Although GABA activates synaptic (αβγ) GABAA receptors with high efficacy, partial agonist activation of αβγ isoforms and GABA activation of the primary extrasynaptic (αβδ) GABAA receptors are limited to low-efficacy activity, characterized by minimal desensitization and brief openings. The unusual sensitivity of αβδ receptor channels to neurosteroid modulation prompted investigation of whether this high sensitivity was dependent on the δ subunit or the low-efficacy channel function that it confers. We show that the isoform specificity (αβδ > αβγ) of neurosteroid modulation could be reversed by conditions that reversed isoform-specific activity modes, including the use of β-alanine to achieve increased efficacy with αβδ receptors and taurine to render αβγ receptors low efficacy. We suggest that neurosteroids preferentially enhance low-efficacy GABAA receptor activity independent of subunit composition. Allosteric conversion of partial to full agonism may be a general mechanism for reversibly scaling the efficacy of GABAA receptors to endogenous partial agonists.
Many GABAA receptor modulators exhibit clear subunit selectivity (Olsen and Macdonald, 2002). GABAA receptor pharmacological studies have focused on “structural” determinants of modulators, such as the subunit dependence of benzodiazepines (Sigel and Buhr, 1997) and the proposed “transduction” element at the outer mouth of the second transmembrane domain (TM2) (Wingrove et al., 1994; Stevenson et al., 1995; Halliwell et al., 1999; Thompson et al., 1999). “Knock-in” mutations of the benzodiazepine binding site emphasized the importance of characterizing the isoform preferences of allosteric modulators (Rudolph et al., 1999; Low et al., 2000; McKernan et al., 2000).
In addition to the rich isoform-specific pharmacology of GABAA receptor channels, there is clear evidence that biophysical properties, such as desensitization and gating efficacy, also depend on subunit composition (Fisher and Macdonald, 1997; Haas and Macdonald, 1999). One of the potential confounding factors associated with investigating subunit-specific pharmacology is that “functional” differences among isoforms, as opposed to simply the presence or absence of modulator binding sites, may play a significant role in determining pharmacological profiles. Therefore, the observation of subunit-dependent modulation might in some cases be an epiphenomenon, in which the action of the modulator was dependent on a difference in a functional property (not a strictly structural one, per se) that was itself subunit-dependent.
The idea that allosterism could also depend on functional differences would offer yet another mechanism for specificity among isoforms, even if the binding site and coupling machinery of the modulator (the often sited basis for subunit specificity) were present in every isoform. It is even more intriguing to consider the potential plasticity of allosteric modulation given the observation of agonist-dependent functional properties of GABAA receptors. For example, partial agonists and so-called nondesensitizing agonists have been characterized, and some of these compounds (such as taurine) are present in the brain and may be endogenous ligands for GABAA receptors (Sakai et al., 1985; Lerma et al., 1986; Huxtable, 1989). Although the physiological relevance of endogenous partial agonists remains poorly understood, the possibility of reversible augmentation of partial agonist efficacy by endogenous modulators raises the interesting possibility of plasticity at the level of agonist-dependent gating.
One class of compounds that shows a clear GABAA receptor isoform preference is the neurosteroids (Mellon and Griffin, 2002), which exert their actions in the CNS in part through interaction with synaptic GABAA receptors (Harrison et al., 1987; Lambert et al., 1995; Cooper et al., 1999; Fancsik et al., 2000). The GABAA receptor δ subunit is of particular importance for behavioral responses to neurosteroids (Mihalek et al., 1999), and the endogenous neurosteroid tetrahydrodeoxycorticosterone (THDOC) preferentially enhanced αβδ over αβγ receptors (Adkins et al., 2001; Brown et al., 2002; Wohlfarth et al., 2002). Interestingly, THDOC modulation produced substantially larger currents than the maximal currents produced by GABA alone for α1β3δ (but not α1β3γ2L) receptors, suggesting that the low-efficacy activity of αβδ receptors (Fisher and Macdonald, 1997) could be overcome by neurosteroids (Wohlfarth et al., 2002). The present study was designed to determine whether the enhanced neurosteroid actions depended on the presence of the δ subunit or the associated functional properties of minimal desensitization and low-efficacy gating.
