration tested for each receptor combination without inducing direct activation: 50 M for the ␣1␤2␥2 and ␣1(G223F)␤2␥2 and 5 M for the ␣1␤2(G219F)␥2 combinations. In all three receptor combinations, pretreatment with propofol was necessary to significantly enhance peak currents induced by GABA (wild type: p Ͻ 0.01, ␣-mutant and ␤-mutant: p Ͻ 0.001). The modulating effect of propofol was significantly smaller (p Ͻ 0.05) for the ␣1(G223F)␤2␥2 combination as compared with the ␣1␤2␥2 combination at both 10 and 50 M propofol (Table II and Fig. 4). The ␣1␤2(G219F)␥2 receptor showed significantly smaller modulation of GABA-induced currents when comparing 1 and 5 M propofol for the ␤2 mutant with 10 and 50 M propofol for the wild type (p Ͻ 0.05). There was no significant difference in the modulating effect of propofol between the ␣1(G223F)␤2␥2 and the ␣1␤2(G219F)␥2 receptors (Table II). Neither the rise times nor the end currents remaining after 5-s application were significantly altered by propofol in any of the receptor combinations, and furthermore no significant differences were found in the rise times between the different receptor combinations (data not shown). Due to the slow time course of current decay during GABA application at EC 20 the corresponding time constants could not be calculated.
Mutation of TM1 Glycine on ␤2 Subunit Increases Anestheticinduced Direct Activation and Alters Desensitization-As shown previously (15), the ␣1␤2(G219F)␥2 receptor demonstrated a biphasic concentration-response curve and was significantly more sensitive than the ␣1␤2␥2 combination to the direct effect of pentobarbital. The concentration-response relationships for pentobarbital in the wild type and mutant receptors are shown in Fig. 5A. For the ␤2-mutant receptor, the peak currents at 500 M (p Ͻ 0.001) and 1500 M (p Ͻ 0.05) pentobarbital were significantly higher than at 50 M pentobarbital. There was no significant difference between the peak currents of the ␣1␤2␥2 and the ␣1(G223F)␤2␥2 receptors at any concentration of pentobarbital tested.
The kinetics of pentobarbital-activated currents in the ␤2mutant was investigated by employing 50, 500, and 1500 M pentobarbital (Fig. 6). The rise times were significantly shorter at 1500 M pentobarbital than 50 (p Ͻ 0.01) and 500 M (p Ͻ 0.05) (Fig. 6A), as expected for a concentration-dependent activation. The current remaining after application of 500 M pentobarbital was significantly larger than the current remaining after application of 50 (p Ͻ 0.01) and 1500 M pentobarbital (p Ͻ 0.05) (Fig. 6B). From the peak, the current faded monoexponentially for all the pentobarbital concentrations tested (Fig. 6C). The time constants for the current fade elicited by 500 M pentobarbital were significantly longer than the time constants for 50 and 1500 M pentobarbital (p Ͻ 0.01). Deactivation values for 50 and 500 M pentobarbital were not significantly different from each other (see Fig. 8D below). For 1500 M pentobarbital, termination of the agonist application gave rise to a transient current peak (off-current) in all of 10 cells tested. Therefore, there was no estimation of the deactivation value at this higher concentration.
For propofol, the ␣1␤2␥2 receptor showed no significant direct activation up to 1 mM (Fig. 5B), consistent with another study, which showed that the ␤2-containing GABA A receptors are not an efficient substrate for propofol modulation (19). The ␣1(G223F)␤2␥2 receptor followed the same pattern as the wild type. The ␣1␤2(G219F)␥2 receptor, on the other hand, demonstrated a monophasic concentration-dependent activation by propofol. The kinetics of this direct activation were investigated by comparing rise time, end current, and time constant at two propofol concentrations. There was no significant difference between the rise times at 300 and 1000 M propofol (Fig.  7A). The end current remaining was smaller for 1000 M propofol as compared with 300 M propofol (p Ͻ 0.05, Fig. 7B), whereas the time courses of desensitization for both concentrations were monoexponential with time constants that were not significantly different from each other (Fig. 7C). Deactivation was significantly smaller with 1000 M propofol than 300 M propofol (p Ͻ 0.05, Fig. 7D).

Role of GABA A Receptor TM1 in Desensitization
significantly different, whereas the peak response of 500 M pentobarbital on the ␣1␤2(G219F)␥2 receptor was significantly larger than any of these (p Ͻ 0.001 in all cases). The rise-time for the wild type and the ␣1-mutant receptors at 500 M pentobarbital and for the ␤2-mutant receptor at 50 and 500 M pentobarbital did not significantly differ from each other (Fig. 8A).
