J Physiol Volume 578, Number 3, 655-676, February 1, 2007 DOI: 10.1113/jphysiol.2006.122135
Enhanced macroscopic desensitization shapes the response of 
4 subtype-containing GABAA receptors to synaptic and extrasynaptic GABA
Andre H. Lagrange1,
Emmanuel J. Botzolakis4 and
Robert L. Macdonald1,2,3
Departments of
1 Neurology
2 Molecular Physiology & Biophysics
3 Pharmacology
4 Program in Neuroscience,Vanderbilt University, Nashville, TN 37212, USA
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Abstract
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Up-regulation of the GABAA receptor 
4 subunit subtype has been consistently shown in multiple animal models of chronic epilepsy. This isoform is expressed in both thalamus and hippocampus and is likely to play a significant role in regulating corticothalamic and hippocampal rhythms. However, little is known about its physiological properties, thus limiting understanding of the role of 4 subtype-containing GABAA receptors in normal and abnormal physiology. We used rapid GABA application to recombinant GABAA receptors expressed in HEK293T cells to compare the macroscopic kinetic properties of 4
3
2L receptors to those of the more widely distributed 132L receptors. These receptor currents had similar peak current amplitudes and GABA EC50 values. However, 432L currents activated more slowly when exposed to submaximal GABA concentrations, had more fast desensitization (
= 15–100 ms), and had less residual current during long GABA applications. In addition, 432L currents deactivated more slowly than 132L currents. Peak currents evoked by repetitive, brief GABA applications were more strongly attenuated for 432L currents than 132L currents. Moreover, the time required to recover from desensitization was prolonged in 432L currents compared to 132L currents. We also found that exposure to prolonged low levels of GABA, similar to those that might be present in the extrasynaptic space, greatly suppressed the response of 432L currents to higher concentrations of GABA, while 132L currents were less affected by exposure to low levels of GABA. Taken together, these data suggest that 432L receptors have unique kinetic properties that limit the range of GABA applications to which they can respond maximally. While similar to 132L receptors in their ability to respond to brief and low frequency synaptic inputs, 432L receptors are less efficacious when exposed to prolonged tonic GABA or during repetitive stimulation, as may occur during learning and seizures.
(Received 6 October 2006;
accepted after revision 22 November 2006;
first published online 23 November 2006)
Corresponding author A. Lagrange: Vanderbilt University Medical Centre, 6140 Medical Research Building III, 465 21st Ave, South, Nashville, TN 37232-8552, USA. Email: andre.h.lagrange{at}vanderbilt.edu
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Introduction
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GABAA receptors are pentameric cys-loop receptors composed primarily of two subunits, two subunits, and either a 
or a subunit selected from six , three , one , and three subunit subtypes. The distribution of specific subtypes is highly brain region and cell type specific, and varies during development and in certain disease states. The presence of a specific subunit subtype confers different pharmacological and physiological properties to receptor isoforms. For example, subtypes strongly influence GABAA receptor pharmacology. When assembled with and subunits, GABAA receptors containing 1, 2, 3 or 5 subtypes are highly diazepam sensitive. However, 1 subtype-containing receptors are much more sensitive to zolpidem than receptors containing 2 or 3 subtypes, and those containing 5 subtypes are completely insensitive to this drug. In contrast, GABAA receptors containing 4 or 6 subtypes are insensitive to both diazepam and zolpidem. Furthermore, the imidazobenzodiazepine Ro 15-4513, which is an inverse benzodiazepine receptor agonist at GABAA receptors containing 1, 2, 3 or 5 subtypes, actually enhances currents from GABAA receptors containing 4 or 6 subtypes.
