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J. Biol. Chem., Vol. 277, Issue 20, 17438-17447, May 17, 2002

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Point Mutation in the First Transmembrane Region of the 2 Subunit of the -Aminobutyric Acid Type A Receptor Alters Desensitization Kinetics of -Aminobutyric Acid- and Anesthetic-induced Channel Gating^*

A. Christine Engblom, Berit X. Carlson, Richard W. Olsen^§, Arne Schousboe, and Uffe Kristiansen^¶

From the  Department of Pharmacology, The Royal Danish School of Pharmacy, Copenhagen 2100, Denmark and the ^§ Department of Molecular and Medical Pharmacology, School of Medicine, University of California at Los Angeles, Los Angeles, California 90095-1735

Received for publication, November 26, 2001, and in revised form, March 1, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A conserved glycine residue in the first transmembrane (TM1) domain of the 2 subunit has been identified to be involved with desensitization induced by -aminobutyric acid (GABA) and anesthetics. Recombinant GABA_A 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 2 subunit (G219F) was to slow the time constant () of desensitization. At GABA concentrations eliciting maximum currents, the corresponding median  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 122, 1(G223F)22, and 12(G219F)2, respectively. The  value for the 2-mutant receptor was significantly longer than 122 (p < 0.01) and 1(G223F)22 (p < 0.05). For pentobarbital-induced currents (500 µM), the corresponding median  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 122, 1(G223F)22, and 12(G219F)2, respectively. The  value for the 2-mutant receptor was significantly longer than that for 122 (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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 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, 122, and mutant GABA_A receptors, 1(G223F)22 and 12(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.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Sequence alignment of the pre-TM1/TM1 amino acids of GABAA receptor subunits. The glycine residue (G, in boldface) is conserved in all of the subunits except the 1 (as indicated by the symbol #), which has a phenylalanine (F, in boldface and underlined). 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 GenBankTM and Cutting et al. (25).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

Electrophysiological Recording-- The experiments were performed essentially as described earlier (15). Briefly, the Sf9 cell cultures were used in experiments after incubation with virus for 27-30 h. They were placed in an artificial balanced salt solution (ABSS) composed of (in millimolar): NaCl 162.5, KCl 3.5, Na₂HPO₄ 1.25, MgSO₄ 2, CaCl₂ 2, glucose 10, and HEPES 10. pH was 7.35 at 22 °C. Membrane currents were recorded in the whole-cell configuration of the patch-clamp technique (17). The intrapipette solution contained (in millimolar): KCl 160, MgCl₂ 1, CaCl₂ 1, EGTA 10, MgATP 2, and HEPES 10; pH 7.3 at 22 °C. A holding potential of 40 mV was used. Series resistance was 60% compensated.

Drug Applications-- Stock solutions of GABA (Sigma) and pentobarbital sodium (DAK, Denmark) were dissolved to a concentration at least 100× greater than that required for perfusion and premixed by diluting solutions in ABSS. Propofol (Tocris) was dissolved in Me₂SO and diluted in ABSS; the content of Me₂SO 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) ~100 µm 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 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) < 0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (122) GABA_A receptor and the mutated 1(G223F)22 and 12(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 EC₅₀ 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)22 receptors, and 0.2 mM for the 12(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 122 receptor. The time constant for solution exchange was 52 ms (37; 67 ms).

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Examples of current traces (gray) showing GABA-induced currents at saturating concentrations in the 122, 1(G223F)22, and 12(G219F)2 receptor combinations. The corresponding fits of the desensitization and deactivation phases are shown as black curves. The time constants for desensitization (desens) are: 122, 807 ms; 1(G223F)22, 873 ms; 12(G219F)2, 1.34 s. The time constants for deactivation (deact) are: 122, 452 ms; 1(G223F)22, 728 ms; 12(G219F)2, 717 ms. Please refer to Fig. 3 for a summary of the data set of  values with saturating GABA concentrations.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Comparison of GABA kinetics at maximum GABA currents in the 122 (2 mM GABA), 1(G223F)22 (2 mM GABA), and 12(G219F)2 (0.2 mM and 2 mM GABA) GABAA receptor combinations. A, the 10-90% rise time. Each column represents the median ± 25-75% interval of 6-26 Sf9 cells. B, the proportion of peak current remaining after 5 s of GABA application. The end current of the 12(G219F)2 receptor was significantly smaller at 2 mM GABA than at 0.2 mM. Each column represents the means ± S.E. of 6-22 Sf9 cells (*, p < 0.05). C, time constants (desens) of desensitization. The median (n = 6-22 cells)  for the 2-mutant at 0.2 mM GABA was significantly slower than at 2 mM GABA and for the wild type and 1 mutant receptors (*, p < 0.05; **, p < 0.01). D, time constants of deactivation (deact) shown as median ± 25-75% interval of 7-22 cells. At 2 mM GABA deact was significantly longer for the 12(G219F)2 than for the 1(G223F)22 receptor combinations (**, p < 0.01).