Materials and Methods
Expression of recombinant GABAA receptors. Human embryonic kidney 293T (HEK293T) cells (a gift from P. Connely, COR Therapeutics, San Francisco, CA) were maintained in DMEM and supplemented with 10% fetal bovine serum at 37°C in 5% CO2/95% air. Cells were transiently transfected with 4 μg each of α1 and β3 subunits, together with either a δ, γ2L, or mutated δ subunit (all subcloned into the pCMVneo expression vector), using the calcium phosphate precipitation technique (Angelotti et al., 1993). Point mutants were generated using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). Cotransfection of the pHook plasmid (Invitrogen, Carlsbad, CA) enables selection of transfected cells by immunomagnetic bead separation 24 hr later (Greenfield et al., 1997). The next day, whole-cell patch-clamp recordings were performed at room temperature.
Electrophysiology and drug application. Patch-clamp recordings were performed on transfected fibroblasts bathed in an external solution consisting of the following (in mm): 142 NaCl, 8 KCl, 6 MgCl2, 1 CaCl2, 10 HEPES, 10 glucose, pH 7.4, 325 mOsm. Low-resistance electrodes (0.8-1.5 MΩ; World Precision Instruments, Pittsburgh, PA) were pulled with a Flaming Brown electrode puller (Sutter Instruments, San Rafael, CA) and fire-polished. The internal solution consisted of the following (in mm): 153 KCl, 1 MgCl2, 2 MgATP, 10 HEPES, 5 EGTA, pH 7.3, 300 mOsm. The combination of internal and external solutions produced a chloride equilibrium potential near 0 mV. Patch-clamped cells were gently lifted from the recording dish to increase solution-exchange efficiency. Cells were voltage-clamped at -10 to -50 mV, and no voltage-dependent effects of desensitization or neurosteroid modulation were observed in this range. For experiments involving excised patches, thick-walled borosilicate glass was used with resistances of 5-15 MΩ, and cells were plated on collagen-treated culture dishes.
THDOC (Sigma, St. Louis, MO) was prepared as a 10 mm stock in dimethylsulfoxide (DMSO) and kept frozen. The THDOC stock was dissolved in external solution containing DMSO at a final concentration of 0.1%. piperidine-4-sulfonic acid (P4S) (Sigma) prepared as a 100 mm stock in water. GABA was prepared as a 1 m stock in water. Drugs were applied via gravity using a rapid perfusion apparatus (Warner Instruments, Hamden, CT) connected to multibarrelled square glass tubing pulled to a final barrel size of ∼250 μm. Solution exchange time measured with an open electrode tip was 0.3-1.5 msec, depending on the flow rate (with faster range used for excised patch experiments), although slower exchange probably occurred around whole cells.
Analysis of currents. Whole-cell currents were low-pass filtered at 2-5 kHz, digitized at 10 kHz, and analyzed using the pCLAMP8 software suite (Axon Instruments, Foster City, CA). To avoid underestimating the effects of THDOC on peak current amplitude resulting from current rundown, control measurements (GABA alone) were made before and after THDOC application, and the average response was used. For THDOC modulation, the small “direct” activation current observed during the preapplication period was subtracted from the peak current in the presence of GABA and THDOC. The desensitization and deactivation time courses of GABAA receptor currents elicited with the concentration-jump technique were fit using the Levenberg-Marquardt least squares method with one, two, or three component exponential functions of the form Σ ane (-t/τn), where n is the best number of exponential components, a is the relative amplitude of the component, t is time, and τ is the time constant. Additional components were accepted only if they significantly improved the fit, as determined by an F test on the sum of squared residuals. For comparison of deactivation time courses, a weighted summation of the fast and slow decay components (af · τf + as · τs) was used. Single-channel data were digitized at 20 kHz, filtered at 2 kHz via the internal Axon 200A (Axon Instruments) amplifier filter, and stored on VHS videotape for off-line analysis. Stretches of single-channel activity were analyzed using the 50% threshold detection method of Fetchan 6.0 (pClamp 8.0). Overlapped openings and bursts were not included in the analysis. Events with durations <150 μsec (1.5 times the system dead time) were shown in the histogram but were not considered in the fitting routine. Logarithmic binning was used as described previously (Haas and Macdonald, 1999) and fitted with a maximum likelihood routine by the Interval5 software (Dr. Barry Pallotta, University of North Carolina, Chapel Hill, NC). The number of exponential functions required to fit the distributions was incremented until additional exponentials failed to significantly improve the fit. Data reduction was implemented for figure display purposes only. Numerical data were expressed as mean ± SEM. Statistical significance using Student's t test (paired or unpaired, as appropriate) was taken as p < 0.05. All data sets were normally distributed.