Unlike the pharmacological properties of GABAA receptors, relatively little is known about the kinetic properties of different subtypes. This limits our understanding of GABAA receptor physiology, as receptor kinetics play an important role in shaping the postsynaptic response to GABA. For example, in synapses, GABAergic inhibitory postsynaptic current (IPSC) time courses are shaped by the rates of activation, desensitization and deactivation. During IPSCs, GABAA receptor channels must activate rapidly and deactivate slowly to provide significant charge transfer during the very brief (
1 ms) pulses of GABA present in the synaptic cleft. GABAA receptor subtypes thought to be expressed in synapses are also often highly desensitizing, which may be linked to the slow deactivation that is crucial for effective synaptic neurotransmission (Jones & Westbrook, 1995). In contrast, extrasynaptic GABAA receptors should be highly sensitive during prolonged exposure to low levels of GABA. As long as they maintain a steady-state level of charge transfer, they need not be rapidly activating, highly desensitizing, or slowly deactivating. Between these two extreme examples, subsets of GABAA receptors may have different rates of activation, desensitization and deactivation, thereby allowing for maximal responses to specific frequencies, durations and concentrations of local GABA.
As a result, altered expression and distribution of certain GABAA receptor isoforms has the potential to profoundly affect inhibitory neurotransmission. Perturbed expression of GABAA receptors has been particularly well studied in epilepsy. Several animal models have consistently found an up-regulation of 4 subtype protein expression in animals with experimental epilepsy (Schwarzer et al. 1997; Sperk et al. 1998; Brooks-Kayal et al. 1998). Although the predominant GABAA receptor isoform in the central nervous system is the 122 isoform, and 4 receptors comprise only a small minority of all native GABAA receptors, the 4 subtype is relatively abundant in brain regions involved in both partial and generalized epilepsies, including cortex, hippocampus and thalamus (Pirker et al. 2000). Furthermore, this subtype may be involved in both synaptic and extrasynaptic neurotransmission, depending on the brain region and physiological state of the animal (Hsu et al. 2003; Belelli et al. 2005; Cope et al. 2005). Nonetheless, the role of increased 4 subtype expression in the pathophysiology of epilepsy remains unclear. Without knowing the kinetic properties of GABAA receptors containing the 4 subtype, it is difficult to predict whether increased expression of this GABAA receptor subtype causes epilepsy or is simply a compensatory response to seizures. Thus, in the present study we compared the physiological and pharmacological properties of recombinant 432 and 132 receptor isoforms using GABA application protocols that mimicked synaptic and extrasynaptic conditions.
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Methods
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Expression of recombinant GABAA receptors
GABAA receptor 1, 4, 3 and 2L subtype cDNAs were individually subcloned into the mammalian expression vector pCMV-neo. Deletion of an extraneous genomic sequence in the 5' untranslated region of the 4 subtype cDNA resulted in improved expression, as previously described (Wallner et al. 2003). All cDNAs were sequenced by the Vanderbilt University Medical Centre sequencing core to confirm that they matched the published sequences for mature rat peptides corresponding to accession numbers NP_899155, NP_542154, P63079 and NP_899156 for the 1, 4, 3 and 2 proteins, respectively.
Human embryonic kidney (HEK293T) cells were plated at a density of 200 000–400 000 cells per 60 mm culture dish and maintained in Dulbecco's modified Eagle's wedium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 100 IU ml–1 each of penicillin and streptomycin (Invitrogen) at 37°C in 5% CO2–95% O2. On day one, cells were transfected using a previously established calcium phosphate precipitation technique (Angelotti et al. 1993). A total of 12 µg GABAA receptor subunit-containing DNA, either with 4 µg of each subunit plasmid (ratio 1 : 1 : 1) for 32L receptors or with 6 µg of each subunit plasmid (ratio 1 : 1) for 3 receptors. Two micrograms of pHook-1 (Invitrogen) was also added so that immunomagnetic bead selection could be performed on day 2 (Greenfield et al. 1997). Following selection, the cells were plated on 35 mm dishes, and recordings were made on day 3, approximately 18–36 h after selection. The cells used for macropatches were treated using the same protocol but were plated on 35 mm dishes that had been previously collagenized.
