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NEUROPHARMACOLOGY

Identification of Structures within GABA_A Receptor Subunits That Regulate the Agonist Action of Pentobarbital

Department of Pharmacology, Physiology, and Neuroscience, University of South Carolina School of Medicine, Columbia, South Carolina

Received March 17, 2006; accepted May 23, 2006.

	Abstract

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Barbiturates act on GABA_A receptors (GABARs) through three distinct mechanisms, resulting in positive allosteric modulation, direct activation, and inhibition. These effects are observed at different concentrations and are differentially affected by some mutations and by the receptor's subunit composition. Mammalian GABARs can be formed from a combination of 16 different subunit subtypes. Although the effect of barbiturates depends largely on the subunit, their agonist activity is substantially influenced by the subunit subtype. Pentobarbital is a more effective agonist than GABA only when receptors contain an 6 subunit. Results from chimeric 1/6 subunits suggested that structural differences within the extracellular N-terminal domain were responsible for this characteristic. Within this domain, we examined 15 amino acid residues unique to the 6 subtype. Each of these sites was individually mutated in the 6 subunit to the corresponding residue of the 1 subunit. The effect of the mutation on direct activation by pentobarbital was determined with whole-cell electrophysiological recordings. Our results indicate that only one of these mutations, 6(T69K), altered pentobarbital efficacy. This single mutation reduced the response to pentobarbital to a level intermediate to the wild-type 112L and 612L isoforms. The mutation did not affect the sensitivity of the receptor to GABA but did reduce the efficacy of etomidate, another i.v. anesthetic with activity similar to pentobarbital. The reverse mutation in the 1 subunit (K70T) did not alter the response to pentobarbital. This is the first identification of a structural difference in GABAR subtypes that regulates direct activation by barbiturates.

The GABARs are pentameric, ligand-gated chloride channels responsible for most fast inhibitory neurotransmission in the mammalian brain. These receptors can be constructed from a diverse array of GABAR subunits. In mammals, seven subunit families and 16 subunit subtypes have been reported (Whiting et al., 1999). Formation of a functional receptor requires both and subunits, and there is considerable diversity in the physiological and pharmacological properties conferred by the six different and three different subtypes (Korpi et al., 2002).

Both and subunits are known to contribute to the effects of barbiturates on GABAR subunits (Thompson et al., 1996; Wafford et al., 1996; Belelli et al., 1999). In general, the 2 and 3 subtypes confer greater sensitivity and efficacy to pentobarbital than the 1 subtype for both its allosteric and agonist actions. A number of residues within the first and second transmembrane domains (TMs) of the subunit have been identified that influence sensitivity to positive modulation and/or direct activation by several anesthetics, including the barbiturates (Birnir et al., 1997; Dalziel et al., 1999; Pistis et al., 1999; Carlson et al., 2000; Cestari et al., 2000; Serafini et al., 2000; Chang et al., 2003). The contribution from structures within the subunits to the activity of barbiturates is less well studied, although the identity of the subtype has a substantial effect on direct activation by pentobarbital. Pentobarbital has the greatest efficacy at receptors containing an 6 subunit and is a more effective agonist than GABA only at these receptors (Thompson et al., 1996). With receptors containing any of the other five subtypes, in combination with a and subunit, pentobarbital is a less effective agonist compared with GABA (Thompson et al., 1996; Wafford et al., 1996; Akk et al., 2004). In contrast to the substantial contribution from the subtype to the agonist action of pentobarbital, it has little impact on the positive allosteric modulation observed at lower concentrations (Thompson et al., 1996).

The goal of this work was to identify the structures within the 6 subunit responsible for conferring high barbiturate efficacy. We compared the responses of recombinant receptors containing each of the six different subtypes to direct activation by pentobarbital and confirmed the unique responsiveness of the 6 subunit. Previous studies suggested that the extracellular N-terminal domain of the 6 subunit regulates pentobarbital efficacy (Fisher et al., 1997). We confirmed that finding and extended these studies to determine which amino acids within this domain contributed to the functional difference. The wild-type, chimeric, or mutated subunits were transiently transfected into L929 fibroblasts along with wild-type 1 and 2L subunits, and the ability of pentobarbital to activate the receptor was measured with whole-cell patch-clamp recordings.