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 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 < 0.01) and the 1-mutant (p < 0.05) receptors. Increasing the GABA concentration to 2 mM on the 2-mutant decreased _desens (p < 0.05) and the end current (p < 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 < 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 EC₂₀ 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 µM for the 122 and 1(G223F)22 and 5 µM for the 12(G219F)2 combinations. In all three receptor combinations, pretreatment with propofol was necessary to significantly enhance peak currents induced by GABA (wild type: p < 0.01, -mutant and -mutant: p < 0.001). The modulating effect of propofol was significantly smaller (p < 0.05) for the 1(G223F)22 combination as compared with the 122 combination at both 10 and 50 µM propofol (Table II and Fig. 4). The 12(G219F)2 receptor showed significantly smaller modulation of GABA-induced currents when comparing 1 and 5 µM propofol for the 2 mutant with 10 and 50 µM propofol for the wild type (p < 0.05). There was no significant difference in the modulating effect of propofol between the 1(G223F)22 and the 12(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 EC₂₀ the corresponding time constants could not be calculated.

View this table: [in this window] [in a new window]   Table II Modulation of GABA-induced peak currents by propofol in GABAA receptor combinations Data shown are the mean ± S.E. of modulated currents in percent of GABA EC20 for 5-21 Sf9 cells. EC20 values were calculated from the data in Table I: 122, 12 µM; 1(G223F)22, 10 µM; and 12(G219F)2, 1.2 µM. The increased (percent of GABA at EC20) peak current for the 122 (50 µM propofol) receptor combination was significantly larger than for the 1(G223F)22 (50 µM propofol) and 12(G219F)2 receptor combinations (5 µM propofol). Concentration-response data for propofol-modulated currents in wild type and 12(G219F)2 receptors have been presented previously in Carlson et al. (15). For concentration-response data for 1(G223F)22 receptors, please refer to Fig. 4 legend.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Representative current traces showing the modulating effect of propofol on currents induced by GABA at EC20 in the 122 and 1(G223F)22 receptor combinations. The increased current variation in the beginning of the propofol-modulated traces is caused by transient voltage pulses used to monitor cell membrane conductance and capacitance. These pulses are suspended ~5 s before agonist application. The corresponding peak currents (percent of GABA EC20) for 122 and 1(G223F)22 receptors were (means ± S.E.): 10 µM propofol, 231 ± 21 (n = 8) and 117 ± 6 (n = 7), respectively; 50 µM propofol, 285 ± 58 (n = 5) and 166 ± 12 (n = 9), respectively. The increased peak current for 1(G223F)22 receptors was significantly less than that in wild type receptors for both 10 µM (p < 0.001) and 50 µM (p < 0.05) propofol.

Although equivalent concentrations of GABA with respect to response amplitude (EC₂₀) were chosen for the modulation experiments, the corresponding deactivation time constants differed between the receptor combinations. The deactivation time constants of the unmodulated responses were (median and 25-75% interval in milliseconds): 122, 268 (219; 385); 1(G223F)22, 247 (207; 385); 12(G219F)2, 471 (358; 551). The deactivation time constant of the 2-mutant receptor was significantly longer (p < 0.01) than those of the wild type and the 1-mutant receptors, whereas the latter two were similar. Due to these absolute differences, the effects of the anesthetics on the deactivation time constant was estimated by calculating the ratio of _deact of the modulated and unmodulated responses in each cell. The propofol-modulated _deact relative to unmodulated were (median and 25-75% interval in percent of unmodulated): 122, 151 (121; 157); 1(G223F)22, 108 (101; 118); 12(G219F)2, 118 (105; 129). The relative _deact on the wild type receptor was significantly greater than 100% (p < 0.01). The effect of propofol was to slow the _deact on the wild type receptor. In addition, pentobarbital-modulated _deact relative to unmodulated were assessed: 122, 116 (95; 134); 1(G223F)22, 102 (98; 108); 12(G219F)2, 86 (84; 100). For pentobarbital, none of the relative _deact differed significantly from 100%. Pentobarbital did not alter the _deact as compared with unmodulated _deact.

