The Journal of Neuroscience, November 26, 2003, 23(34):10934-10943
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Cellular/Molecular
Neurosteroids Shift Partial Agonist Activation of GABAA Receptor Channels from Low- to High-Efficacy Gating Patterns
Matt T. Bianchi1 and
Robert L. Macdonald2
1Neuroscience Graduate Program, University of Michigan, Ann Arbor, Michigan 48104-1687, and 2Departments of Neurology, Molecular Physiology and Biophysics, and Pharmacology, Vanderbilt University, Nashville, Tennessee 37212
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Abstract
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Although GABA activates synaptic (
) GABAA receptors with high efficacy, partial agonist activation of isoforms and GABA activation of the primary extrasynaptic (
) GABAA receptors are limited to low-efficacy activity, characterized by minimal desensitization and brief openings. The unusual sensitivity of receptor channels to neurosteroid modulation prompted investigation of whether this high sensitivity was dependent on the subunit or the low-efficacy channel function that it confers. We show that the isoform specificity ( > ) of neurosteroid modulation could be reversed by conditions that reversed isoform-specific activity modes, including the use of -alanine to achieve increased efficacy with receptors and taurine to render 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.
Key words: GABAA receptor; neurosteroid; modal gating; desensitization; subunit; taurine; -alanine
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Introduction
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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 subunit is of particular importance for behavioral responses to neurosteroids (Mihalek et al., 1999), and the endogenous neurosteroid tetrahydrodeoxycorticosterone (THDOC) preferentially enhanced over 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 13 (but not 132L) receptors, suggesting that the low-efficacy activity of 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 subunit or the associated functional properties of minimal desensitization and low-efficacy gating.
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Materials and Methods
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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 1 and 3 subunits, together with either a , 2L, or mutated 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 M
; 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 M, 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 
ane (-t/
n), where n is the best number of exponential components, a is the relative amplitude of the component, t is time, and 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 · f + as · s) 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.
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Results
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THDOC preferentially enhanced 13 over 132L GABAA receptor currents
The neurosteroid THDOC has been shown to preferentially enhance GABAA receptors containing the subunit over those containing the 2L 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 13 and 132L receptors expressed in HEK293T cells (see Materials and Methods). 132L 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). 13 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 132L 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 13 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 132L receptor currents evoked by saturating GABA (1 mM). However, neurosteroids are well known to enhance submaximal 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). 132L receptor single-channel currents are known to exhibit higher efficacy gating patterns (longer open times in long complex bursts) than 13 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, 132L 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 132L receptors but also the apparent isoform preference of 13 over 132L receptors. We tested this hypothesis in the following sections.
Increased THDOC enhancement of 132L receptor currents evoked by a partial agonist
One complication of using low concentrations of GABA to favor a low-efficacy 132L 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 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 132L receptors with low concentrations of GABA and also to those observed with 13 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 132L receptors. Figure 2A shows the response of 132L 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 132L receptors, similar to the effects of modulators on GABAA receptor partial agonists shown previously (Maksay et al., 2000). Indeed, P4S-evoked 132L 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 13 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).
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Figure 2. 132L GABAA receptor currents evoked by the low-efficacy agonist P4S were enhanced by THDOC. A, Current traces recorded from the same cell expressing 132L GABAA receptors in response to 1 mM GABA (left) or 1 mM P4S, a low-efficacy GABAA receptor agonist (right). B, 132L receptor currents evoked by P4S (solid bar, left) were markedly enhanced by pre-applied THDOC (hatched bar, right). The response to GABA alone (1 mM) in the same cell is scaled and overlaid (gray) with the P4S plus THDOC current for comparison. The first 200 msec (indicated by the dotted oval) was expanded in the inset. C, Comparison of currents evoked by GABA, P4S, and P4S plus THDOC. For each cell, the P4S amplitude was <20% of the GABA-evoked current (left bar), the P4S plus THDOC amplitude was 600% of the P4S amplitude (middle bar), and the P4S plus THDOC amplitude was not different from the GABA alone amplitude (right bar). The dotted line indicates 100%. D, The extent of desensitization is shown for currents evoked by GABA alone (G), P4S alone, and P4S plus THDOC (T). Asterisk indicates significant difference from GABA alone and GABA plus THDOC (p < 0.0001).