THDOC preferentially enhanced α1β3δ over α1β3γ2L GABAA receptor currents
The neurosteroid THDOC has been shown to preferentially enhance GABAA receptors containing the δ subunit over those containing the γ2L subunit (Adkins et al., 2001; Brown et al., 2002; Wohlfarth et al., 2002). This effect is shown in Figure 1. Currents evoked by a saturating 1 mm GABA concentration were compared with and without pre-applied THDOC (1 μm; 2-3 sec) for α1β3δ and α1β3γ2L receptors expressed in HEK293T cells (see Materials and Methods). α1β3γ2L receptor currents desensitized rapidly and extensively during a 6 sec application of GABA (1 mm) (Fig. 1A, left). In the presence of pre-applied THDOC (1 μm), peak currents were slightly inhibited (92.6 ± 9.9% of control amplitude; n = 7), although this was not significant (Fig. 1C), which is consistent with previous studies of neurons (Le Foll et al., 1997; Zhu and Vicini, 1997). Macroscopic desensitization in the presence of THDOC tended to be slightly faster, although this too was variable and neither the rates nor the extents of desensitization differed significantly from control values (Fig. 1D; Table 1). α1β3δ receptor currents were substantially and reversibly (data not shown) enhanced by pre-applied THDOC (1290 ± 163%; n = 8) (Fig. 1B,C). Although this isoform exhibited minimal, slow desensitization even during saturating (1 mm) GABA application (Haas and Macdonald, 1999) (Table 1), THDOC-modulated currents showed pronounced desensitization during the 6 sec of GABA exposure (57.8 ± 2.6%) (Fig. 1B,D), which is in agreement with our previous results (Bianchi et al., 2002; Wohlfarth et al., 2002). In most cases, this increased desensitization was well described by two exponential functions, with time constants similar to the intermediate and slow time constants fitted to the triphasic desensitization of α1β3γ2L receptor currents evoked by 1 mm GABA (Table 1). The rate of current deactivation was prolonged by THDOC for both isoforms (Table 1). Clearly, THDOC enhanced α1β3δ currents beyond the maximal currents evoked by a saturating concentration of GABA alone, and we showed previously that this was explained in part by the introduction of a longer duration, third, open state. In contrast, THDOC failed to increase the amplitude of α1β3γ2L receptor currents evoked by saturating GABA (1 mm). However, neurosteroids are well known to enhance submaximal αβγ receptor currents, and thus they clearly bind to the receptor (Puia et al., 1990; Lan et al., 1991; Adkins et al., 2001; Wohlfarth et al., 2002). Although this apparent GABA concentration dependence was similar to modulation by benzodiazepines (that only enhance submaximal currents), neurosteroids have been shown to increase the gating efficacy of GABAA receptor single-channel currents in a manner that is unlikely to be explained simply by altered GABA binding (Mistry and Cottrell, 1990; Twyman et al., 1992). α1β3γ2L receptor single-channel currents are known to exhibit higher efficacy gating patterns (longer open times in long complex bursts) than α1β3δ receptors [brief openings and brief bursts at both high and low GABA concentrations (Fisher and Macdonald, 1997)] in response to high concentrations of GABA (Fisher and Macdonald, 1997; Haas and Macdonald, 1999). Also, α1β3γ2L receptor single-channel currents show low-efficacy gating when low GABA concentrations are used (Fisher and Macdonald, 1997), and, under these conditions, THDOC enhancement is robust (Wohlfarth et al., 2002). If THDOC acts via stabilization of high-efficacy gating, and therefore selectively enhances GABAA receptors under conditions of low-efficacy gating, this might account not only for the GABA concentration-dependent modulation of α1β3γ2L receptors but also the apparent isoform preference of α1β3δ over α1β3γ2L receptors. We tested this hypothesis in the following sections.