To compare the desensitization kinetics, the fading of direct pentobarbital-induced currents was compared (Fig. 8B). Upon application of 500 M pentobarbital to the ␤2-mutant receptor, a significantly larger (p Ͻ 0.01) end current remained than with 500 M pentobarbital for the wild type and the ␣1-mutant receptors. For all combinations, the time course of current fade was described by one exponential component. The desens value for 500 M pentobarbital in the ␤2-mutant receptor was significantly larger (p Ͻ 0.01) than 50 M in the same receptor and 500 M pentobarbital in the wild type receptor (Fig. 8C).
The deactivation time course of the pentobarbital-elicited currents were also adequately described by one exponential component (Fig. 8D), with no significant differences between deact values for the wild type and mutant receptors at the same concentrations.

␤2(G219F)-mutant Receptors and GABA-induced Currents-
The present study examined the effect of a point mutation in the TM1 region of the ␣1 and ␤2 GABA A receptor subunits on the kinetics of GABA-mediated Cl Ϫ currents. As indicated previously (15), the apparent affinity for GABA was increased in the ␣1␤2(G219F)␥2 receptors.
Using ultra-fast agonist application to GABA A receptors in outside-out membrane patches, it is possible to achieve 10 -90% rise times of ϳ1 ms at saturating GABA concentrations (e.g. ␣1␤1␥2 receptors (20)). In the present experiments the maximum activation rate and peak current were limited by the speed of agonist application. This limit comes into effect at the GABA concentrations giving rise to maximum peak currents where the rise times reach the lower limit set by the extracellular solution exchange rate (Fig. 3). Accordingly, an increase of the GABA concentration from 0.2 to 2 mM for the ␤-mutant did not further decrease the rise time.
As measured by the end-current remaining during a saturating GABA application, the degree of desensitization was not different between the wild type and the two mutant receptors. Part of the current fade may be due to the Cl Ϫ current causing a shift of the Cl Ϫ gradient across the cell membrane (21), but because of the similar extent of current fade for the three receptor combinations tested, it is likely that the contribution of Cl Ϫ shift is of similar magnitude. The time constant for desensitization, however, was significantly longer for 200 M GABA in the ␤2-mutant receptor. This slower desensitization could be significantly accelerated by raising the GABA concentration at the ␤2(G219F) combination to 2 mM (as used for the other combinations), whereby a time constant comparable to the other combinations was achieved. At the same time, the amount of desensitization increased significantly.
Ultra-fast agonist application to GABA A receptors in outside-out membrane patches often reveals a desensitization time course with two exponential components (e.g. a fast component of Ͻ 10 ms and a slower component of ϳ 150 ms for ␣1␤3␥2 receptors) (22), although some investigations have found monoexponential desensitization (e.g. ϳ 500 ms, ␣1␤1␥2 receptors) (20). A fast component of desensitization would not be resolved in the present experiments due to the limited rate of extracellular solution exchange. Even though our experiments do not reveal the true magnitude and time constants of desensitization, the differences observed between the receptor combinations reflect actual differences in desensitization kinetics. Although the approximately 10-fold decrease of EC 50 and the concentration required to achieve the minimum rise time and maximum peak current could be explained by a selective increase in agonist binding rate for the ␤2mutant receptor, the changes of the magnitude and time constant of desensitization imply that the TM1 glycine on the ␤2 subunit is part of the desensitization machinery of GABA A receptors.
How a decrease in functional EC 50 for GABA is associated with an increase in the value for desensitization is difficult to resolve. However, it should be noted that the GABA A receptors have a lower EC 50 for GABA than ␣1␤2␥2 receptors, and receptors do not desensitize or desensitize at an extremely slow rate (23,24). Moreover, this TM1 glycine residue is conserved across all GABA A receptor subunits, except the subunit, which has a phenylalanine (25). A working theory on channel gating described by Akabas and Karlin (16), hypothesizes that the N-terminal region of the TM1 domain works in tandem with the TM2 domain to elicit the conformational events of gating (i.e. activation, desensitization, and deactivation). Because glycine residues allow for conformational flexibility (26), subunits containing this residue may transfer the agonist binding energy more readily to the conformational state of desensitization than the bulkier hydrophobic residue, phenylalanine. Thus, upon channel activation, the ␤2 mutant receptors have increased channel currents due to the slowing of desensitization.