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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 13 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 13 receptors, because it evoked smaller currents than GABA (data not shown), similar to a recent report using 43 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 132L 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 13 receptor currents (and P4S-evoked 132L 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 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 13 (Fig. 3A) or 13 (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 13 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 13 (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 132L receptors (Fisher and Macdonald, 1997; Haas and Macdonald, 1999), indicating that the mutation caused a clear shift toward higher efficacy gating.
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Figure 3. 13 (L9'S) GABAA receptors exhibited increased gating efficacy. A, B, Concentration-jump experiments (400 msec) performed on outside-out patches demonstrated minimal desensitization in response to GABA (1 mM) for 13 (A) and 13 (L9'S) (B) isoforms. C, Representative single-channel records are presented from 13 (top trace) and 13 (L9'S) receptors. A portion of the middle trace, indicated by the open bar, is expanded in the bottom trace. D, Event histogram of 13 (L9'S) receptor single-channel openings evoked by steady state application of GABA (1 mM). Data were pooled from two patches. The distribution was best described by the sum of three exponential functions. The individual fitted functions, as well as their sum, are shown as curves on the plot. See Results for time constants and relative areas. E, F, Currents evoked by GABA (1 mM; solid bar) applied to cells expressing 13 (L9'S) (E) and 13 (L9'F) (F) in the absence (left traces) and presence (right traces) of THDOC (1 µM; hatched bar). G, Extent of desensitization (percentage) for 6 sec applications of GABA in the absence (solid bars) or presence of pre-applied THDOC (1 µM; hatched bars).
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Because 13 (L9'S) GABAA receptors showed high-efficacy gating (like 132L receptors) but unaltered macroscopic desensitization (like 13 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 13 (L9'S) receptors should still be robustly enhanced. However, if low-efficacy (13-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 (132L-like) gating. We found that THDOC enhancement of 13 (L9'S) receptors was reduced more than fivefold compared with wild-type 13 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 13 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, 13 (L9'F). THDOC enhancement of this isoform was robust and indistinguishable from enhancement of 13 receptors (Fig. 3F,G) (1043 ± 81%; n = 5). Although we have not investigated the single-channel gating characteristics of receptor channels with the (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 13 (L9'F) receptor currents by THDOC excluded a nonspecific requirement for the 9' leucine as an explanation for the 13 (L9'S) results. Desensitization of 13 (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 13 and 13 (L9'S) receptor currents. We also used P4S (1 mM) to evoke currents from the high-efficacy 13 (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 13 (L9'S) receptors was approximately twice the enhancement observed with currents evoked by 1 mM GABA (data not shown).
Decreased neurosteroid enhancement of 13 receptor currents evoked by -alanine
Despite the clear reduction in THDOC enhancement of 13 (L9'S) receptor currents, we endeavored to alter the gating efficacy of 13 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 43 receptors (but not 432 receptors), suggesting that GABA might not be a full agonist at receptors. Although we observed similar results with THIP on 13 receptors (data not shown), we found even greater "superagonism" using the endogenous amino acid -alanine. -alanine did not show increased efficacy compared with GABA at 132L receptors (data not shown). The -alanine concentration-response relationship for 13 receptors is shown in Figure 4A, normalized to the maximum GABA response (evoked by 1 mM GABA) in each cell. Clearly a higher efficacy 13 receptor gating pattern was accessible with -alanine than with GABA, because high concentrations (200 mM) of -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, -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 13 receptor currents evoked by low-concentration -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 -alanine were enhanced 2000% by THDOC (Fig. 4B). Our prediction was that currents evoked by a higher concentration of -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 13 currents evoked by 50 mM -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 13 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 13 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 132L 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 132L receptor currents under a condition of low-gating efficacy. Therefore, we evoked 132L 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 13 and 132L receptors. 132L 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 13 receptor currents, and they were markedly enhanced (2030 ± 440%; n = 4) (Fig. 6B,D). In contrast to 132L receptors, the maximal THDOC-modulated 13 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 63 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 63 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.
