Introduction

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Molecular events leading to anesthesia have been attributed to the modulation of the excitatory or inhibitory ligand-gated ion channels (Nicoll, 1972; Nicoll et al., 1975; Barker and Ransom, 1978; Franks and Lieb, 1994). The primary target for a number of anesthetic compounds, including pentobarbital, are the -aminobutyric acid type A (GABA_A) receptor channels (Schulz and Macdonald, 1981; Gage and Robertson, 1985; Parker et al., 1986; MacIver et al., 1991; Tanelian et al., 1993), the key components of synaptic inhibition in the central nervous system. GABA-gated chloride channels are heterooligomeric or homooligomeric pentamers composed of numerous combinations of homologous , , , , or  classes of subunits (Macdonald and Olsen, 1994). These subunits have the potential for creating a great number of GABA receptor channels with distinct pharmacology (Macdonald and Olsen, 1994). For example, GABA-evoked currents for heterooligomeric (GABA_A; Schofield et al., 1987; Levitan et al., 1988; Macdonald and Olsen, 1994) and homooligomeric  receptor channels (Blair et al., 1988; Sanna et al., 1995) are potentiated in the presence of low concentrations of pentobarbital (modulatory action). Moreover, pentobarbital at higher concentrations can directly activate these channels (agonistic action; Mathers and Barker, 1980; Nicoll and Wojtowicz, 1980; Akaike et al., 1991; Sanna et al., 1995; Rho et al., 1996). In contrast, the homooligomeric ₁ receptor channel (GABA_C; Cutting et al., 1991) is insensitive to pentobarbital (Shimada et al., 1992).

In this study, experiments were conducted to gain insight into the mechanisms of barbiturate modulation and activation of GABA-gated chloride channels. Results with - chimeras and site-directed mutagenesis of ₁ indicate that hydrophobic amino acid substitution for Trp328 within the third transmembrane domain (TM3) imparts modulatory and agonistic properties of pentobarbital to ₁ homooligomeric receptor channel. In addition, residue 328 plays an important role in agonist-dependent activation. Collectively, these results provide important clues concerning the mechanism of barbiturate action on GABA-gated ion channels.

	Materials and Methods

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All chimeras were constructed using either conserved restriction sites between the ₁ and ₂ subunits (e.g., HincII for 346/305), or synthetic oligonucleotides containing designed restriction enzyme sites and polymerase chain reaction. Special care was taken not to alter the relative position of the conserved amino acids on both sides of the junction (with the exception of 405/399). The DNA sequence of all chimeras was verified by DNA sequencing.

The cDNAs corresponding to ₁ and ₂ were cloned into the pSELECT vector (Promega, Madison, WI) and oligonucleotide-mediated site-directed mutagenesis was achieved according to the manufacturer's protocol (Altered Sites; Promega). Successful mutagenesis was verified by DNA sequencing. The cDNAs were linearized with NheI leaving a several-hundred base pair tail (3'). These additional sequences at the 3' end may increase cRNA stability in the oocyte. The cRNA was transcribed from the linearized cDNAs by standard in vitro transcription procedures (Megascript; Ambion, Austin, TX).

Xenopus laevis (Xenopus I; Ann Arbor MI) were anesthetized by hypothermia and oocytes were surgically removed from the frog and placed in Oocyte Ringer (OR₂) that consisted of: 82.5 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM Na₂HPO₄, 50 U/ml penicillin, and 50 µg/ml streptomycin, pH 7.5. Oocytes were dispersed and then incubated in OR₂ minus Ca²⁺ plus 0.3% collagenase A (Boehringer Mannheim, Indianapolis, IN) for approximately 2 h. After isolation, the oocytes were thoroughly rinsed with OR₂. Stage VI oocytes were separated and maintained overnight at 18°C.