	Materials and Methods

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Construction of Chimeric and Mutated Subunits. The 1/6 chimeric subunits were constructed with a splice site in the first transmembrane domain as described by Fisher et al. (1997). The commercially available QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used to generate the point mutations. Incorporation of the desired mutation was verified by sequencing (University of South Carolina DNA Core).

Transient Expression of Recombinant Receptors. Full-length cDNAs for the wild-type rat or human (2) GABAR subunits in mammalian expression vectors were obtained from Dr. Robert Macdonald (Vanderbilt University, Nashville, TN). Recombinant receptors were transiently expressed in the L929 mouse fibroblast cell line. The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% horse serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were passaged with a 0.05% trypsin/0.53 mM EDTA solution in phosphate-buffered saline (10 mM Na₂HPO₄, and 150 mM NaCl, pH 7.3).

L929 cells were transfected using calcium phosphate precipitation (Fisher et al., 1997). Plasmids encoding the selected GABAR subunit cDNAs were added to the cells in 1:1:1 ratios (::) of 4 µg each. To isolate the transfected cells, 2 µg of the Capture-Tec pHook-1 (Invitrogen, Carlsbad, CA) plasmid encoding a surface antibody, sFv, was also transfected.

The isolation procedure for the transfected cells was conducted 20 to 28 h later (see Drafts and Fisher, 2004). The cells were first passaged with trypsin and then mixed for 40 to 50 min with magnetic beads (approximately 7.5 x 10⁵ beads) coated with antigen specific for the pHook antibody. Bead-bound cells were separated with a magnetic stand, resuspended into Dulbecco's modified Eagle's medium, and plated onto coverslips coated with polylysine and collagen. Cells were used for recording 20 to 28 h later.

Electrophysiological Recording Techniques. All recordings were performed in the whole-cell configuration. The external bath solution contained 142 mM NaCl, 8.1 mM KCl, 6 mM MgCl₂, 1 mM CaCl₂, and 10 mM HEPES, with a pH of 7.4 and osmolarity adjusted to 295 to 305 mOsm. Recording electrodes were filled with internal solution composed of 153 mM KCl, 1 mM MgCl₂, 5 mM K-EGTA, and 10 mM HEPES (pH 7.4, 295–305 mOsm). A Narishige PP-830 electrode puller (Narishige, Tokyo, Japan) was used to pull recording electrodes to a resistance of 5 to 10 M from thick-walled borosilicate glass with an internal filament (World Precision Instruments, Sarasota, FL).

Drugs were diluted into external solution and applied to the cells using a computer-controlled stepper solution exchanger (SF-77B; Warner Instruments, Hamden, CT), allowing solution changes to the cell in <50 ms (open tip). There was a continuous flow of external solution through the chamber. Chloride currents were recorded at –50 mV with an Axon 200B patch-clamp amplifier and the pClamp 8 suite of software programs (Axon Instruments Inc., Union City, CA). The recordings were stored on a computer hard drive for off-line analysis.

Analysis of Whole-Cell Currents. Whole-cell current recordings were analyzed using the pClamp8 suite (Axon Instruments Inc.) and GraphPad Prism (GraphPad Software Inc., San Diego, CA). To determine GABA concentration-response relationships, peak current amplitudes were normalized to the maximal current elicited by 1 mM GABA for each cell. Normalized concentration-response data were fit with a four-parameter logistic equation: current = [minimal current + (maximal current – minimal current)]/[1 + (10 (log EC₅₀ – log [GABA]) x n), where n represents the Hill number. Statistical comparisons were performed using the Tukey-Kramer multiple comparisons test (Instat; GraphPad Software Inc.) with a significance level of p < 0.05.