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Figure 6. THDOC enhancement of GABAA receptor currents evoked by the endogenous partial agonist taurine. A, 132L receptor current evoked by 20 mM taurine alone (solid bar, left trace) or with pre-applied 1 µM THDOC (hatched bar, middle trace). The maximal GABA-evoked current is shown in the same cell for comparison (1 mM; solid bar, right trace). Scale bars apply to all three traces, and the time bar applies to other panels (except C, right trace). B, 13 receptor current evoked by 20 mM taurine alone (solid bar, left trace) or with pre-applied 1 µM THDOC (hatched bar, middle trace). The maximal current evoked by 1 mM GABA is shown from the same cell (solid bar, right trace). C, 63 receptor current evoked by 10 µM taurine alone (solid bar, left trace) or with pre-applied 100 nM THDOC (hatched bar, middle trace). The response to 1 µM GABA from the same cell is shown (right trace; note the different horizontal and vertical scale bars). The dotted line (left trace) is a baseline reference for the small taurine-evoked current. D, Summary plot indicating peak current enhancement by THDOC for the various conditions (n = 4 cells for each).
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It is worth noting that Hamann et al. (2002) suggested that 6x 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 63 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 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.
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Discussion
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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 13 receptors, accompanied by a shift in gating toward the high-efficacy bursting pattern observed with 132L receptors (Wohlfarth et al., 2002). In contrast, 132L 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 ( vs ) 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 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 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 isoforms even at very high concentrations. Shifting the gating efficacy of 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 13 and 132L receptors was coincidental or represented a coupling of these processes was unknown. THDOC increased both 13 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 132L receptors, consistent with this hypothesis. However, several other observations from this study were consistent with a dissociation of these processes. The (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 (L9'S) mutation, whereas minimal desensitization was observed despite more than 10-fold increases in peak amplitude for the (L9'F) mutant and taurine-evoked currents from wild-type 13 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 isoforms was specifically suggested by the observation that THIP (Adkins et al., 2001; Brown et al., 2002) and -alanine (this study) evoked currents that were larger than those evoked by GABA alone. The increased modulation 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 11 but not 112 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.
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Footnotes
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Received July 23, 2003;
revised September 12, 2003;
accepted September 22, 2003.
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: Robert.Macdonald{at}vanderbilt.edu.
Copyright © 2003 Society for Neuroscience 0270-6474/03/2310934-10$15.00/0
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References
|
|---|
Adkins CE, Pillai GV, Kerby J, Bonnert TP, Haldon C, McKernan RM, Gonzalez JE, Oades K, Whiting PJ, Simpson PB (2001) 43 GABAA receptors characterized by fluorescence resonance energy transfer-derived measurements of membrane potential. J Biol Chem 276: 38934-38939.[Abstract/Free Full Text]