Micropipettes for injecting cRNA were fabricated on a Narishige PP-83 puller (Narishige USA, Greenvale, NY) and the tips were cut off with microscissors. The cRNA in diethylpyrocarbonate-treated water was drawn up into the micropipette with negative pressure and then injected into the oocytes by applying positive pressure using a Picospritzer II (General Valve Corporation, Fairfield, NJ). The oocytes were incubated in OR₂ at 18°C for 2 to 3 days before the experiment. To ensure that equal concentrations of cRNA for each construct were injected (especially important for comparison of maximum GABA-activated currents), set dilutions of cRNA from mutants were electrophoresed on a 1% formaldehyde-containing agarose gel. The amount of cRNA was judged and matched by interpolation of lanes containing different dilutions of the corresponding cRNA. In addition, for nearly all mutants, two independent isolates were characterized and tested.

Two to 3 days after injection, oocytes were placed on a nylon mesh suspended in a small volume chamber (~75 µl). The chamber has an inlet in the top and an outlet in the bottom that allows continuous and rapid perfusion. Twenty separate reservoirs (100-ml glass containers) were connected to four six-way valves and the outlet of each of these six-way valves (the sixth position was connected to the reservoir containing the control solution) was connected to one four-way valve. The outlet of the four-way valve lead to the chamber. In this way, up to 20 different solutions could be introduced to an individual oocyte. Switching between the different solutions was controlled manually. The oocyte was continuously perfused with recording OR₂ (OR₂ without antibiotics and the 1 mM Na₂HPO₄ replaced with 1 mM NaCl) and briefly switched to the test solution containing drug.

Recording microelectrodes were fabricated with a Narishige PP-83 puller and filled with 3 M KCl. Electrodes with resistances of 0.6-1 M were used. Standard two-electrode voltage-clamp techniques (Turbo TEC-05 npi; Adams and List, Westbury, NY) were used to record currents in response to application of drugs. In all cases, membrane potential was clamped to 70 mV. Data were played out on a Gould EasyGraf chart recorder (Gould Inc., Glen Burnie, MD) during the experiment and recorded on a VCR (Instrutech PA10b; Instrutech Labs., Plymouth Meeting, PA) for off-line analysis.

The EC₅₀, and Hill numbers were estimated by fitting the concentration-response relationships to the following equation: [I = I_max/(1 [EC₅₀/(A)]ⁿ )] using computer software provided by Dr. David S. Weiss, where I is the peak current at a given concentration of agonist A, I_max is the maximum current, EC₅₀ is the concentration of agonist yielding a current half the maximum, and n is the Hill coefficient.