Increased THDOC enhancement of α1β3γ2L receptor currents evoked by a partial agonist
One complication of using low concentrations of GABA to favor a low-efficacy α1β3γ2L receptor gating is that modulators such as THDOC may alter GABA binding in addition to the gating pattern. We reasoned that a partial agonist (restricted to low-efficacy activation despite saturating concentration) would be a useful alternative to assess of the role of gating efficacy in GABAA receptor modulation by THDOC. The compound P4S has been characterized as a partial agonist acting at the GABA binding site. P4S evoked smaller amplitude currents than GABA on αβγ receptors, even at the saturating concentration of 1 mm (EC50 was similar to GABA) (Krogsgaard-Larsen et al., 1980, 1981; Ebert et al., 1997). Single-channel recording indicated previously that P4S evoked brief duration openings (Steinbach and Akk, 2001), similar to those observed from α1β3γ2L receptors with low concentrations of GABA and also to those observed with α1β3δ receptor currents (even at high GABA concentration) (Fisher and Macdonald, 1997; Haas and Macdonald, 1999). Thus, P4S could be used to saturate the GABA binding site(s), yet induce only the low-efficacy gating pattern of α1β3γ2L receptors. Figure 2A shows the response of α1β3γ2L receptors to a saturating concentration of GABA or P4S from the same cell. Currents evoked by P4S (1 mm) were always smaller than currents evoked by GABA (1 mm) from the same cells (16.8 ± 2.8% of control; n = 4) and showed minimal extent of desensitization (19.7 ± 3.7%) (Fig. 2D). If gating efficacy was a critical factor for THDOC enhancement, then the low-efficacy gating favored by P4S (relative to 1 mm GABA) should be markedly increased by THDOC modulation of α1β3γ2L receptors, similar to the effects of modulators on GABAA receptor partial agonists shown previously (Maksay et al., 2000). Indeed, P4S-evoked α1β3γ2L receptor currents were strongly potentiated by pre-applied THDOC (598 ± 86%; n = 4) (Fig. 2B,C). This potentiation was accompanied by increased desensitization (Fig. 2D), similar to the effect of THDOC on α1β3δ receptor currents (Fig. 1D), although the rate of desensitization was not as fast as that observed with GABA (1 mm) application to the same cell (Fig. 2B, inset; Table 1). The extent of desensitization during the 6 sec application of P4S in the presence of THDOC was similar to that observed for applications of GABA in the same cells (90.4 ± 2.1, compared with 82.3 ± 3.6%) (Fig. 2D). Interestingly, deactivation after removal of P4S was not significantly enhanced by THDOC (45.8 ± 13.2 msec; with THDOC, 58.0 ± 4.5 msec).
The current amplitudes recorded with GABA (1 mm) alone were not different from those recorded with P4S and THDOC together within individual cells (Fig. 2C). Identical experiments were performed on α1β3δ receptors to determine whether P4S-evoked currents could be modulated to amplitudes greater than those observed with GABA alone. We confirmed that P4S is also a partial agonist at α1β3δ receptors, because it evoked smaller currents than GABA (data not shown), similar to a recent report using α4β3δ receptors (Brown et al., 2002). THDOC strongly enhanced P4S-evoked currents, which exceeded the amplitude of GABA-evoked (1 mm) currents (in the same cells) by ∼10-fold (data not shown).
Gating efficacy, not desensitization, is a critical determinant of THDOC modulation of GABAA receptor currents
Despite the clear enhancement by THDOC of α1β3γ2L receptor currents evoked by P4S, we could not rule out the possibility that the minimal desensitization, not the low-gating efficacy, was the critical “permissive” factor involved in THDOC enhancement of GABAA receptor currents. In fact, desensitized states have been suggested to be important for THDOC modulation of GABAA receptors (Leidenheimer and Chapell, 1997; Zhu and Vicini, 1997). If α1β3δ receptor currents (and P4S-evoked α1β3γ2L receptor currents) were selectively enhanced by THDOC because of their characteristically minimal desensitization, then one would predict that a high-efficacy, nondesensitizing, GABAA receptor isoform should also be markedly enhanced by THDOC. In an unrelated set of experiments, we found that a L9'S mutation in TM2 of the δ subunit clearly increased single-channel gating efficacy but did not alter macroscopic desensitization (Fig. 3). The currents evoked by a 400 msec application of 1 mm GABA to outside-out patches (to ensure resolution of possible fast phases) containing either α1β3δ (Fig. 3A) or α1β3δ (L9'S) receptors (Fig. 3B) demonstrated the minimal desensitization for both receptors. Single-channel analysis revealed longer duration openings that tended to occur in bursts (Fig. 3C), similar to the effects of L9'S-T mutations in GABAA receptor and nACh receptor channels (Filatov and White, 1995; Labarca et al., 1995; Bianchi and Macdonald, 2001). In contrast to α1β3δ receptors that open to one of two brief-duration open states in response to 1 mm GABA (Fisher and Macdonald, 1997; Haas and Macdonald, 1999), the distribution of open durations for α1β3δ (L9'S) receptors required three exponential functions with time constants of 0.54, 1.36, and 4.74 msec, with relative areas of 0.25, 0.57, and 0.18, respectively (Fig. 3D). The time constants and their fractional contributions were similar to our previous reports for α1β3γ2L receptors (Fisher and Macdonald, 1997; Haas and Macdonald, 1999), indicating that the mutation caused a clear shift toward higher efficacy gating.