Angelotti TP, Uhler MD, Macdonald RL (1993) Assembly of GABAA receptor subunits: analysis of transient single-cell expression utilizing a fluorescent substrate/marker gene technique. J Neurosci 13: 1418-1428.[Abstract]
Bai D, Zhu G, Pennefather P, Jackson MF, MacDonald JF, Orser BA (2001) Distinct functional and pharmacological properties of tonic and quantal inhibitory postsynaptic currents mediated by gamma-aminobutyric acid(A) receptors in hippocampal neurons. Mol Pharmacol 59: 814-824.[Abstract/Free Full Text]
Bianchi MT, Macdonald RL (2001) Mutation of the 9' leucine in the GABAA receptor 2L subunit produces an apparent decrease in desensitization by stabilizing open states without altering desensitized states. Neuropharmacology 41: 737-744.[Web of Science][Medline]
Bianchi MT, Haas KF, Macdonald RL (2002) alpha1 and alpha6 subunits specify distinct desensitization, deactivation and neurosteroid modulation of GABA(A) receptors containing the delta subunit. Neuropharmacology 43: 492-502.[Web of Science][Medline]
Brickley SG, Cull-Candy SG, Farrant M (1996) Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors. J Physiol (Lond) 497: 753-759.[Abstract/Free Full Text]
Brickley SG, Revilla V, Cull-Candy SG, Wisden W, Farrant M (2001) Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance. Nature 409: 88-92.[Medline]
Brown N, Kerby J, Bonnert TP, Whiting PJ, Wafford KA (2002) Pharmacological characterization of a novel cell line expressing human alpha(4)beta(3)delta GABA(A) receptors. Br J Pharmacol 136: 965-974.[Web of Science][Medline]
Cooper EJ, Johnston GAR, Edwards FA (1999) Effects of naturally occurring neurosteroid on GABAA IPSCs during development in rat hippocampal or cerebellar slices. J Physiol (Lond) 521: 437-449.[Abstract/Free Full Text]
Delcour AH, Tsien RW (1993) Altered prevalence of gating modes in neurotransmitter inhibition of N-type calcium channels. Science 259: 980-984.[Abstract]
Ebert B, Thompson SA, Saounatsou K, McKernan R, Krogsgaard-Larsen P, Wafford KA (1997) Differences in agonist/antagonist binding affinity and receptor transduction using recombinant human gamma-aminobutyric acid type A receptors. Mol Pharmacol 52: 1150-1156.[Abstract/Free Full Text]
Fancsik A, Linn DM, Tasker JG (2000) Neurosteroid modulation of GABA IPSCs is phosphorylation dependent. J Neurosci 20: 3067-3075.[Abstract/Free Full Text]
Filatov GN, White MW (1995) The role of conserved leucines in the M2 domain of the acetylcholine receptor in channel gating. Mol Pharmacol 48: 379-384.[Abstract]
Fisher JL, Macdonald RL (1997) Single channel properties of GABAA receptors containing 2 or subtypes expressed with 1 and 3 subtypes in L929 cells. J Physiol (Lond) 505: 283-297.[Abstract/Free Full Text]
Greenfield Jr LJ, Sun F, Neelands TR, Burgard EC, Donnelly JL, Macdonald RL (1997) Expression of functional GABAA receptors in transfected L929 cells isolated by immunomagnetic bead separation. Neuropharmacology 36: 63-73.[Web of Science][Medline]
Haas KF, Macdonald RL (1999) GABAA receptor subunit 2 and subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts. J Physiol (Lond) 514: 27-45.[Abstract/Free Full Text]
Halliwell RF, Thomas P, Patten D, James CH, Martinez-Torres A, Miledi R, Smart TG (1999) Subunit-selective modulation of GABAA receptors by the non-steroidal anti-inflammatory agent, mefenamic acid. Eur J Neurosci 11: 2897-2905.[Web of Science][Medline]
Hamann M, Rossi DJ, Attwell D (2002) Tonic and spillover inhibition of granule cells control information flow through cerebellar cortex. Neuron 33: 625-633.[Web of Science][Medline]
Harrison NL, Vicini S, Barker JL (1987) A steroid anesthetic prolongs inhibitory postsynaptic currents in cultured rat hippocampal neurons. J Neurosci 7: 604-609.[Abstract]