Electrophysiological recording and drug application
Whole cell voltage-clamp recordings were performed on transfected HEK293T cells. All experiments were performed using at least two separate transfected batches of cells from at least two separate days of recording. Cells were bathed in an external solution consisting of (mM): NaCl 142, CaCl2 1, KCl 8, MgCl2 6, glucose 10, Hepes 10 (pH 7.4, 
320–340 mosmol l–1) throughout the duration of the experiment. All recordings were done at room temperature. Glass micropipettes were formed from thin-walled borosilicate glass with a filament (World Precision Instruments, Sarasota, FL, USA) with a P2000 laser electrode puller (Sutter Instruments, San Rafael, CA, USA) and fire polished with a microforge (Narishige, East Meadow, NY, USA). Microelectrodes used for lifted cell recording had resistances of 1–2 M
when filled with an internal solution consisting of (mM): KCl 153, MgCl2 1, Hepes 10, EGTA 5, Mg2+-ATP 2 (pH 7.3, 300–310 mosmol l–1). This combination of external and internal solutions produced a chloride equilibrium potential (ECl) of approximately 0 mV. Electrodes used for macropatch recording were fire polished to achieve resistances of 1.5–4 M when filled with the same internal solution.
Membrane voltages were clamped at –20 mV using an Axopatch 200A amplifier (Axon Instruments, Union City, CA, USA) amplifier. GABA was applied to the lifted cells via a three-, four-, or six-barrelled square glass (Friedrich and Dimmock, Millville, NJ, USA). These multibarrelled pipettes were pulled on a P-87 Flaming-Brown (Sutter Instruments, San Rafael, CA, USA) electrode puller with custom made platinum–iridium filament and sanded to a final diameter of 200–400 µm for each barrel. The multibarrelled pipettes were attached to a Warner SF-77B Perfusion Fast-Step (Warner Instrument Corporation, Hamden, CT, USA), allowing for rapid solution changes. All GABA application protocols began with a cell positioned in the flow of external bath solution from which the multibarrelled array was repositioned such that the unmoved cell and electrode were now exposed to GABA. The drug application was initiated by an analog pulse triggered by the pCLAMP 9 software (Axon Instruments) that caused the motor of the Warner Fast-Step to reposition the multibarrelled array from one barrel to another (e.g. external solution to GABA). Exchange times were routinely measured and always found to be 0.3–0.7 ms at an open electrode tip by stepping from control to dilute external solution. However, all GABA concentrations > 1 mM were applied using barrels with exchange times < 0.45 ms. The exchange around an intact cell was measured in a subset of cells by stepping into 10 µM GABA and then another step into 10 µM GABA in external solution in which NaCl was replaced by NaSCN. The resulting current had a 10–90% rise time of 0.9 ± 0.1 ms (n
= 9) using a drug application pipette with 0.35 ms open tip exchange time.
For generation of concentration–response relationships, peak GABAA receptor currents evoked by randomly sequenced concentrations of GABA were recorded with at least 45 s of wash between each application. This time was empirically determined to be sufficient for complete recovery from desensitization. The preapplication concentration–response curves were generated by exposing the cells to 45 s of 1 µM GABA between briefer (1–4 s) applications of higher concentrations of GABA. To assess possible changes in the transmembrane chloride ion concentration gradient, pCLAMP 9 generated a 500 ms ramp voltage step from –50 mV to +50 mV. The current–voltage relationship was determined at the beginning and end of each prolonged exposure to 1 µM GABA, as well as at the end of the wash in external solution. The use of six-barrelled drug application pipettes allowed us to test several concentrations of GABA with each sweep of the pClamp protocol. The responses during concentration–response determinations and preapplication studies were normalized to the current elicited by 1 mM GABA after a prolonged wash in external solution during each sweep. Data were excluded if there was a greater than 10% rundown of the maximal response between sweeps.