	Results

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TM3 of ₂ Subunit Is Sufficient To Impart Pentobarbital Sensitivity to ₁. To determine the crucial domain(s) for the dual agonistic and modulatory action of pentobarbital, chimeric human ₁ and rat ₂ subunits were constructed. The cRNA from the different - chimeras were expressed in Xenopus oocytes and the responses of these receptor channels were electrophysiologically recorded in the presence of GABA, pentobarbital, and a combination of both. The summary of these experiments is depicted in Fig. 1A (see also Tables 1 and 2). The most striking result was the role of the TM3 from the ₂ subunit in conferring pentobarbital sensitivity (compare the 324/283 and 346/305). The ₁ receptor channel containing both the TM3 and the TM4 from the ₂ subunit displayed marked sensitivity to pentobarbital. In contrast, deletion of the sequences corresponding to the TM3 of the ₂ within the 324/283 chimera and replacement with the TM3 of the ₁ subunit abolished pentobarbital sensitivity in the resulting receptor channels (Fig. 1A, 346/305 and 405/399).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Determination of crucial residue in conferring pentobarbital-sensitivity to 1 receptor channel. A, chimeras between human 1 and rat 2 subunits were constructed. Special care was taken not to alter relative position of conserved amino acids on both sides of junction (with exception of 405/399). cRNAs from different - chimeras were expressed in Xenopus oocytes and resulting receptor channels were examined using GABA (up to 20 mM), pentobarbital (up to 2.5 mM), or both; * indicates spontaneously open channels (see Results). In these channels (*), chloride leak (judged based on reversal potential for chloride) was directly proportional to amount of injected cRNA (data not shown). 2 receptor channels had a severe depression in maxima when tested with GABA alone. All receptor channels that responded to GABA or pentobarbital or both are scored with +. All numbers indicate amino acid position for 1 and 2 subunit, respectively. Many constructed chimeras did not yield functional channels (data not shown). It is important to note that rat 2 subunit is highly suited to form heterooligomeric receptor channels. This is corroborated by nearly three orders of magnitude greater maximal current when equivalent cRNA concentrations for 2 subunit is coexpressed with  and  subunit. B, TM3 (but not TM1) of 2 subunit confers pentobarbital sensitivity to 1 receptor channel. C, alignment of amino acid sequences corresponding to TM3 of human 1, rat 2, 1, 2, and Drosophila (DRC) GABA subunits. Boxed residues represent amino acids that were mutated in this study. D, pentobarbital-dependent modulation of GABA responses from 1 and W328M receptor channels. Mutation of Trp328 to Met, imparted pentobarbital sensitivity to 1 receptor channel. For W328M receptor channel, GABA (0.3 µM) responses were markedly potentiated in presence of 50 µM pentobarbital. In contrast, GABA currents (0.6 µM) for 1 receptor channel were not altered in presence of equivalent concentration of pentobarbital. Thick line above each current trace represents duration of GABA application or coapplication of GABA and pentobarbital.

View this table: [in this window] [in a new window]   TABLE 2 Parameters determined from fitting Hill equation to pentobarbital concentration-response relationships. Numbers in parentheses indicate number of oocytes tested. For all pentobarbital-sensitive mutants, 2.5 mM pentobarbital was used (with exception of Y198S/W328M and Y198S/WVS328-330MGC receptor channels, where 1 mM pentobarbital was used) to obtain pentobarbital Imax (Pb Imax). Concentration of pentobarbital required for half-maximal activation (EC50), Hill coefficient and Pb Imax/GABA Imax are mean ± S.D.

Methionine Substitution for Trp328 Within TM3 of ₁ Confers Pentobarbital Sensitivity. Comparison of the amino acid sequences encoding the TM3 of ₁ and ₂ subunits revealed nonconserved differences with respect to size and hydrophobicity mainly at three positions, Trp328, Val329, and Ser330. The corresponding residues within the ₂ subunit were Met286, Gly287, and Cys288 (Fig. 1C). Using site-directed mutagenesis, the Trp328, Val329, and Ser330, within the ₁ subunit, singly or in combination, were mutated to Met, Gly, and Cys, respectively. All mutant receptor channels that included Trp328 to Met substitution (W328M, WV328,329 MG, and WVS328-330 MGC) were sensitive to pentobarbital. For these mutant receptor channels, pentobarbital at low concentrations mediated the potentiation of the GABA responses (see below, e.g., Fig. 1D) and displayed agonistic properties at higher concentrations (Table 2). In contrast, V329G, S330C, or VS329,330GC receptor channels were insensitive to both modulatory and agonistic action of pentobarbital (Table 2). The specificity of position 328 in conferring pentobarbital sensitivity is corroborated by the lack of response of the V329G, S330C, or VS329,330GC receptor channels to pentobarbital, given the proximity of Val and Ser residues to Trp328.