Herlitze S, Zhong H, Scheuer T, Catterall WA (2001) Allosteric modulation of Ca2+ channels by G proteins, voltage-dependent facilitation, protein kinase C, and Ca(v)beta subunits. Proc Natl Acad Sci USA 98: 4699-4704.[Abstract/Free Full Text]
Hess P, Lansman JB, Tsien RW (1984) Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists. Nature 311: 538-544.[Medline]
Huxtable RJ (1989) Taurine in the central nervous system and the mammalian actions of taurine. Prog Neurobiol 32: 471-533.[Web of Science][Medline]
Khakh BS, Proctor WR, Dunwiddie TV, Labarca C, Lester HA (1999) Allosteric control of gating and kinetics at P2X(4) receptor channels. J Neurosci 19: 7289-7299.[Abstract/Free Full Text]
Kristiansen U, Lambert JD (1996) Benzodiazepine and barbiturate ligands modulate responses of cultured hippocampal neurones to the GABAA receptor partial agonist, 4-PIOL. Neuropharmacology 35: 1181-1191.[Web of Science][Medline]
Krogsgaard-Larsen P, Falch E, Schousboe A, Curtis DR, Lodge D (1980) Piperidine-4-sulphonic acid, a new specific GABA agonist. J Neurochem 34: 756-759.[Web of Science][Medline]
Krogsgaard-Larsen P, Snowman A, Lummis SC, Olsen RW (1981) Characterization of the binding of the GABA agonist [3H]piperidine-4-sulphonic acid to bovine brain synaptic membranes. J Neurochem 37: 401-409.[Web of Science][Medline]
Labarca C, Nowak MW, Zhang H, Tang L, Deshpande P, Lester HA (1995) Channel gating governed symmetrically by conserved leucine residues in the M2 domain of nicotinic receptors. Nature 376: 514-516.[Medline]
Lambert JJ, Belelli D, Hill-Venning C, Peters JA (1995) Neurosteroids and GABAA receptor function. Trends Pharmacol Sci 16: 295-303.[Medline]
Lan NC, Gee KW, Bolger MB, Chen JS (1991) Differential responses of expressed recombinant human -aminobutyric acidA receptors to neurosteroids. J Neurochem 57: 1818-1821.[Web of Science][Medline]
Le Foll F, Castel H, Louiset E, Vaudry H, Cazin L (1997) Multiple modulatory effects of the neuroactive steroid pregnanolone on GABAA receptor in frog pituitary melanotrophs. J Physiol (Lond) 504: 387-400.[Abstract/Free Full Text]
Lees G, Edwards MD (1998) Modulation of recombination human gamma-aminobutyric acidA receptors by isoflurane: influence of the delta subunit. Anesthesiology 88: 206-217.[Web of Science][Medline]
Leidenheimer NJ, Chapell R (1997) Effects of PKC activation and receptor desensitization on neurosteroid modulation of GABA(A) receptors. Brain Res Mol Brain Res 52: 173-181.[Medline]
Lerma J, Herranz AS, Herreras O, Abraira V, Martin DR (1986) In vivo determination of extracellular concentration of amino acids in the rat hippocampus. A method based on brain dialysis and computerized analysis. Brain Res 384: 145-155.[Web of Science][Medline]
Low K, Crestani F, Keist R, Benke D, Brunig I, Benson JA, Fritschy JM, Rulicke T, Bluethmann H, Mohler H, Rudolph U (2000) Molecular and neuronal substrate for the selective attenuation of anxiety. Science 290: 131-134.[Abstract/Free Full Text]
Macdonald RL, Olsen RW (1994) GABAA receptor channels. Annu Rev Neurosci 17: 569-602.[Web of Science][Medline]
Maksay G, Thompson SA, Wafford KA (2000) Allosteric modulators affect the efficacy of partial agonists for recombinant GABA(A) receptors. Br J Pharmacol 129: 1794-1800.[Web of Science][Medline]
Marrion NV (1993) Selective reduction of one mode of M-channel gating by muscarine in sympathetic neurons. Neuron 11: 77-84.[Web of Science][Medline]
Marrion NV (1996) Calcineurin regulates M channel modal gating in sympathetic neurons. Neuron 16: 163-173.[Web of Science][Medline]
McKernan RM, Rosahl TW, Reynolds DS, Sur C, Wafford KA, Atack JR, Farrar S, Myers J, Cook G, Ferris P, Garrett L, Bristow L, Marshall G, Macaulay A, Brown N, Howell O, Moore KW, Carling RW, Street LJ, Castro JL, et al. (2000) Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype. Nat Neurosci 3: 587-592.[Web of Science][Medline]