Data analysis
Currents were low-pass filtered at 2 kHz, digitized at 5–10 kHz, and analysed using the pCLAMP 9 software suite. For those cells with very small (< 50 pA) currents, rise time, desensitization and deactivation were not determined. Current amplitudes and 10–90% rise times were measured using the Axon Instruments Clampfit 9 software package. The desensitization and deactivation time courses of GABAA receptor currents were fitted using the Levenberg-Marquardt least squares method with up to six component exponential functions of the form 
ane(–t/n)
+
C, where t is time, n is the best number of exponential components, an is the relative amplitude of the nth component, n is the time constant of the nth component, and C is the residual current at the end of the GABA application. Additional components were accepted only if they significantly improved the fit, as determined by an F-test automatically performed by the analysis software on the sum of squared residuals. The time course of deactivation was summarized as a weighted time constant, defined by the following expression: 
.
Repetitive stimulation experiments applied 10 ms of 1 mM GABA at 10 Hz four times. The ratio of the fourth peak response to the first peak response was determined for each cell. Recovery from desensitization was studied as previously described (Overstreet et al. 2000). In brief, pairs of 5 ms applications of 1 mM GABA with variable wash intervals between the first and second GABA applications were used. Recovery from desensitization was defined as: 
, where Residual is the remaining current immediately before the second application of GABA, and Peak1 and Peak2 refer to the peak current during the first and second GABA applications, respectively. GraphPad Prism 4 (GraphPad Software Inc, San Diego, CA, USA) was used to fit the concentration–response results to a sigmoidal function using the equation:
| (1) |
where I is the peak current at a given GABA concentration, and Imax is the maximal peak current. Numerical data were expressed as means ±
S.E.M. Statistical analysis was performed using GraphPad Prism 4. Data were compared using a Mann-Whitney test for pairs of data, or a Kruskal-Wallis test for comparing three or more groups. Statistical significance was taken as P < 0.05.
Kinetic modelling
Using QuB version 1.4 (http://www.qub.buffalo.edu), a modified version of the 132L GABA receptor model proposed by Haas & Macdonald (1999) was fitted to representative 132L and 432L whole cell currents. For each GABA concentration used in the fitting process, a representative current was generated by averaging the responses of three to eight cells whose peak currents were normalized to the 1 mM GABA peak current. Before using this averaged current for fitting, it was verified against the average kinetic properties of individual cells at the same GABA concentration.
Since mono-liganded states should have negligible occupancy in the presence of saturating concentrations of GABA, we first fitted an averaged current elicited by 10 mM GABA to a version of the model including only the di-liganded states. This reduced the number of free parameters, thus decreasing the time required for each fitting iteration. To further reduce the number of free parameters, the exit rates from di-liganded open states were fixed so that mean open times would be consistent with previously published single channel data for these receptor isoforms (Haas & Macdonald, 1999; Akk et al. 2004). Once an adequate fit was obtained (i.e. when the log likelihood ratio changed by less than 0.5 between iterations), the currents were refitted to a model with scaled rate constants to obtain open time distributions consistent with the published single channel data. Closed time distributions were not considered during the fitting process, as the longest components tend to be artificially shortened due to the presences of multiple channels in most patches. Nonetheless, it should be noted that the di-liganded portion of our model contains five closed states. This gives rise to a closed time distribution with five components, a number consistent with published reports for both receptor isoforms.
After fitting the model to currents elicited by 10 mM GABA, all di-liganded rate constants were fixed, and mono- and unliganded non-conducting states were added to generate two GABA binding steps. The two GABA binding sites were assumed have equal affinity. To obtain an estimate of the unbinding rates, this model was first fitted to the time course of current deactivation following a brief GABA application (5 ms, 1 mM). Mono-liganded open and desensitized states were then added, and the model was refitted to currents elicited by the lowest GABA concentration that permitted significant activation and macroscopic desensitization (3 and 10 µM for 432 and 132 receptor isoforms, respectively). The final kinetic model for each receptor isoform was verified by generating theoretical currents with the differential equation solving program Berkeley-Madonna 8.0 (http://www.berkeleymadonna.com) using the fourth-order Runge-Kutta method and time intervals of 10–100 µs.