	Results

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Pentobarbital Is a More Effective Agonist Than GABA Only at Receptors Containing the 6 Subtype. To examine the role of the GABAR subtype in the agonist activity of pentobarbital, L929 fibroblasts were transiently transfected with wild-type 1 and 2L subunits and one of the six subtypes. Although this may not represent a common native isoform for all of the subtypes (McKernan and Whiting, 1996), this combination of and subtypes was selected to provide a standard background for comparison. The subtype can influence the response to pentobarbital, and we chose the 1 subtype because it provided the greatest contrast in responses between 1- and 6-containing receptors (see below). The 2 subunit is the most widely expressed of the subunits and probably contributes to the majority of postsynaptic receptors (McKernan and Whiting, 1996).

Whole-cell currents were recorded in response to 5-s applications of GABA or pentobarbital from cells voltage clamped at –50 mV. Based on full concentration-response relationships for the 112L and 612L isoforms (see Fig. 4D), we selected 300 µM pentobarbital as a concentration for comparison among receptors containing different subtypes. This concentration produced near-maximal responses for pentobarbital and showed less of the inhibitory, channel block action observed at higher concentrations that might obscure measurement of the peak current. The amplitude of the response to pentobarbital alone was compared with that seen with 1 mM GABA in the same cell. This represented a maximally effective GABA concentration for all receptor isoforms.

View larger version (26K): [in this window] [in a new window]   Fig. 4. The 6(T69K) mutation affects the agonist efficacy of pentobarbital but does not alter GABA sensitivity. A, alignment of rat sequence for the subunits from Tyndale et al. (1995). Mutated site is boxed and indicated by bold type. Numbering from mature rat sequence. B, threonine 69 of the 6 subunit was mutated to each of the residues (lysine, histidine, and isoleucine) found at the homologous site in each of the subtypes. Wild-type residues in the 1 and 4 subunits were mutated to threonine. Bars, mean ± S.E.M. of the response to 300 µM pentobarbital as a percentage of the response to 1 mM GABA. Number of cells (n) is given by the numbers in parentheses. Data for wild-type subunits are from Fig. 1 and for 6(T69K) are from Fig. 3. **, p < 0.001, indicates a significant difference from 612L. The responses of receptors containing the mutated 1(K70T) and 4(I69T) subunits were not significantly different from their wild-type receptors (p > 0.05). C, representative whole-cell current responses to 1 mM GABA and 300 µM pentobarbital (PB) are shown for receptors containing mutated 6(T69K) or 1(K70T) subunits. Cells were voltage-clamped at –50 mV, and the drugs were applied for 5 s, indicated by the bar. D, concentration-response relationships for direct responses to pentobarbital were constructed for receptors containing wild-type or mutated 6(T69K) subunits. The mutation caused a reduction in pentobarbital efficacy to a level intermediate between the wild-type receptors. The inhibitory effect of pentobarbital, apparent at millimolar concentrations, prevented accurate fitting of the data points, but there was no obvious effect on pentobarbital EC50. E, GABA concentration-response relationships were determined for receptors containing wild-type or mutated subunits. The wild-type 6 subunit confers a greater sensitivity to GABA compared with the 1 subunit. The point mutations in the 1 or 6 subunits had no effect on GABA EC50.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Pentobarbital agonist efficacy depends upon the and subtypes. A, mouse L929 fibroblasts were transfected with each of the subunits along with 1 and 2L. GABA (1 mM) or 300 µM pentobarbital (PB) was applied for 5 s (bar) to cells voltage clamped at –50 mV. Representative whole-cell currents obtained for each agonist from the same cell are shown for each isoform. B, responses to 300 µM pentobarbital were expressed as a percentage of the response to 1 mM GABA. Bars, mean ± S.E.M., with the number of cells (n) given by the number in parentheses. Dotted line, normalized response to 1 mM GABA (100%). Pentobarbital produces a greater current response than GABA only when the receptors contain an 6 subunit. C, subunit subtype influences the efficacy of pentobarbital at receptors containing an 1 or 4 subunit but not those containing an 6 subunit. All receptors also contain the 2L subunit. Data are presented as in B.