Hydrophobic Residues at Position 328 Impart Pentobarbital Sensitivity to ₁ Receptor Channel. The Trp328 within the ₁ subunit was replaced with an array of diverse amino acids differing in hydropathy index (HI) (Kyte and Doolittle, 1982), charge, and size. Remarkably, as with Met (HI = 1.9), substitutions of Trp328 (HI = 0.9) with other hydrophobic residues such as Leu, Ile, and Val (HI of 3.8, 4.5, and 4.2, respectively) and Ala (Fig. 1C, found at the corresponding position on the ₁ subunit, HI = 1.8), imparted both the agonistic and modulatory properties of pentobarbital to the mutated ₁ receptor channels. GABA responses from the ₁ receptor channel containing the Phe substitution (HI = 2.5), however, were only weakly enhanced by pentobarbital. The homooligomeric Drosophila GABA receptor channel responds to the modulatory action of pentobarbital at high concentrations (Chen et al., 1994). The TM3 of the Drosophila subunit contains Gly (HI = 0.4), Thr, and Cys at the equivalent position with respect to the TM3 of the ₁ (Fig. 1C). The corresponding residues (Trp, Val, and Ser) within 1 receptor channel were mutated to Gly, Thr, and Cys. Pentobarbital potentiated the GABA-evoked currents from the WVS328,330GTC receptor channel (data not shown) and was also found to be an agonist for this mutant receptor channel (depressed maximum, Table 2). In contrast, the Tyr (HI = 1.3) substitution (W328Y) yielded receptor channel with a pharmacological profile resembling that of the wild type. Pentobarbital neither directly activated nor modulated the GABA-evoked currents for the W328Y receptor channel (see below). Finally, substitutions of Trp328 by Glu or Ser (Fig. 1C, found at the corresponding position on the ₂ subunit) or Pro (HI of 3.5, 0.8, and 1.6, respectively) failed to yield functional channel when tested with GABA (Table 1, up to 30 mM) or pentobarbital (Table 2, 2.5 mM). Thus, the HI of the substituted amino acid at position 328 appears to be crucial not only in imparting pentobarbital sensitivity, but also in GABA-dependent activation.

Mutation of Trp328 Transforms GABA Sensitivity. Figure 2 shows the current traces elicited by different concentrations of GABA, as well as the GABA concentration-response relationships from oocytes expressing ₁, W328L, W328I, W328V, W328M, W328A, W328F, and W328Y receptor channels. GABA activated these mutants with similar efficacy (with the exception of W328Y, with the maximum current of approximately 10% of the wild type), when matched cRNA concentrations (see Materials and Methods) for these individual mutants were injected into oocytes and their maximal GABA-evoked currents were compared. These mutations, however, induced marked transformation in the GABA potency (Table 1). The W328L and W328I receptor channels displayed approximately a 3-fold increase in the sensitivity. The GABA EC₅₀s for W328L and W328I were 0.35 and 0.39 µM, respectively (in comparison with 1.03 µM for the wild type). In contrast, the Phe and Ala substitution produced a drastic 30-fold reduction in GABA sensitivity, when compared with ₁ receptor channel. The EC₅₀ values estimated form GABA concentration-response relationships for W328A and W328F receptor channels were 32 and 30 µM, respectively. The GABA concentration-response relationship for WVS328-330GTC receptor channel was also altered (Table 1). In comparison with wild type, the GABA EC₅₀ for the WVS328-330GTC receptor channel was increased by approximately 8-fold. Finally, the Met, Val, and Tyr substitutions at position 328 produced receptor channels with similar GABA sensitivity. The EC₅₀ values for these receptor channels deviated 20 to 30% from the EC₅₀ value for wild type. These results suggest that position 328 is not only crucial in conferring pentobarbital sensitivity, but also plays a key role in GABA-dependent activation.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   GABA-dependent activation of 1 Trp328 mutant receptor channels. A, current traces evoked by different concentrations of GABA for 1 and 1 328 mutants. Thick line above each current trace represents duration of GABA application. Symbol representing each mutant in B (concentration-response relationships) is shown on left of current traces corresponding to that mutant. B, GABA concentration-response relationship for 1 Trp328 mutants. Each plot represents average of normalized peak currents versus GABA concentrations from three oocytes expressing 1 or 1 Trp328 mutants. Lines are best fit of Hill equation to data points, and error bars represent S.D. (n = 3). Note that there are approximately two orders of magnitude variation in concentration of GABA required to elicit half-maximal currents (EC50) among these mutants.