Because α1β3δ (L9'S) GABAA receptors showed high-efficacy gating (like α1β3γ2L receptors) but unaltered macroscopic desensitization (like α1β3δ receptors), this construct provided an ideal tool for dissecting the potential roles of gating efficacy and desensitization in THDOC modulation. If THDOC enhancement was somehow limited by receptor desensitization (or an associated process), then the minimally desensitizing α1β3δ (L9'S) receptors should still be robustly enhanced. However, if low-efficacy (α1β3δ-like) gating was a prerequisite for enhancement, then THDOC effects on peak current should be reduced by the L9'S mutation because of its high efficacy (α1β3γ2L-like) gating. We found that THDOC enhancement of α1β3δ (L9'S) receptors was reduced more than fivefold compared with wild-type α1β3δ receptors (262 ± 55%; n = 5) (Fig. 3E,G), consistent with the importance of low-efficacy gating (not minimal desensitization) for neurosteroid modulation. No additional enhancement was observed with increased THDOC concentration (3 μm; data not shown). Interestingly, despite the attenuated enhancement of peak current, the extent of desensitization was increased to 50.3 ± 2.8%, indistinguishable from the effect of THDOC on α1β3δ receptor current desensitization (Fig. 3H). Because it was possible that the decreased THDOC enhancement was because of a structural requirement for the conserved 9′ leucine (and not because of the higher efficacy gating of the L9'S mutation), we studied an additional mutation at that site, α1β3δ (L9′F). THDOC enhancement of this isoform was robust and indistinguishable from enhancement of α1β3δ receptors (Fig. 3F,G) (1043 ± 81%; n = 5). Although we have not investigated the single-channel gating characteristics of receptor channels with the δ (L9′F) mutation, the unaltered deactivation rate argued against an increase in gating efficacy for this mutant (Table 1). In any case, the clear enhancement of α1β3δ (L9′F) receptor currents by THDOC excluded a nonspecific requirement for the 9′ leucine as an explanation for the α1β3δ (L9'S) results. Desensitization of α1β3δ (L9′F) receptor currents was not significantly increased in the presence of THDOC (Fig. 3H) (12.9 ± 7.5% with GABA alone; 19.1 ± 4.3% with THDOC), in contrast to the effect of THDOC on α1β3δ and α1β3δ (L9'S) receptor currents. We also used P4S (1 mm) to evoke currents from the high-efficacy α1β3δ (L9'S) receptors and observed partial agonism with maximal currents that were ∼50% smaller than those evoked by GABA (1 mm) for that isoform. Accordingly, THDOC (1 μm) enhancement of P4S-evoked currents using α1β3δ (L9'S) receptors was approximately twice the enhancement observed with currents evoked by 1 mm GABA (data not shown).