Mehta AK, Ticku MK (1999) An update on GABAA receptors. Brain Res Brain Res Rev 29: 196-217.[Medline]
Mellon SH, Griffin LD (2002) Neurosteroids: biochemistry and clinical significance. Trends Endocrinol Metab 13: 35-43.[Web of Science][Medline]
Mihalek RM, Banerjee PK, Korpi ER, Quinlan JJ, Firestone LL, Mi ZP, Lagenaur C, Tretter V, Sieghart W, Anagnostaras G, Sage JR, Fanselow MS, Guidotti A, Spigelman I, Li Z, DeLorey TM, Olsen RW, Homanics GE (1999) Attenuated sensitivity to neuroactive steroids in -aminobutyrate type A receptor delta subunit knock-out mice. Proc Natl Acad Sci USA 96: 12905-12910.[Abstract/Free Full Text]
Milone M, Wang HL, Ohno K, Prince R, Fukudome T, Shen XM, Brengman JM, Griggs RC, Sine SM, Engel AG (1998) Mode switching kinetics produced by a naturally occurring mutation in the cytoplasmic loop of the human acetylcholine receptor epsilon subunit. Neuron 20: 575-588.[Web of Science][Medline]
Mistry DK, Cottrell GA (1990) Actions of steroids and bemegride on the GABAA receptor of mouse spinal neurones in culture. Exp Physiology 75: 199-209.[Abstract]
Naranjo D, Brehm P (1993) Modal shifts in acetylcholine receptor channel gating confer subunit-dependent desensitization. Science 260: 1811-1814.[Abstract/Free Full Text]
Nusser Z, Sieghart W, Somogyi P (1998) Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. J Neurosci 18: 1693-1703.[Abstract/Free Full Text]
Olsen RW, Macdonald RL (2002) GABAA receptor complex: structure and function. In: Glutamate and GABA receptors and transporters: structure, function, and pharmacology, Sec 9 (Egebjerg J, Schousboe A, Krogsgaard-Larsen P, eds), pp 203-235. London: Taylor and Francis.
O'Shea SM, Wong LC, Harrison NL (2000) Propofol increases agonist efficacy at the GABA(A) receptor. Brain Res 852: 344-348.[Web of Science][Medline]
Paul SM, Purdy RH (1992) Neuroactive steroids. FASEB J 6: 2311-2322.[Abstract]
Puia G, Santi MR, Vicini S, Pritchett DB, Purdy RH, Paul SM, Seeburg PH, Costa E (1990) Neurosteroids act on recombinant human GABAA receptor. Neuron 4: 759-765.[Web of Science][Medline]
Rossi DJ, Hamann M (1998) Spillover-mediated transmission at inhibitory synapses promoted by high affinity alpha6 subunit GABA(A) receptors and glomerular geometry. Neuron 20: 783-795.[Web of Science][Medline]
Rudolph U, Crestani F, Benke D, Brunig I, Benson JA, Fritschy JM, Martin JR, Bluethmann H, Mohler H (1999) Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes. Nature 401: 796-800.[Medline]
Sakai Y, Okamoto K, Kimura H (1985) Pharmacological evidence for taurine as an inhibitory neurotransmitter in the cerebellum. Prog Clin Biol Res 179: 313-319.[Medline]
Schonherr R, Mannuzzu L, Isacoff E, Heinemann S (2002) Conformational switch between slow and fast gating modes. Allosteric regulation of voltage sensor mobility in the EAG K(+) channel. Neuron 35: 935.[Web of Science][Medline]
Sigel E, Buhr A (1997) The benzodiazepine binding site of GABAA receptors. Trends Pharmacol 18: 425-429.[Medline]
Steinbach JH, Akk G (2001) Modulation of GABA(A) receptor channel gating by pentobarbital. J Physiol (Lond) 537: 715-733.[Abstract/Free Full Text]
Stell BM, Mody I (2002) Receptors with different affinities mediate phasic and tonic GABA(A) conductances in hippocampal neurons. J Neurosci 22: RC223.[Abstract/Free Full Text]
Stevenson A, Wingrove PB, Whiting PJ, Wafford KA (1995) beta-Carboline gamma-aminobutyric acidA receptor inverse agonists modulate gamma-aminobutyric acid via the loreclezole binding site as well as the benzodiazepine site. Mol Pharmacol 48: 965-969.[Abstract]
Thompson SA, Arden SA, Marshall G, Wingrove PB, Whiting PJ, Wafford KA (1999) Residues in transmembrane domains I and II determine gamma-aminobutyric acid type A receptor subtype-selective antagonism by furosemide. Mol Pharmacol 55: 993-999.[Abstract/Free Full Text]