Some of the variation in responsiveness reported by different laboratories may be associated with the identity of the subtype, which also influences pentobarbital efficacy. In general, compared with 1, the 2 and 3 subtypes increase the effectiveness of barbiturates both as allosteric modulators and as agonists (Thompson et al., 1996). We examined the effect of the subtype when coexpressed with 2L and 1, 4, or 6. We found that the 3 subtype was associated with greater pentobarbital efficacy compared with the 1 subtype when coexpressed with either the 1 or 4 subunits (Fig. 1C). However, in combination with the 6 subunit, the identity of the subtype did not alter the response to pentobarbital. These results are consistent with previous studies that showed that the subtype influenced barbiturate efficacy at 1- but not 6-containing receptors (Thompson et al., 1996). Because the x12L isoforms produced the greatest contrast in responses among the different receptors, we used these subunit combinations to examine the role of the subtype.

Higher Efficacy of Pentobarbital Is Associated with the N-Terminal Domain of the 6 Subunit. The 6 subtype is apparently unique among the subtypes in its ability to confer a greater response to pentobarbital than to GABA. To identify the structural domains within the subunit responsible for this characteristic, we examined the properties of receptors containing chimeric subunits that exchanged the N-terminal extracellular domains of the 1 and 6 subunits. We previously coexpressed these chimeric constructs with the 3 and 2L subunits and found that the N-terminal domain conferred pentobarbital efficacy (Fisher et al., 1997). We repeated these experiments with the 1 and 2L subunits and confirmed that when the subunit contains the extracellular N-terminal domain from the 6 subunit (6/1), pentobarbital acts as a high-efficacy agonist compared with GABA, as it does with receptors containing the 6 subunit (Fig. 2). Conversely, when this domain is contributed from the 1 subunit (1/6), pentobarbital is not as effective an agonist as GABA. The response of the receptors containing the 6/1 chimera was not significantly different from the wild-type 612L receptors, whereas those with the 1/6 chimera did not differ from the 112L receptors (p > 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 2. High pentobarbital efficacy is conferred by the extracellular N-terminal domain of the 6 subunit. A, chimeric subunits were constructed that exchanged the extracellular N-terminal domains of the 1 and 6 subunits (Fisher et al., 1997). The 1/6 chimera contains the N-terminal domain of the 1 subunit, with the remainder of the structure contributed from the 6 subunit. Conversely, the 6/1 chimera contains the N-terminal domain of the 6 subunit. Representative traces show the whole-cell responses of the same cell to 5-s applications of either 1 mM GABA or 300 µM pentobarbital (PB). B, peak amplitude of the whole-cell current in response to 300 µM pentobarbital was divided by the response of the same cell to 1 mM GABA. Bars, mean ± S.E.M., with n indicated by the number in parentheses. Symbols indicate a significant difference (p < 0.001) from 612L (**) or 112L (++).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Effect of single point mutations in the 6 subunit on pentobarbital agonist activity. Fifteen different single mutations were made in the 6 subunit at sites of heterogeneity among the subtypes. The 6 residue was mutated to the homologous 1 residue in each case. The agonist efficacy of pentobarbital was determined by comparing the peak amplitude of the whole-cell response to 300 µM pentobarbital to the current produced by 1 mM GABA in the same cell. Bars, mean ± S.E.M. n is shown by the numbers in parentheses. Data for wild-type subunits are from Fig. 1. Dashed lines, responses of the wild-type 112L and 612L receptor isoforms. **, p < 0.001, indicates a significant difference from 612L.