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Results
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To investigate the role of 4 subtypes in determining the physiological properties of GABAA receptor currents, we used whole cell voltage clamp recording and a rapid drug delivery system to apply GABA to lifted HEK293T cells that had been cotransfected with 3, 2L, and either 1 or 4 subtypes. We chose the combination of 4, 3 and 2L subunits because rodent brain 4 subtypes coprecipitate with 2/3 subunits (Bencsits et al. 1999) and are among the subtypes coexpressed in thalamus and hippocampus (Pirker et al. 2000). Moreover, multiple models of epilepsy have found a consistent up-regulation of 4 subtype expression in the hippocampal dentate gyrus, where 3 is the predominant subunit subtype. Furthermore, low levels of endogenous 3 subtypes have been detected by RT-PCR in HEK293 cells (Kirkness & Fraser, 1993; Davies et al. 2000). We therefore used the 3 subtype to prevent possible contamination with multiple subtypes. Although 122 receptors are the most abundant native receptors in mammalian brain, we used 132L receptors to permit comparison of the effects of different subtypes on receptor current kinetic properties, without the possible confound of different subtypes.
1,43 receptors yielded small currents with altered kinetic properties
We confirmed that cells transfected with , , and cDNAs actually form ternary (i.e. ) complexes. Previous work has demonstrated that transfection of fibroblasts with the , , or subunits alone does not produce currents and neither does cotransfection of cells with and subunits (Zezula et al. 1996). There have been inconsistent reports of immortalized cells expressing GABAA receptors (Verdoorn et al. 1990). The kinetic properties and zinc sensitivity of these currents, however, were very similar to cells expressing GABAA receptors (Verdoorn et al. 1990; Draguhn et al. 1990), suggesting that these receptors probably included the endogenous 3 subunit expressed in HEK293 cells (Davies et al. 2000). However, 13 subunits have been shown to form functional currents (Angelotti & Macdonald, 1993; Angelotti et al. 1993), and there is evidence from immunoprecipitation studies that 4x receptors may be present in rat brain (Bencsits et al. 1999). We found that application of a synaptically relevant GABA concentration (1 mM) consistently evoked currents from cells expressing either 13 or 43 receptors (Fig. 1); however, these currents activated more slowly, and the maximal current amplitudes were five to 10 times smaller than those recorded from cells expressing 132L or 432L receptors (Table 1, Imax). Furthermore, 13 receptor currents desensitized much more rapidly than 132L receptor currents, although the overall residual current at the end of 4 s GABA application was not different (Fig. 1A; Table 1). In contrast, 43 receptor currents desensitized more slowly and less extensively than 432L receptor currents (Fig. 1B; Table 1).
With the exception of 13 currents, these currents generally required four components to accurately fit the desensitization time course. These complex kinetics might suggest that 132L currents are actually mediated by a mixed population of GABAA receptors. However, cells transfected with 1, x, x, xx, or 1x subtype combinations do not generally produce current (Verdoorn et al. 1990; Angelotti & Macdonald, 1993; Tretter et al. 1997; Davies et al. 2000). Moreover, previous studies have found that in cells transfected with , and subunits there is a preferential assembly of all three subunits into functional pentamers with a stoichiometry of two , two and one subunits per receptor (Angelotti & Macdonald, 1993; Chang et al. 1996; Zezula et al. 1996; Tretter et al. 1997; Farrar et al. 1999). Finally, given the small amplitudes of the currents, we concluded that the majority of the current from transfected cells was from ternary receptors.
432L currents desensitized more rapidly and extensively than 132L currents
To characterize the effect of subtypes on current desensitization, 432L and 132L currents were recorded during prolonged (4 s) applications of a high concentration of GABA (1 mM). The 432L currents desensitized more rapidly and extensively than 132L currents (Fig. 2A and B). All currents were fitted best with a three or four component exponential function (online Supplemental material, Figure S1) with time constants that fell into four discrete groups, 1 (< 15 ms), 2 (20–100 ms), 3 (110–800), and 4 (800–4800 ms) (Fig. 2C). The 432L currents had a greater contribution of fast (< 100 ms) desensitization than 132L receptors (Fig. 2C), although the actual time constants of desensitization were similar for both receptors. Moreover, 432L receptors had more overall desensitization, as assessed by the residual current at the end of the 4 s GABA application.