Decreased neurosteroid enhancement of α1β3δ receptor currents evoked by β-alanine
Despite the clear reduction in THDOC enhancement of α1β3δ (L9'S) receptor currents, we endeavored to alter the gating efficacy of α1β3δ receptors without introducing any mutations. Two recent studies (Adkins et al., 2001; Brown et al., 2002) demonstrated that the synthetic GABA analog tetrahydroisoxazolopyridinol (THIP) evoked maximal responses that were ∼70% larger than those evoked by saturating GABA for α4β3δ receptors (but not α4β3γ2 receptors), suggesting that GABA might not be a full agonist at αβδ receptors. Although we observed similar results with THIP on α1β3δ receptors (data not shown), we found even greater “superagonism” using the endogenous amino acid β-alanine. β-alanine did not show increased efficacy compared with GABA at α1β3γ2L receptors (data not shown). The β-alanine concentration-response relationship for α1β3δ receptors is shown in Figure 4A, normalized to the maximum GABA response (evoked by 1 mm GABA) in each cell. Clearly a higher efficacy α1β3δ receptor gating pattern was accessible with β-alanine than with GABA, because high concentrations (200 mm) of β-alanine evoked currents that were over 600% of the maximal GABA-evoked current amplitude (n = 3). Although these concentrations were much higher than are likely to occur in the brain, β-alanine provided an additional tool to test the hypothesis that THDOC preferentially enhanced GABAA receptor currents under conditions that favored low-efficacy gating. First, we confirmed that THDOC robustly enhanced α1β3δ receptor currents evoked by low-concentration β-alanine (presumed to be low-efficacy, on the basis of similar macroscopic current amplitude and desensitization to currents evoked by 1 mm GABA). Indeed, currents evoked by 2 mm β-alanine were enhanced ∼2000% by THDOC (Fig. 4B). Our prediction was that currents evoked by a higher concentration of β-alanine (a condition of high-efficacy gating, on the basis of peak currents, relative to currents evoked by GABA) would be less sensitive to THDOC modulation. THDOC enhancement of α1β3δ currents evoked by 50 mm β-alanine was significantly decreased (393.5 ± 21.8%; n = 4), consistent with attenuated THDOC modulation of high-efficacy GABAA receptor currents. The extent of desensitization was increased from 14.5 ± 3.8 to 59.0 ± 8.0% by THDOC, similar to the effect of THDOC on GABA-evoked currents.
Are allosteric shifts in α1β3δ receptor gating efficacy limited to neurosteroids?
To determine whether gating efficacy might be a general target for GABAA receptor modulation, we investigated the effects of an additional compound, mefenamic acid, a nonsteroidal anti-inflammatory drug known to modulate GABAA receptor currents (Halliwell et al., 1999). Mefenamic acid (MFA) (30 μm) enhanced maximal GABA-evoked α1β3δ receptor currents by more than 1000%, similar to the effects of THDOC. In contrast, pre-applied MFA had little effect on maximal GABA-evoked currents for α1β3γ2L receptors. Although these results were consistent with MFA targeting reluctant GABAA receptors, additional evidence for modulation of gating efficacy would be to restore MFA enhancement of α1β3γ2L receptor currents under a condition of low-gating efficacy. Therefore, we evoked α1β3γ2L receptor currents with a low concentration of GABA, known to elicit small macroscopic currents and primarily brief-duration (low-efficacy), single-channel openings (Fisher and Macdonald, 1997). MFA markedly enhanced these currents (Fig. 5C), consistent with the idea that shifts in gating efficacy may be a general mechanism for regulation of GABAA receptor function.
THDOC enhancement of GABAA receptor currents evoked by the endogenous partial agonist taurine
Taurine is an endogenous amino acid that has partial agonist activity at GABAA receptors in addition to agonist activity at glycine receptors. Its basal extracellular concentration is thought to be ∼10-20 μm (Lerma et al., 1986). We investigated whether THDOC could also enhance the small currents evoked by taurine for α1β3δ and α1β3γ2L receptors. α1β3γ2L receptor currents evoked by a saturating concentration of taurine (20 mm) were markedly enhanced by THDOC (515 ± 104%; n = 4) (Fig. 6A,D). A maximal current evoked by GABA (1 mm) from the same cell is shown for comparison. The same protocol was applied to α1β3δ receptor currents, and they were markedly enhanced (2030 ± 440%; n = 4) (Fig. 6B,D). In contrast to α1β3γ2L receptors, the maximal THDOC-modulated α1β3δ receptor currents were clearly larger than the maximal GABA-evoked current in the same cells. Finally, we used lower concentrations of taurine (10 μm) and THDOC (100 nm) to more closely approximate physiological conditions under which THDOC modulation of GABAA receptors might occur (Paul and Purdy, 1992). Experiments were performed on high-affinity α6β3δ receptors, known to be expressed exclusively in extrasynaptic membrane in the cerebellum (Nusser et al., 1998), where they contribute to tonic forms of cerebellar inhibition (Rossi and Hamann, 1998; Hamann et al., 2002). Taurine evoked small amplitude currents from α6β3δ receptors (<20 pA) (Fig. 6C, left trace, D). In the presence of THDOC, taurine current amplitudes were enhanced 449 ± 36% (n = 4) (Fig. 6C, middle trace). The direct agonist action of 100 nm THDOC could be observed during the pre-application, consistent with our previous study indicating a high apparent affinity of this isoform for THDOC (Wohlfarth et al., 2002). For comparison, the response to 1 μm GABA was shown in the same cell to indicate that the currents evoked by taurine, even modulated by THDOC, were small compared with GABA-evoked currents.