Thompson SA, Wingrove PB, Connelly L, Whiting PJ, Wafford KA (2002) Tracazolate reveals a novel type of allosteric interaction with recombinant gamma-aminobutyric acid(A) receptors. Mol Pharmacol 61: 861-869.[Abstract/Free Full Text]
Twyman RE, Macdonald RL (1992) Neurosteroid regulation of GABAA receptor single-channel kinetic properties of mouse spinal cord neurons in culture. J Physiol (Lond) 456: 215-245.[Abstract/Free Full Text]
Twyman RE, Rogers CJ, Macdonald RL (1989) Pentobarbital and picrotoxin have reciprocal actions on single GABAA receptor channels. Neurosci Lett 96: 89-95.[Web of Science][Medline]
Wang HL, Ohno K, Milone M, Brengman JM, Evoli A, Batocchi AP, Middleton LT, Christodoulou K, Engel AG, Sine SM (2000) Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome. J Gen Physiol 116: 449-462.[Abstract/Free Full Text]
Wingrove PB, Wafford KA, Bain C, Whiting PJ (1994) The modulatory action of loreclezole at the gamma-aminobutyric acid type A receptor is determined by a single amino acid in the beta 2 and beta 3 subunit. Proc Natl Acad Sci USA 91: 4569-4573.[Abstract/Free Full Text]
Wohlfarth KM, Bianchi MT, Macdonald RL (2002) Enhanced neurosteroid potentiation of ternary GABAA receptors containing the subunit. J Neurosci 22: 1541-1549.[Abstract/Free Full Text]
Wu Y, Wang W, Richerson GB (2003) Vigabatrin induces tonic inhibition via GABA transporter reversal without increasing vesicular GABA release. J Neurophysiol 89: 2021-2034.[Abstract/Free Full Text]
Yue DT, Herzig S, Marban E (1990) Beta-adrenergic stimulation of calcium channels occurs by potentiation of high-activity gating modes. Proc Natl Acad Sci USA 87: 753-757.[Abstract/Free Full Text]
Zhong H, Li B, Scheuer T, Catterall WA (2001) Control of gating mode by a single amino acid residue in transmembrane segment IS3 of the N-type Ca2+ channel. Proc Natl Acad Sci USA 98: 4705-4709.[Abstract/Free Full Text]
Zhou JY, Potts JF, Trimmer JS, Agnew WS, Sigworth FJ (1991) Multiple gating modes and the effect of modulating factors on the microI sodium channel. Neuron 7: 775-785.[Web of Science][Medline]
Zhu WJ, Vicini S (1997) Neurosteroid prolongs GABAA channel deactivation by altering kinetics of desensitized states. J Neurosci 17: 4022-4031.[Abstract/Free Full Text]
Zhu WJ, Wang JF, Krueger KE, Vicini S (1996) Delta subunit neurosteroid modulation of GABAA receptors. J Neurosci 16: 6648-6656.[Abstract/Free Full Text]
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[Abstract]
[Full Text]
[PDF]
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J. Liang, E. Cagetti, R. W. Olsen, and I. Spigelman
Altered Pharmacology of Synaptic and Extrasynaptic GABAA Receptors on CA1 Hippocampal Neurons Is Consistent with Subunit Changes in a Model of Alcohol Withdrawal and Dependence
J. Pharmacol. Exp. Ther.,
September 1, 2004;
310(3):
1234 - 1245.
[Abstract]
[Full Text]
[PDF]
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G. Akk, J. R. Bracamontes, D. F. Covey, A. Evers, T. Dao, and J. H. Steinbach
Neuroactive steroids have multiple actions to potentiate GABAA receptors
J. Physiol.,
July 1, 2004;
558(1):
59 - 74.
[Abstract]
[Full Text]
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C. Leroy, P. Poisbeau, A. F. Keller, and A. Nehlig
Pharmacological plasticity of GABAA receptors at dentate gyrus synapses in a rat model of temporal lobe epilepsy
J. Physiol.,
June 1, 2004;
557(2):
473 - 487.
[Abstract]
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D. Rusch, H. Zhong, and S. A. Forman
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J. Biol. Chem.,
May 14, 2004;
279(20):
20982 - 20992.
[Abstract]
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Top
Abstract
Introduction