Only one of the mutations in the 6 subunit, threonine to lysine at residue 69, significantly reduced the efficacy of 300 µM pentobarbital, producing a response smaller in amplitude than that seen with 1 mM GABA (p < 0.001 compared with 612L) (Figs. 3 and 4). However, this single mutation did not fully produce an 1-like response. Instead, the efficacy of pentobarbital was intermediate between the wild-type isoforms (Fig. 4). Mutations were also made in the 6 subunit to the homologous residues found in the 3 (histidine) and 4 (isoleucine) subunits (Fig. 4A). These point mutations also significantly reduced pentobarbital efficacy (Fig. 4B). Reverse mutations were made to convert the wild-type residues to leucine in the 1 (Lys70) and 4 (Iso69) subunits. However, neither of these mutations increased the efficacy of pentobarbital (Fig. 4, B and C). This suggests that, although the threonine residue in this location may be necessary for the high-efficacy response associated with the 6 subunits, other structural differences must combine with it to generate the full response to pentobarbital.

The T69K mutation did not affect GABA sensitivity of the receptor, with an average GABA EC₅₀ for 6(T69K)12L of 3.1 ± 0.5 µM (n = 5) comparable with that of the wild-type 612L isoform (2.3 ± 0.1 µM, n = 5, p > 0.05) (Fig. 4E). The mutation at the homologous site in the 1 subunit (K70T) also did not alter GABA sensitivity, with similar average EC₅₀s for receptors containing wild-type (17.2 ± 3.2 µM, n = 4) and mutated (18.1 ± 0.6 µM, n = 4, p > 0.05) subunits (Fig. 4E). This is consistent with earlier studies suggesting that GABA and pentobarbital activate the receptor through separate sites, with distinct structural requirements and pharmacological properties (Amin and Weiss, 1993; Thompson et al., 1996).

The 6(T69K) Mutation Alters Direct Activation But Not Allosteric Modulation by Pentobarbital. Most studies suggest that the positive allosteric effect of pentobarbital and its direct agonist activity occur through separate sites (Serafini et al., 2000). Mutations in the first and second transmembrane domains of subunits that reduce allosteric modulation have less (Chang et al., 2003) or no (Dalziel et al., 1999) impact on direct activation by barbiturates. This conclusion is also supported by the observation that the subtype has little influence on the ability of pentobarbital to enhance the response to GABA at concentrations that do not produce direct activation (Thompson et al., 1996). We measured the effect of the 6(T69K) mutation on potentiation of GABA-activated currents by 10 µM pentobarbital, a concentration that produced little direct activation for any of the receptors. Pentobarbital was similarly effective (p > 0.05) in increasing the response to 1 µM GABA for receptors containing the wild-type 6 (178.8 ± 7.3% GABA alone, n = 6) or the mutated 6(T69K) (171.6 ± 8.1%, n = 6) (Fig. 5) subunit. This supports previous conclusions that the allosteric and agonist actions of pentobarbital have distinct structural requirements.

View larger version (13K): [in this window] [in a new window]   Fig. 5. The 6(T69K) mutation does not reduce positive allosteric modulation by pentobarbital. Pentobarbital enhances the response to GABA at concentrations lower than those required for direct activation. Whole-cell current responses to 1 µM GABA or 1 µM GABA + 10 µM pentobarbital are shown for receptors containing the wild-type or mutated 6(T69K) subunit. Cells were voltage-clamped at –50 mV, and the drugs were applied for 5 s, indicated by the bar.

Role of Thr69 in Direct Activation by Etomidate. Many of the i.v. anesthetics, including pentobarbital, etomidate, and propofol, show similar patterns of activity at the GABARs, with positive modulation, direct activation, and inhibition occurring at increasing concentrations (Hill-Venning et al., 1997; Sanna et al., 1997; Hong and Wang, 2005). In addition, most mutations in subunits that alter pentobarbital effects also influence responsiveness to these drugs, suggesting common binding or transduction pathways (Belelli et al., 1997; Moody et al., 1998; Carlson et al., 2000). Some of the i.v. anesthetics also show similar subtype dependence, with the 6 subtype conferring a higher efficacy for direct activation (Hill-Venning et al., 1997; Belelli et al., 1999). To determine whether Thr69 of the 6 subunit has a general role in regulating the agonist activity of drugs similar to pentobarbital, we examined the effect of the mutation on efficacy of the general anesthetic etomidate.