The rapidly changing membrane conductance during GABA application introduces a transient series resistance error that cannot be compensated with our recording system. In theory, this could cause us to underestimate the true peak amplitude and degree of rapid desensitization. However, if this were the case, we would expect those cells with larger currents to have a smaller fraction of fast desensitization. Similar to previous work from our laboratory (Bianchi & Macdonald, 2002), we found no such correlation between current amplitude and desensitization (data not shown). Moreover, since the peak amplitudes of 132L and 432L are the same, any transient series resistance errors should be the same in both groups.
432L currents deactivated more slowly than 132L currents
Synaptic inhibitory neurotransmission involves very brief exposure to high concentrations of GABA. To characterize the response of these receptors to a more synaptically relevant application of GABA, cells were exposed to 1 mM GABA for 5 ms, the briefest application that we were able to achieve reproducibly with our drug delivery system. The currents were fitted to multiexponential functions (Fig. 3B). Both 132L and 432L receptor currents decayed slowly after a brief exposure to GABA (Fig. 3A). However, 432L currents deactivated more slowly, with a weighted time constant of 371 ± 54 ms (n
= 18) versus 200 ± 24 ms (n
= 13, P < 0.05) for 132L currents. Deactivation of 132L current was relatively simple, with two (1 of 13 cells), three (6 of 13 cells), or four (6 of 13 cells) time constants, but the major part of the deactivation was due to a single component (3, 100–300 ms). In contrast, 432L current deactivation tended to be more complex, requiring three (7 of 18 cells), four (8 of 18 cells) or five (3 of 18 cells) components to accurately fit the deactivation time course. Furthermore, unlike 132L current deactivation, no single component dominated the deactivation time course. Interestingly, in most published reports, only one or two component exponential functions are generally used to fit IPSC decay. The basis for this difference is uncertain, but may be due to the fact that our recombinant currents were significantly larger than most IPSCs, which may have allowed us to detect greater kinetic complexity than would be apparent from the smaller IPSC currents. Different fitting techniques are another potential source of confusion when comparing studies among laboratories. To address this issue, all of the currents in these studies were also fitted with a two-component exponential function. Although this approach provided less precise fits of the data, there was no change in the overall weighted time constant compared to the more complicated fits described above (data not shown).
To further improve the speed of GABA application, 1 mM GABA was applied to macropatches. Similar to data from lifted cells, the peak current amplitudes were similar (543 ± 212 pA versus 351 ± 138 pA for 132L and 432L currents, respectively) but the rise times were prolonged for 432L currents (1.78 ± 0.21 ms, n
= 9, P < 0.05) compared to 132L (1.30 ± 0.44 ms, n
= 8). Both combinations had greater extents of fast desensitization than those recorded using lifted cells, although 432L currents still had more fast desensitization (fraction of 1
= 0.47 ± 0.04, P < 0.05) than 132L currents (0.38 ± 0.04). Finally, 432L currents still had less residual current during application of 1 mM GABA for 4 s (0.02 ± 0.01, P < 0.05) than 132L currents (0.07 ± 0.02). Interestingly, unlike data from lifted cells, there was no difference in the rate of deactivation following application of 1 mM GABA for 5 ms (weighted
= 143 ± 18 ms, n
= 8 and 109 ± 5 ms, n
= 6 for 132L and 432L currents, respectively). The reason for the difference between macropatch and whole cell currents is not entirely clear. One explanation might be that there is improved GABA exchange around a macropatch. The enhanced fast desensitization and briefer deactivation is consistent with that interpretation. However, we measured the solution exchange around a whole cell and found it to be consistently faster than 1 ms. Furthermore, the fastest kinetic feature, current activation, was not different between macropatches and lifted cells. Another possible explanation is that the smaller macropatch currents were less distorted by series resistance error, thereby producing faster desensitization and deactivation compared to currents from lifted cells. However, similar to the lifted cell data, we saw no correlation between current amplitude and desensitization kinetics. Moreover, neither improved drug application speed or reduced series resistance error explain why despite enhanced fast desensitization of 432L receptor currents, currents from 432L macropatches did not have prolonged deactivation time constants compared to 132L currents, as seen in lifted cells. We favour the interpretation that the act of pulling macropatches alters the function of GABAA receptors, possibly a consequence of losing modifying cytoplasmic proteins. Therefore, the remaining studies were performed using lifted cells.