It is worth noting that Hamann et al. (2002) suggested that α6βxδ receptors that appear to mediate tonic inhibition in the cerebellum are insensitive to neurosteroids, consistent with initial studies by Zhu et al. (1996) but in contrast to this and other studies (Adkins et al., 2001; Brown et al., 2002; Wohlfarth et al., 2002). One possible explanation for the results of Hamann et al. (2002) is the use of prolonged applications of THDOC (10-30 sec) in their neuronal preparation. We have shown that THDOC causes rapid and nearly complete desensitization of α6β3δ receptor currents within 6 sec (using 1 mm GABA and 1 μm THDOC), raising the possibility that extensive desensitization developed within the time course of their delivery of THDOC, off-setting potential enhancement. However, we cannot rule out the possibility that endogenous δ subunit-containing receptors behave differently than those expressed in recombinant systems (Cooper et al., 1999; Fancsik et al., 2000), possibly attributable to phosphorylation state or interactions with other membrane or cytoplasmic proteins.
Although many pharmacological agents exhibit subunit specificity for GABAA receptors (Macdonald and Olsen, 1994; Mehta and Ticku, 1999), the basis for this selectivity may, in some instances, be more complex than the presence or absence of the binding site(s). The neurosteroid THDOC caused a marked increase in the maximal GABA-evoked currents of α1β3δ receptors, accompanied by a shift in gating toward the high-efficacy bursting pattern observed with α1β3γ2L receptors (Wohlfarth et al., 2002). In contrast, α1β3γ2L receptor peak currents were not significantly modulated by THDOC when currents were evoked by saturating GABA but were enhanced when evoked by low GABA concentrations. This prompted us to investigate whether the observed selectivity of THDOC modulation depended on subunit composition or the distinct functional properties of each isoform. We exploited differences in gating efficacy that were dependent not only on receptor subunit composition (αβδ vs αβγ) but also on the type of agonist (partial vs full) used to activate the receptors. Our results suggested that THDOC (and perhaps other modulators) acted by producing a shift in channel activity from low-efficacy to high-efficacy gating patterns. The observations (from this and previous studies) that GABAA receptor efficacy is a function not only of subunit composition but also of the concentration and identity of the agonist used suggested that THDOC modulation can effectively “distinguish” receptor populations on the basis of subunit composition as well as on functional behavior within a given receptor population.
THDOC modulation: targeting GABAA receptor-gating activity independent of subunit composition
Much attention has focused on generation and characterization of subunit-specific GABAA receptor modulators. Allosteric modulation, on the basis of functional differences (such as gating efficacy or desensitization) rather than differences in primary structure per se, may represent an alternative basis for isoform-specific modulation. Although subunit composition can influence functional properties, channel behavior is also dependent on agonist identity, concentration, and allosteric modulation. Thus, GABAA receptor function is not uniquely specified by subunit composition, and targeting receptors on the basis of gating efficacy may represent a novel cross-section of isoforms. Also, neurosteroid modulation has the potential to regulate a given isoform differently, depending on its level of activation. The observations that both endogenous (THDOC) and exogenous (MFA) modulators are capable of scaling the efficacy of αβδ GABAA receptor function has important implications for the in vivo regulation of tonic inhibition as well as possible therapeutic targeting of this inhibition. Tonic forms of inhibition are likely to be mediated by low concentrations of GABA or other GABA-mimetics present in the extracellular space (Lerma et al., 1986). Because αβδ isoforms are thought to mediate tonic inhibition by sensing extrasynaptic neurotransmitter (Nusser et al., 1998), their low-efficacy gating may allow for a dynamic regulation of neuronal excitability through allosteric modulators. Increasing the concentration of GABA or taurine may be a relatively inefficient mechanism for increasing tonic inhibitory drive, in part because these agonists fail to fully activate αβδ isoforms even at very high concentrations. Shifting the gating efficacy of αβδ receptors through the release of endogenous modulators like THDOC may present an alternative mechanism for regulating inhibition.