Etomidate was not as effective an agonist as GABA or pentobarbital at either 1- or 6-containing receptors (Fig. 6). However, an subtype dependence similar to that of pentobarbital was observed, with a significantly higher efficacy at 612L receptors. The responses of receptors containing the 1/6 or 6/1 chimeric subunits showed that the extracellular N-terminal domain conferred this difference in etomidate efficacy, as it does for pentobarbital (Fig. 6B). The T69K mutation in the 6 subunit also influenced the ability of etomidate to activate the receptor because the response to 300 µM etomidate was significantly reduced compared with the wild-type receptor (Fig. 6). As was seen with pentobarbital, the mutation produced behavior intermediate to the 112L and 612L wild-type receptors, suggesting that other sites contribute to the effect of the subunit on direct activation by etomidate.

View larger version (10K): [in this window] [in a new window]   Fig. 6. The 6(T69K) mutation also reduces etomidate efficacy. A, representative traces show the whole-cell responses of the same cell to 5-s applications of either 1 mM GABA or 300 µM etomidate. In all cases, the current in response to etomidate is smaller than that produced by GABA. B, peak amplitude of the whole-cell current in response to 300 µM etomidate was divided by the response of the same cell to 1 mM GABA. Bars, mean ± S.E.M., with the n indicated by the number in parentheses. Symbols, significant difference from 612L (**, p < 0.01; ***, p < 0.001).

	Discussion

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Structural models of the N-terminal domain of the Cys-loop ligand-gated channels have been modified to describe the GABA_A receptor subunits (Holden and Czajkowski, 2002; Ernst et al., 2005). According to these structural estimates, residue 69 of the 6 subunit lies at the end of the strand identified as binding loop D. The GABA binding pocket is formed through the interaction of the and subunits. The subunit is believed to contribute to the ligand binding site via its "plus side," which includes loops A and B. The "minus side" is formed by the subunits and includes loops D and E. Therefore, this residue is in a location that suggests a role in pentobarbital binding or in transduction of the binding signal. Topological representations of the locations of these structures and their proposed contributions to the ligand binding site can be found in Holden and Czajkowski (2002) and Ernst et al. (2005). Cysteine substitution studies of loop D of the 1 subunit showed that binding of GABA or a competitive antagonist did not alter the accessibility of lysine 70 (Holden and Czajkowski, 2002), consistent with our results that a mutation at this site did not affect GABA sensitivity. These studies also reported differences in the effects of pentobarbital and GABA on the accessibility of some residues in this region. It would be interesting to determine with these techniques whether pentobarbital might have different effects in the 6 subunit, compared with the 1 subunit, because variations in induced conformational changes could underlie agonist efficacy. Exchanging threonine and lysine at this location replaces a smaller, polar side chain with a bulkier, positively charged residue. Because mutations to isoleucine and histidine also reduced pentobarbital efficacy, it is possible that the volume of the side chain at this location is an important factor. Mutations to a variety of residues with different chemical properties may provide further clues into the role that this structure plays in receptor function.