Other studies using chimeras and site-directed mutagenesis have shown that the TM1 region of the ␤2 and ␥2 subunits harbor important residues for fast desensitization (22,27). Interestingly, two TM1 residues on the ␥2 subunit, directly adjacent to the conserved TM1 glycine residue that is mutated in the present study, have been shown to be important for fast desensitization (22). Fast desensitization in the ␥2-containing GABA A receptors was not eliminated by mutating these ␥2 TM1 residues alone (22), but other structural determinants in the extracellular N-terminal are most likely required, indicating that there are multiple determinants on the ␥2 subunit and perhaps other subunits for fast desensitization. The data with our ␣1␤2(G219F)␥2 receptor support the claim that the Nterminal end of TM1 domain of the ␤ subunit is involved with desensitization. Fast desensitization has been shown to correlate with prolonged deactivation, probably due to reopening of channels after leaving the long-lived desensitized states (28). Desensitization and deactivation can, however, be uncoupled by mutation of the above-mentioned two amino acids in the TM1 region of the GABA A receptor ␥2 subunit, which selectively accelerated deactivation without altering desensitization (22). Although our agonist application rate does not allow us to resolve fast desensitization, the resulting states still become populated during agonist application and would be expected to influence the time course of deactivation. Indeed, an increase in GABA FIG. 7. Propofol kinetics for direct activation of the ␣1␤2(G219F)␥2 GABA A -mutated receptor. A, the 10 -90% rise time. Each column represents the median Ϯ 25-75% interval of 6 -10 Sf9 cells. B, the end-current (after 5-s application of propofol relative to the peak current) was significantly smaller for 1000 M than for 300 M propofol (*, p Ͻ 0.05). Each column represents the means Ϯ S.E. of 9 -10 Sf9 cells. C, the time constants of desensitization ( desens ) for 300 and 1000 M were not significantly different from each other. Each column represents the median Ϯ 25-75% interval of 3-5 Sf9 cells. D, the time constant of deactivation ( deact ) was significantly smaller for 1000 M as compared with 300 M propofol (*, p Ͻ 0.05). Each column represents the median Ϯ 25-75% interval of 6 -9 Sf9 cells. concentration from EC 20 to a saturating GABA concentration prolonged deactivation for all three receptor types in the present investigation. The ␤2(G219F) mutation gave rise to a significant prolongation of deact at EC 20 concentrations compared with the wild type and ␣-mutant receptors, but this difference vanished at the saturating GABA concentrations. Further increase in the GABA concentration from 200 M to 2 mM with the ␤2-mutant receptor resulted in both increased amount of desensitization and prolongation of the deactivation (Fig. 3). Thus, the ␣1(G223F) and ␤2(G219F) mutations did not have any prominent effect on desensitization-deactivation coupling.
TM1 Glycine and Anesthetic-modulated GABA Currents-Because propofol can induce direct activation of GABA A receptors, modulation experiments with propofol were conducted with concentrations that elicited only enhancement and were not confounded with direct activation effects. For the ␣1and ␤2-mutant receptors, propofol-induced enhancement of GABA currents was diminished. A similar finding for the ␤2-mutant receptor has been shown for pentobarbital-modulated GABA currents (15). Upon analyzing the decay of the propofol-modulated currents, it was assessed that there was no change in the magnitude of desensitization for both the ␣1and ␤2-mutant receptors as compared with wild type receptors. The same results were also assessed for pentobarbital-modulated GABA currents (results not shown). Desensitization time constants could not be determined for the modulation experiments due to the slow decay of current elicited by GABA (EC 20 ). Other studies have shown that the mechanism by which anesthetics modulate GABA currents is achieved by slowing the desensitization and deactivation rates (4,6). For the wild type receptor we observed that deactivation was slowed significantly by propofol, whereas for the ␣1and ␤2-mutant receptors, the effect on deact was insignificant. The reduced effect of propofol on deactivation in the two mutant receptors thus parallels the reduced enhancement of peak current. A reduced dissociation rate of GABA from the receptor has been suggested as one reason for the slowing of deactivation (4), which may also contribute to the increase in deact and peak current by propofol in the wild type receptor in this study (although other mechanisms are possible). For pentobarbital-modulated currents, the deact was not altered in any receptor combination tested, suggesting that other mechanisms that perhaps include desensitization are A decrease in anesthetic-modulation observed with the TM1 mutant receptors is consistent with two other studies which showed that the sensitivity to GABA was enhanced with point mutations at the TM2 9Ј leucine (␤2L259) and the 15Ј serine (␤1S265 and ␣2S270) (29,30). In addition, positive allosteric potentiation was reduced (29,30). A decrease in GABA-induced desensitization with the TM2 9Ј point mutation was demonstrated, as well (29). Because in the present study the TM1 ␤2(G219F) point mutation significantly decreased the EC 50 for GABA, the conformational changes needed to allosterically potentiate GABA on these receptors are most likely at or near its intrinsic maximum and thus cannot be modulated any further. However, it is important to note that the TM2 9Ј and 15Ј point mutations created spontaneously active channels and that all positive allosteric modulators, including benzodiazepines, were affected. The TM1 point mutation in the present study did not create spontaneously active channels, and in our previous study, benzodiazepine potentiation of GABA A currents in the ␤2(G219F) receptor combination was shown to be unchanged (15).