Pentobarbital-Dependent Potentiation Versus Inhibition of GABA Responses Occurs Over a Narrow Range of GABA Concentration for ₁ 328 Mutants. Enhancement of the GABA-evoked currents by pentobarbital from the homooligomeric Trp328 mutants was dependent on GABA concentration. Figure 3A shows the pentobarbital-mediated (50 µM) modulation of GABA-evoked currents at different agonist concentrations for W328L, W328I, W328V, W328M, W328A, and W328F receptor channels. It is noteworthy that pentobarbital alone at a concentration (50 µM) applied in this experiment does not activate these Trp328 mutants.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Pentobarbital modulation of GABA-evoked currents for Trp328 mutant receptor channels. A, pentobarbital modulation of GABA-evoked currents (at different concentrations of GABA) for W328L, W328I, W328V, W328M, W328A, and W328Y receptor channels. Note that pentobarbital potentiation versus inhibition occurs over a narrow range of GABA concentrations. Magnitude of disrupted current traces for W328M and W328V at 4 µM GABA are 3.0 and 2.1 µA, respectively. Thick line above each current trace represents duration of GABA application or coapplication of GABA and pentobarbital. B, plot of pentobarbital potentiation for W328M receptor channel in presence of 0.2, 0.3, and 0.5 µM GABA versus ratio of GABA concentrations to EC50 for W328M. Note exponential relationship between relative potentiation by pentobarbital and GABA concentrations. Pentobarbital potantiation occurs at GABA concentrations below GABA EC50 value for W328M. Error bars are S.D.s (n = 3).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   A, pentobarbital potentiation of isoguvacine-evoked currents for W328I and W328L. Pentobarbital (50 µM) markedly potentiated isoguvacine (8 µM) responses for W328I and W328L receptor channels. Concentration of isoguvacine (8 µM) used in this study is equivalent to fraction of isoguvacine EC50s for W328I and W328L receptor channels. B, pentobarbital modulation of heterooligomeric 122 receptor channel. GABA concentrations used were equivalent (with respect to their EC50s) to those used in Fig. 3A for W328M receptor channel. Thick line above each current trace represents duration of GABA application or coapplication of GABA and pentobarbital. Note contrast in pentobarbital action between 122 and W328M, where potentiation for 122 receptor channel occurs over a wide range of GABA concentration (relative to EC50 value). C and D, a mutation within GABA activation domain of W328M markedly reduces GABA potency but does not alter GABA concentration-dependent (relative to EC50 value) modulation by pentobarbital. C, at GABA concentration (200 µM) equivalent to a fraction of EC50 value (3331 ± 346.7 µM), pentobarbital (20 µM) markedly enhanced GABA-evoked currents for homooligomeric Y198S/W328M receptor channel. D, pentobarbital depression of GABA responses occurred at concentrations of GABA below EC50 for Y198S/W328M receptor channels. At GABA concentration equivalent to 0.84 of EC50 (2800 µM GABA), pentobarbital (50 µM) inhibited GABA responses and at higher concentration (30,000 µM GABA) caused an apparent block desensitization. Thick line above each current trace represents duration of GABA application, or coapplication of GABA and pentobarbital.

Pentobarbital Potentiation Occurs Over a Wide Range of GABA Concentrations for ₁₂₂ Receptor Channel. Figure 4B shows the pentobarbital modulation of the heterooligomeric ₁₂₂ receptor channel in the presence of different concentrations of GABA. The GABA concentrations used were equivalent (with respect to their EC₅₀s) to those used for the W328M receptor channel (see Fig. 3A). For the ₁₂₂ receptor channel (Table 1, GABA EC₅₀ = 46.5 ± 4.7 µM), pentobarbital at a concentration of 50 µM markedly potentiated the GABA responses evoked by 7, 10, 17, and 34 µM GABA (Fig. 4B). Moreover, at concentrations of GABA (135 µM) approximately three times the EC₅₀, moderate enhancement of the GABA response was still present. This experiment demonstrates that for the heterooligomeric ₁₂₂ receptor channel, the potentiation by pentobarbital occurs over a much wider range of GABA concentrations (relative to EC₅₀) than pentobarbital-sensitive Trp328 mutants. The contrast in pentobarbital mode of modulation for the Trp328 ₁ mutants and ₁₂₂ receptor channels is intriguing, given that these two classes of receptor channels may be activated differently by GABA (Amin and Weiss, 1996; see Discussion).