Mutation of TM1 Glycine on 2 Subunit Increases Anesthetic-induced Direct Activation and Alters Desensitization-- As shown previously (15), the 12(G219F)2 receptor demonstrated a biphasic concentration-response curve and was significantly more sensitive than the 122 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 µM (p < 0.001) and 1500 µM (p < 0.05) pentobarbital were significantly higher than at 50 µM pentobarbital. There was no significant difference between the peak currents of the 122 and the 1(G223F)22 receptors at any concentration of pentobarbital tested.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Concentration-response curves for directly pentobarbital- and propofol-activated peak currents. Peak currents were normalized to a maximum GABA peak current in each cell and shown as means ± S.E. (n = 4-40 cells). The concentrations used to elicit maximum GABA responses were: 2 mM GABA, 122 and 1(G223F)22; 0.2 mM GABA, 12(G219F)2. A, pentobarbital. The peak currents at 15, 50, and 500 µM pentobarbital were significantly higher at the 12(G219F)2 receptors than at the 122 and the 1(G223F)22 receptors (**, p < 0.01; ***, p < 0.001). B, propofol. The peak currents at 30 µM propofol and higher concentrations were significantly larger with the 12(G219F)2 receptor than the 122 and the 1(G223F)22 receptors (**, p < 0.01; ***, p < 0.001). Part of the data in this figure has been published previously (15).

The kinetics of pentobarbital-activated currents in the 2-mutant was investigated by employing 50, 500, and 1500 µM pentobarbital (Fig. 6). The rise times were significantly shorter at 1500 µM pentobarbital than 50 (p < 0.01) and 500 µM (p < 0.05) (Fig. 6A), as expected for a concentration-dependent activation. The current remaining after application of 500 µM pentobarbital was significantly larger than the current remaining after application of 50 (p < 0.01) and 1500 µM pentobarbital (p < 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 µM pentobarbital were significantly longer than the time constants for 50 and 1500 µM pentobarbital (p < 0.01). Deactivation  values for 50 and 500 µM pentobarbital were not significantly different from each other (see Fig. 8D below). For 1500 µM 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.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Pentobarbital (PB) kinetics for direct activation of the 12(G219F)2 GABAA-mutated receptor. A, the 10-90% rise time was significantly shorter for 1500 µM pentobarbital than for 50 and 500 µM. Each column represents the median ± 25-75% interval of 9-26 Sf9 cells (*, p < 0.05; **, p < 0.01). B, the end-current (after 5-s application of pentobarbital relative to the peak current) was significantly smaller for 50 and 1500 µM pentobarbital than for 500 µM. Each column represents the means ± S.E. of 10-27 Sf9 cells (*, p < 0.05; **, p < 0.01). C, the time constant of desensitization (desens) for 50 and 1500 µM pentobarbital was significantly smaller than for 500 µM. Each column represents the median ± 25-75% interval of 10-26 Sf9 cells (**, p < 0.01).

For propofol, the 122 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)22 receptor followed the same pattern as the wild type. The 12(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 µM propofol (Fig. 7A). The end current remaining was smaller for 1000 µM propofol as compared with 300 µM propofol (p < 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 µM propofol than 300 µM propofol (p < 0.05, Fig. 7D).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Propofol kinetics for direct activation of the 12(G219F)2 GABAA-mutated receptor. A, the 10-90% rise time. Each column represents the median ± 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 µM than for 300 µM propofol (*, p < 0.05). Each column represents the means ± S.E. of 9-10 Sf9 cells. C, the time constants of desensitization (desens) for 300 and 1000 µM were not significantly different from each other. Each column represents the median ± 25-75% interval of 3-5 Sf9 cells. D, the time constant of deactivation (deact) was significantly smaller for 1000 µM as compared with 300 µM propofol (*, p < 0.05). Each column represents the median ± 25-75% interval of 6-9 Sf9 cells.

Comparison of Anesthetic Kinetics of Wild Type and TM1 Glycine-mutated GABA_A Receptors in the Absence of GABA-- As illustrated in Fig. 5A, the peak response levels of 50 µM pentobarbital on the 12(G219F)2 receptor and 500 µM pentobarbital on the 122 and 1(G223F)22 receptors were not significantly different, whereas the peak response of 500 µM pentobarbital on the 12(G219F)2 receptor was significantly larger than any of these (p < 0.001 in all cases). The rise-time for the wild type and the 1-mutant receptors at 500 µM pentobarbital and for the 2-mutant receptor at 50 and 500 µM pentobarbital did not significantly differ from each other (Fig. 8A).