With the ␣1(G223F)␤2␥2 receptors, the EC 50 for GABA was not altered, yet propofol-induced potentiation was significantly less than in the wild type receptors, whereas pentobarbitalinduced potentiation was not altered by the ␣1 point mutation (15). Perhaps, the TM1 glycine residue on the ␣1 subunit may be a component of the binding pocket for propofol. This suggestion is supported by the finding that propofol-induced enhancement of GABA A agonist binding was also reduced in this same mutant receptor complex (15). These findings indicate that the same conserved glycine residue on the ␣1 and ␤2 subunit may contribute in different ways to the conformational events elicited by different anesthetics.
The ␤2 TM1 Glycine and the Kinetics of Anesthetic-induced Direct Activation of GABA A Receptors-Although the activation of GABA-gated currents at high concentrations were effectively limited by the application system, the observed rise times and desensitization time constants for anesthetic-gated currents were considerably larger than for GABA-gated currents. Although this does not exclude the existence of unresolved fast components, it allowed us to resolve some differences on a slower time scale, which reflect actual kinetic differences between the receptor combinations. For pentobarbital-induced direct activation of the GABA A chloride channel, the ␣1␤2(G219F)␥2 receptors demonstrated a biphasic concentration-response curve. The first phase was shifted leftward relative to the concentration-response curves of the ␣1␤2␥2 and ␣1(G223F)␤2␥2 receptors, and the peak current, rise time, end current, and time constants for desensitization and deactivation of 50 M pentobarbital for the ␤2-mutant receptor were similar to the same parameters of 500 M pentobarbital for the ␣1␤2␥2 and ␣1-mutant receptors. Thus the first phase of the concentration-response curve of the ␤2-mutant receptor could be explained by an increased association rate of pentobarbital. The effect of 500 M pentobarbital on the ␤2-mutant receptor was significantly different from that on the other two receptor combinations when comparing peak current, decay, and desens . Specifically, the amount of decay induced by 500 M pentobarbital in the ␤2-mutant receptor was significantly smaller and developed significantly slower than in the ␣1␤2␥2 receptor.
The biphasic nature of the pentobarbital concentration-response curve of the ␣1␤2(G219F)␥2 receptor was further emphasized by the concentration-dependent kinetics. Although a normal concentration-dependent decrease of the rise time was observed, the pattern of current decay was atypical. At 500 M pentobarbital, the decay was less extensive and developed with a longer time constant than at 50 or 1500 M pentobarbital. At the same time, the deactivation time constant tended to become longer with 500 M than with 50 M pentobarbital. It should be noted that for agonists the extent and rate of desensitization normally increase with increasing concentration. The present findings suggest that a second (or additional) binding site with lower affinity for pentobarbital was exposed in the presence of the ␤2(G219F) point mutation and elicited a different pattern of desensitization and that may appear to become uncoupled from deactivation.
For propofol, similar to GABA and pentobarbital, ␣1␤2(G219F)␥2 conferred a receptor that was activated by lower agonist concentrations. Desensitization was more extensive (but with a rate that tended to be slower) and deactivation was faster at a higher concentration as compared with a lower one. Although a larger fraction of receptors were in one of the desensitized states after application of the higher propofol concentration, we cannot determine whether a smaller fraction of these were in states corresponding to fast desensitization and therefore expected to slow deactivation. Thus the intactness of desensitization-deactivation coupling cannot be determined. As seen with the GABA-induced currents, the point mutation (G3 F), in the TM1 domain on the ␤2 subunit, alters the conformational changes involved in desensitization upon direct activation by an agonist, in this case, anesthetics. However, it should be kept in mind that the complete cascade of events for desensitization may not be identical between GABA-and anesthetic-induced direct activation (21).
Concluding Remarks-The molecular basis of desensitization in GABA A receptors is not well understood, but there is increasing evidence, including the present study, in support of the claim that the TM1 domain is involved in mediating conformational changes that lead to desensitization (22,27,31). To this end, the present findings suggest that GABA and anesthetics appear to implement similar conformational events, which elicit desensitization. Because it appears that the allosteric regulation of GABA A receptors by anesthetics is related to, in part, the regulation of desensitization, identifying structural determinants involved with desensitization will be essential to further elucidate the mechanism of anesthetic action.