Impairment of GABA Sensitivity Does Not Alter Unique Pentobarbital Modulation of ₁ 328 Mutants. The ₁ receptor channel is approximately 40 times more sensitive to GABA than ₁₂₂ receptor channel. To examine whether this difference in GABA sensitivity could account for the contrast in pentobarbital modulation between the two classes of receptor channels, a Tyr at position 198 (presumably located with in the extracellular receptor domain) was mutated to Ser within both W328M and WVS328-330 MGC mutant subunits. Previous studies have demonstrated that Tyr198 to Ser substitution within the ₁ receptor channel results in a 2500-fold decrease in GABA sensitivity (Table 1, Y198S, also Amin and Weiss, 1994). Similar to Y198S, both Y198S/W328M and Y198S/WVS328-330MGC receptor channels exhibited a three orders of magnitude reduction in GABA sensitivity (Table 1, EC₅₀s of ~3000 µM). Nevertheless, the mode of pentobarbital modulation for these receptor channels remained the same as other Trp328 ₁ mutants. Pentobarbital at a concentration of 20 µM synergistically potentiated the GABA (200 µM) responses from oocytes expressing Y198S/W328M (Fig. 4C) or Y198S/WVS328-330MGC by 390 ± 14% (n = 3) and 780 ± 160% (n = 3), respectively. However, at concentrations of 750 µM and 2800 µM of GABA (below the EC₅₀ values for Y198S/W328M), pentobarbital displayed antagonistic properties. Moreover, in the presence of 30 mM GABA, the pentobarbital effect was consistent with a channel block (or desensitization; see Fig. 4D for Y198S/W328M).

Pentobarbital at Higher Concentrations Is an Agonist for ₁ Trp328 Mutants. In addition to the modulatory effect, pentobarbital at higher concentrations is also an agonist for W328L, W328I, W328V, W328M, and W328A receptor channels. Figure 5A shows current traces evoked by different concentrations of pentobarbital for W328L and W328M receptor channels. In pentobarbital-direct activation studies with GABA_A receptor channels, the current amplitude increases before returning to the baseline following removal of pentobarbital (at high concentrations, Rho et al., 1996; J. Amin, unpublished observations). This phenomenon was absent in pentobarbital-direct activation of the Trp328 mutants. Note that for W328L and W328M receptor channels, even at the highest concentration of pentobarbital (2.5 mM), the evoked currents did not increase in amplitude following pentobarbital wash.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   A, current traces evoked by different concentrations of pentobarbital for W328M and W328L receptor channels. Thick line above each current trace represents duration of GABA application. B, pentobarbital concentration-response relationship for 1Trp328 mutants. Each plot represents average of normalized peak (to extrapolated maximum) currents versus pentobarbital concentrations from three oocytes (except for W328I, two oocytes) expressing W328L, W328I, W328V, W328M and W328A receptor channels. Lines are best fit of Hill equation to data points, and error bars represent S.D. C, comparison of W328L, W328I, W328V, W328M, and W328A receptor channels' EC50 values for pentobarbital and GABA. Note that for these mutants, there are marked alteration in EC50 values for GABA, while there are only moderate difference in EC50 values for pentobarbital.