Because pentobarbital produces a greater response from 6x2L receptors than does GABA, GABA can be considered a lower efficacy agonist and perhaps even a partial agonist at these receptors in terms of its ability to activate the channel. A comparison of single-channel properties showed that GABA activates primarily short-duration channel openings at 6-containing receptors, in contrast to substantial activation of a long-duration open state for 1-containing receptors (Fisher, 2004). Similar to 6-containing receptors, GABARs containing subunits respond to activation by GABA with predominantly short-duration channel openings (Fisher and Macdonald, 1997; Akk et al., 2004). However, these receptors can produce long-duration openings in the presence of some modulators, such as neurosteroids (Bianchi and Macdonald, 2003), and in response to high-efficacy agonists, like pentobarbital (Akk et al., 2004; Feng et al., 2004). In an analogous fashion, it is possible that pentobarbital could increase transitions into a long-duration state for the 612L receptors, thereby producing the larger macroscopic currents compared with GABA. However, it is not simply a difference in channel gating properties in response to GABA, i.e., a ceiling effect, that is responsible for the higher efficacy of pentobarbital observed at the 612L receptors. Previous studies with the chimeric subunits showed that the extracellular N-terminal domain is not responsible for the different gating patterns associated with the 1 and 6 subunits (Fisher, 2004). That is, receptors containing the 1/6 chimera respond with low-efficacy gating to GABA but are also less responsive to pentobarbital. Conversely, those with the 6/1 chimeric subunit exhibit the long-duration openings characteristic of the 1 subunit but are still more highly activated by pentobarbital. An examination of the response to pentobarbital at the level of the single channel will be necessary to determine the gating mechanisms by which this greater activation occurs.

Several general anesthetics, including barbiturates, etomidate, and propofol, have similar actions at GABARs. Previous studies have demonstrated that the same mutations within the subunits can affect modulation and/or activation by all of these drugs, suggesting that they may use common binding sites or transduction pathways (Belelli et al., 1999; Pistis et al., 1999; Carlson et al., 2000; Cestari et al., 2000; Chang et al., 2003; Siegwart et al., 2003). Our results show that pentobarbital and etomidate may share some common structures in the subunits as well because the T69K mutation in the 6 subunit reduced the efficacy of both of these agonists.

Receptors containing the 6 subunit have distinctive pharmacological properties (Korpi et al., 2002). Some of these characteristics, such as high sensitivity to zinc and furosemide and low sensitivity to benzodiazepine agonists, are shared with the 4 subunit. Others are unique to the 6 subtype, including higher sensitivity to amiloride and GABA and greater responsiveness to barbiturates, neurosteroids, and anesthetics. Previous studies have identified some of the structural differences responsible for these properties (Korpi et al., 2002). Residues located in the extracellular N-terminal domain contribute to GABA, benzodiazepine, and amiloride sensitivity (Wieland et al., 1992; Drafts and Fisher, 2004). Sensitivity to inhibition by zinc is associated with a histidine residue located in the extracellular TM2-TM3 domain of the 6 subunit (Fisher and Macdonald, 1998), and a residue in TM1 confers higher furosemide sensitivity (Thompson et al., 1999). The unique pharmacological properties associated with the 6 subunit along with its restricted expression pattern in the adult brain (Laurie et al., 1992) make it a potentially useful target for selective modulation. Long-term administration of pentobarbital has been shown to increase the expression of the 6 subunit (Ito et al., 1996; Raol et al., 2005), which may be a result of the higher activity level of barbiturates at these receptors. The 6 subunits contribute to both postsynaptic and extrasynaptic receptor populations in cerebellar granule cells and play an important role in both phasic and tonic neurotransmission in this region (Brickley et al., 1996).

A description of the structural differences among the GABAR subunits that give rise to their distinct properties will aid in the development of subunit-selective drugs that may be able to provide targeted treatment of neurological disorders. These studies could also increase our understanding of the mechanisms by which agonist binding is transduced into either low- or high-efficacy gating of the ion channel.

	Acknowledgements

 
We thank Amber Picton for technical assistance and Courtney Pinard for help with site-directed mutagenesis.

	Footnotes

 
This work was supported by National Institutes of Health-National Institute of Neurological Disorders and Stroke (Grant RO1-NS045950), by the PhRMA Foundation, by the Epilepsy Foundation, and by the University of South Carolina School of Medicine Research Development Fund.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.104844.

ABBREVIATIONS: GABAR, GABA_A receptor; TM, transmembrane domain.

Address correspondence to: Dr. Janet L. Fisher, Department of Pharmacology, Physiology, and Neuroscience, University of South Carolina School of Medicine, Columbia, SC 29208. E-mail: jfisher{at}med.sc.edu

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