Dissociating effects of THDOC on gating efficacy and desensitization
Whether the described correlation between gating efficacy and desensitization for α1β3δ and α1β3γ2L receptors was coincidental or represented a coupling of these processes was unknown. THDOC increased both α1β3δ receptor macroscopic desensitization and single-channel gating efficacy, consistent with the coupling of these processes (Wohlfarth et al., 2002). Also, THDOC increased the maximal amplitude and desensitization of P4S-evoked currents from α1β3γ2L receptors, consistent with this hypothesis. However, several other observations from this study were consistent with a dissociation of these processes. The δ (L9'S) mutation clearly increased gating efficacy without altering desensitization, suggesting that the two processes could be dissociated. Also, THDOC could alter peak amplitude and desensitization independently. Desensitization was increased despite relatively small enhancement of peak current for the δ (L9'S) mutation, whereas minimal desensitization was observed despite more than 10-fold increases in peak amplitude for the δ (L9′F) mutant and taurine-evoked currents from wild-type α1β3δ receptors. Evaluation of an isoform exhibiting fast desensitization but low-gating efficacy would support this proposed dissociation. Despite this evidence for independent modulation of gating and desensitization, additional work on this issue is necessary because it is apparent that macroscopic changes in desensitization may not necessarily reflect altered desensitized states per se (Bianchi and Macdonald, 2001).
Gating efficacy as a general target for allosteric modulation of ion channels
GABAA receptor gating efficacy appears to be specified by a combination of subunit composition, agonist identity, agonist concentration, and allosteric modulation. Allosteric control of gating efficacy is observed in many systems, suggesting a general mechanism for tuning channel-mediated electrical signaling. The more generalized phenomenon of modal gating has been described in several types of ligand- and voltage-gated channels, and gating patterns can be altered by subunit composition (Naranjo and Brehm, 1993; Fisher and Macdonald, 1997), mutation (Milone et al., 1998; Wang et al., 2000; Zhong et al., 2001), phosphorylation (Yue et al., 1990; Marrion, 1996), G-protein interaction (Delcour and Tsien, 1993), allosteric modulators (Hess et al., 1984; Twyman et al., 1989; Twyman and Macdonald, 1992; Wohlfarth et al., 2002), and other factors (Zhou et al., 1991; Marrion, 1993; Herlitze et al., 2001; Schonherr et al., 2002).
Allosteric modulation sets the gain of agonist-receptor interactions
It is often assumed that GABA is a full agonist at all GABAA receptor isoforms. Determination of full agonism is a relative one, requiring a comparison with other known agonists. The possibility that GABA is a partial agonist at αβδ isoforms was specifically suggested by the observation that THIP (Adkins et al., 2001; Brown et al., 2002) and β-alanine (this study) evoked currents that were larger than those evoked by GABA alone. The increased modulation αβδ receptors can be explained most simply if GABA is acting as a partial agonist, with larger observed enhancement attributable to the limited baseline level of activation. Consistent with this idea, the volatile anesthetic isofluorane and the nonbenzodiazepine anxiolytic tracazolate were shown to enhance maximal GABA-evoked currents of α1β1δ but not α1β1γ2 receptors (Lees and Edwards, 1998; Thompson et al., 2002). Enhancing the activity of partial agonists with allosteric modulators has been reported for GABAA and ATP-gated receptor channels (Kristiansen and Lambert, 1996; Khakh et al., 1999; Maksay et al., 2000; O'Shea et al., 2000).
Finally, the results suggest a potential mechanism for regulating extrasynaptic GABAA receptor currents evoked by partial agonists present in the extracellular space. The role of taurine in GABAA receptor function has remained elusive in part because of its weak agonism. If endogenous modulators such as neurosteroids augmented the response of native GABAA receptors to partial agonists, they may serve to reversibly regulate the gain of tonic inhibition. It is intriguing to consider the extreme case, in which a very weak partial agonist acted as a competitive antagonist capable of reversibly converting to full agonism by THDOC or other modulators. The role of tonic inhibition for CNS function has been the focus of several recent studies (Brickley et al., 1996, 2001; Bai et al., 2001; Hamann et al., 2002; Stell and Mody, 2002; Wu et al., 2003), but additional work is necessary to clarify the specific isoforms responsible, the physiological and pathophysiological relevance of this inhibition, and the roles of partial agonists and allosteric modulators in the regulation of tonic inhibition.
This work was supported by National Institutes of Health Grant R01-NS33300 (R.L.M.) and National Institute on Drug Abuse Training Fellowship T32-DA07281-03 (M.T.B.).
Correspondence should be addressed to Dr. Robert L. Macdonald, Department of Neurology, Vanderbilt University, 2100 Pierce Avenue, Nashville, TN 37212. E-mail:.
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