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Comparison of pentobarbital (PB) direct activation kinetics of the 122, 1(G223F)22, and 12(G219F) GABAA receptor combinations. A, the 10-90% rise time. Each column represents the median ± 25-75% interval of 5-26 Sf9 cells. B, at 500 µM, the end-current (after 5-s application of pentobarbital relative to the peak current) of the 12(G219F)2 receptor was significantly greater than the end current of the 122 receptor. Each column represents the means ± S.E. of 4-27 Sf9 cells (**, p < 0.01). C, the time constant of desensitization (desens) for 500 µM pentobarbital at the 12(G219F)2 receptor was significantly longer than for 50 µM at the same receptor and 500 µM pentobarbital at the 122 receptor. Each column represents the median ± 25-75% interval of 3-26 Sf9 cells (**, p < 0.01). D, the time constant of deactivation (deact) at 500 µM pentobarbital for all the receptor combinations were not significantly different from each other. For the -mutant receptor, the deact values at 50 and 500 µM pentobarbital were not significantly different from each other. Each column represents the median ± 25-75% interval of 3-20 Sf9 cells.

To compare the desensitization kinetics, the fading of direct pentobarbital-induced currents was compared (Fig. 8B). Upon application of 500 µM pentobarbital to the 2-mutant receptor, a significantly larger (p < 0.01) end current remained than with 500 µM 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 µM pentobarbital in the 2-mutant receptor was significantly larger (p < 0.01) than 50 µM in the same receptor and 500 µM 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.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 currents. As indicated previously (15), the apparent affinity for GABA was increased in the 12(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 ~1 ms at saturating GABA concentrations (e.g. 112 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 current causing a shift of the Cl 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 shift is of similar magnitude. The time constant for desensitization, however, was significantly longer for 200 µM 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  < 10 ms and a slower component of  ~ 150 ms for 132 receptors) (22), although some investigations have found monoexponential desensitization (e.g.  ~ 500 ms, 112 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 EC₅₀ 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 2-mutant 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 EC₅₀ 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 EC₅₀ for GABA than 122 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 12(G219F)2 receptor support the claim that the N-terminal 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 concentration from EC₂₀ 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 EC₂₀ 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 µM 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 1- and 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 1- and 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 (EC₂₀). 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 1- and 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 more important for pentobarbital enhancement of GABA_A currents.

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 9' leucine (2L259) and the 15' serine (1S265 and 2S270) (29, 30). In addition, positive allosteric potentiation was reduced (29, 30). A decrease in GABA-induced desensitization with the TM2 9' point mutation was demonstrated, as well (29). Because in the present study the TM1 2(G219F) point mutation significantly decreased the EC₅₀ 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 9' and 15' 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)22 receptors, the EC₅₀ for GABA was not altered, yet propofol-induced potentiation was significantly less than in the wild type receptors, whereas pentobarbital-induced 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 12(G219F)2 receptors demonstrated a biphasic concentration-response curve. The first phase was shifted leftward relative to the concentration-response curves of the 122 and 1(G223F)22 receptors, and the peak current, rise time, end current, and time constants for desensitization and deactivation of 50 µM pentobarbital for the 2-mutant receptor were similar to the same parameters of 500 µM pentobarbital for the 122 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 µM 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 µM pentobarbital in the 2-mutant receptor was significantly smaller and developed significantly slower than in the 122 receptor.

The biphasic nature of the pentobarbital concentration-response curve of the 12(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 µM pentobarbital, the decay was less extensive and developed with a longer time constant than at 50 or 1500 µM pentobarbital. At the same time, the deactivation time constant tended to become longer with 500 µM than with 50 µM 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, 12(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 (GF), 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.

	ACKNOWLEDGEMENTS

We thank Dr. D. Gallager for wild type GABA_A receptor baculoviruses and Drs. J. Amin and D. Weiss for assistance with subunit mutations.

	FOOTNOTES

^* This work was supported by the Alfred Benzon Foundation (to A. C. E., U. K., B. X. C., and A. S.), by the Academy of Finland (to A. C. E.), by the Danish Medical Research Council, Grant 52-00-1011 (to A. S.), by the Lundbeck foundation (to A. S.), by the Finnish Society of Science and Letters (to A. C. E.), and by National Institutes of Health Grant NS28772 (to R. W. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, The Royal Danish School of Pharmacy, Universitetsparken 2, Copenhagen 2100, Denmark. Tel.: 45-35-30-63-81; Fax: 45-35-30-60-20; E-mail: uk@dfh.dk.

Published, JBC Papers in Press, March 4, 2002, DOI 10.1074/jbc.M111215200

	ABBREVIATIONS

The abbreviations used are: GABA_A, -aminobutyric acid type A; GABA, -aminobutyric acid; TM1-3, transmembrane domains 1-3; Sf9, Spodoptera frugiperda 9; ABSS, artificial balanced salt solution.

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