Thiopental and Phenobarbital Modulation of Trp328 Mutants. Thiopental and phenobarbital were also effective at potentiating GABA responses for pentobarbital-sensitive mutants, albeit with different potencies. As shown in Fig. 6, in the presence of 4 µM GABA, thiopental (50 µM) was nearly as potent as pentobarbital (50 µM) for W328A homooligomeric receptor channel. Thiopental and pentobarbital increased the GABA responses for W328A receptor channel by 659 ± 72.8% (n = 4) and 836.8 ± 90.4% (n = 4), respectively. On the other hand, phenobarbital at twice the concentration (100 µM), was less effective at potentiating W328A's responses to GABA (452.5 ± 16.1%, n = 4). Preliminary results indicate that W328L, W328I, W328V, and W328M receptor channels were also modulated by thiopental and phenobarbital with similar relative potencies (data not shown). The comparative potencies of these barbiturates for Trp328 mutants are consistent, in general, with their relative clinical potencies (Franks and Lieb, 1994).

View larger version (9K): [in this window] [in a new window]   Fig. 6.   Comparison of pentobarbital, phenobarbital, and thiopental in modulating GABA responses from W328A receptor channel. Among tested barbiturates, pentobarbital and thiopental appear to be most potent positive modulators of GABA responses for W328A receptor channels. Thick line above each current trace represents duration of GABA application or coapplication of GABA and barbiturates.

Tryptophan Substitution for Met286 within TM3 of ₂ Subunit Abolishes Pentobarbital Sensitivity. Within the ₂ subunit, the Met at position 286 alone or in combination with Gly287 and Cys288 were mutated to their corresponding amino acid counterparts found in the ₁ subunit. Figure 7 illustrates the currents elicited by bath application of GABA (5 µM) or both GABA (5 µM) and pentobarbital (30 µM) to oocytes expressing ₂ or M286W receptor channel. Similar to ₂, the expression of the cRNA for M286W (or MGC286-288WVS) yielded spontaneously open channels. The magnitude of the chloride ion leak (judged by the reversal potential for chloride) in these ion channels was proportional to the amount of injected cRNA (data not shown). In addition, ₂ wild-type and mutant receptor channels displayed severe depression in the I_max when tested with GABA (see legend to Fig. 1). Nonetheless, in contrast to ₂, coapplication of pentobarbital and GABA to oocytes expressing M286W (or MGC286-288WVS, data not shown) receptor channels failed to increase the GABA-evoked currents (Fig. 7).

View larger version (8K): [in this window] [in a new window]   Fig. 7.   Pentobarbital-dependent modulation of GABA responses from 2 or M286W receptor channel. Mutation of Met 286 to Trp (M286W), abolished pentobarbital sensitivity of 2 receptor channel. On other hand, GABA currents for wild-type 2 receptor channel markedly increased by coapplication of pentobarbital. Thick lines above each current trace represent duration of GABA application or coapplication of GABA and pentobarbital.

	Discussion

Top Summary Introduction Materials and Methods Results Discussion References

The data presented here indicate that replacing residue 328 with a spectrum of amino acid residues can confer barbiturate modulation as well as alter GABA-dependent activation of the mutated ₁ receptor channel. The apparent major determinant for pentobarbital sensitivity of the mutated ₁ receptor channel was, however, the hydrophobicity of the substituted amino acid at position 328. There were also key differences in the pentobarbital modulation between the homooligomeric ₁ 328 mutants and the heterooligomeric receptor channels.

Pentobarbital Versus GABA. The lack of stringency for amino acid side chains (except for hydrophobicity) at position 328 to confer pentobarbital sensitivity is unique. For instance, the Met side chain is different from that of Ala in both size and the constituent elements, whereas the EC₅₀ for pentobarbital between W328M and W328A receptor channels varied by less than 2-fold. Mutational analysis of different ligand-gated ion channels (including GABA) has shown that even conservative amino acid substitutions (such as Tyr to Phe) within the agonist-dependent activation domain can markedly impair the agonist sensitivity (Vandenberg et al., 1992; Amin and Weiss, 1993). The differences in amino acid side chain requirement between the agonist and the pentobarbital activation domains is perhaps best manifested by the nature of the bond they form. The interaction between pentobarbital and its site of action may be mediated through the butyl side chain of the amphipathic pentobarbital molecule via the relatively weak hydrophobic interaction, whereas the GABA agonist may interact with its activation domain by more specific and stronger hydrogen bonding. In the pentobarbital-mediated potentiation of the GABA responses, the apparent sequential release of pentobarbital followed by GABA may attest to this notion, because there appeared to be a direct correlation between the magnitude of current rise following the wash, and the EC₅₀ of the Trp328 mutants (Fig. 3A, smallest rise in the current for W328A and largest for W328L and W328I).

Target-Specific or General Perturbation? The view that general anesthetics indirectly affect membrane-embedded ion channels by altering the fluidity of lipid membranes is being gradually replaced by a target-specific model (Franks and Lieb, 1994). Two chief arguments, namely the identification of protein targets such as luciferase, as well as the discovery of the stereoselectivity of anesthetic agents, support the target-specific model for anesthetic action (Huang and Barker, 1980; MacIver and Roth, 1987; Franks and Lieb, 1991). Recently, several groups (Belelli et al., 1997; Mihic et al., 1997) have shown that the residues within the TM2 and the TM3 appear to be crucial for the action of the general anesthetics etomidate and enflurane. Interestingly, Mihic et al. (1997) have conversely mutated the corresponding 328 residue within the  subunit of the glycine receptor, or  and  subunit of the GABA_A receptor channel to a Trp, to abolish the action of enflurane. Pentobarbital and enflurane are two structurally diverse anesthetics that appear to exert their action through the same site. This notion, together with the lack of amino acid side chain specificity for pentobarbital-dependent modulation, rekindles the debate over the mechanism of anesthetic action. It is tempting to speculate that substitution of the Trp328 to a hydrophobic residue may cause the TM2 (gate) to be readily accessible to the pentobarbital's induced local lateral pressure within the membrane bilayer (Gaines, 1966; Gruner and Shyamsunder, 1991; Cantor, 1997). In this scenario, anesthetics may only need the exposure of the channel's gating component to the membrane bilayer to shift equilibrium between the open and closed states.

Pentobarbital Modulation of ₁ Versus ₁₂₂ Receptor Channels. The contrast in the pentobarbital modulation between homooligomeric ₁ and heterooligomeric ₁₂₂ receptor channels is intriguing given that the ₁ receptor channel shows approximately 40-fold greater sensitivity to GABA than ₁₂₂, and ₁ displays unique activation and deactivation kinetics.

Tryptophan Residue. What architectural features within the ₁ receptor channel might be created by Trp328 substitutions? The Trp residue is unique not only with respect to size, but also because of the indole moiety on its side chain. This residue can potentially anchor the TM3 to the extracellular side of the membrane. In this scenario, mutation of Trp328 to hydrophobic amino acids such as Met, Leu, Ile, Ala, or Val may dislodge the N-terminal amino acids of the TM3 from the interface of the extracellular side of the membrane and subsequently allow the residue 328 to rest deep within the membrane. This structural perturbation in the TM3 may then expose the gate of the channels to the membrane components (see above). Alternatively, the TM3 in the new configuration along with other TMs may constitute a binding cavity for pentobarbital. This phenomenon in which membrane-spanning domains interact to create a binding site, is not unique among membrane-embedded proteins. For example, the interactions of different TMs in the rhodopsin molecule constitute a binding cavity for the retinal molecule (Unger et al., 1997). Finally, Trp328 may impede the interaction of the pentobarbital with its binding/sensor domain solely based on its size. Consistent with this hypothesis, ₁ receptor channels containing the Tyr at position 328 did not respond to pentobarbital. Furthermore, in comparison with other hydrophobic amino acid substitutions, ₁ receptor channel containing the Phe (contains an aromatic ring on its side chain) substitution (W328F) exhibited lower pentobarbital sensitivity.