432L currents desensitized more rapidly than 132L currents during repetitive stimulation
Although 432L receptors deactivated slowly after a brief GABA application, they desensitized rapidly during long GABA applications. GABAA receptor desensitization is thought to involve entry into a long-lived, non-conducting state(s). We therefore tested the hypothesis that during repetitive applications of brief GABA (10 ms applications of 1 mM GABA at 10 Hz), GABAA receptors accumulate in desensitized state(s), resulting in progressively attenuated responses. During repetitive GABA application, current attenuation was much more pronounced for 432L currents than for 132L currents (Fig. 4A). The ratio of the fourth to the first peak response was 0.52 ± 0.06 (n
= 10) for 432L receptors versus 0.75 ± 0.03 (n
= 11) for 132L receptors (P < 0.01). Similar differences were noted when overall normalized charge transfer was compared between these two GABAA receptor combinations (data not shown).
To determine the time courses for recovery from desensitization of 432L and 132L currents, GABA was applied in brief pairs (5 ms, 1 mM GABA) with variable time intervals between applications (Fig. 5A). While these brief GABA applications would not allow these receptors to achieve steady state desensitization, they more closely mimic the response to brief repeated bursts of GABA that are likely to occur in inhibitory synapses. The time course of recovery from desensitization was fitted best by a four-component exponential function for both 132L and 432L currents. The 432L currents were more suppressed during paired GABA applications, and required a longer recovery period between paired applications than 132L currents. The weighted recovery time constant for 432L receptors was 2.29 s (1
= 6 ms (37%), 2
= 65 ms (20%), 3
= 801 ms (25%), 4
= 11.12 s (19%)) compared to the weighted recovery time constant for 132L receptors of 314 ms (1
= 6 ms (55%) and 2
= 35 ms (22%), 3
= 240 ms (14%), 2
= 2.99 ms (9%)). In summary, the highly desensitizing 432L currents were progressively attenuated during high frequency GABA application and also required a prolonged wash period to allow recovery from desensitization after each exposure to GABA.
432L currents activated more slowly than 132L currents
Synaptic GABA is thought to reach high concentrations (1 mM) for only a very brief time ( 1 ms). Given the brief availability of GABA in the synaptic cleft, the rate of activation might shape 432L receptor currents within or near the synapse. We therefore determined if there was a concentration-dependent difference in the activation rates of 432L and 132L currents. Activation rate was defined as the inverse of the 10–90% rise time and plotted as a function of GABA concentration (Fig. 6A). Since the maximal rates of activation at GABA concentrations > 1 mM approach the solution exchange time around the cell, these results may underestimate the true rates. However, the maximal activation rates of 432L and 132L currents were not obviously different and, more importantly, the entire concentration–activation rate curves were shifted to the right for 432L currents (EC50
= 988 µM) compared with 132L currents (EC50
= 377 µM). Thus, at any given submaximal GABA concentration ( 3 mM), the slower activation of 432L currents would favour incomplete activation, and therefore, a truncated peak current.
Despite the very brief availability of GABA within the synaptic cleft, synaptic currents usually last on the order of tens to hundreds of milliseconds. This is thought to occur because receptors rapidly enter long-lived, agonist bound states, from which GABA unbinds slowly. During extremely brief GABA applications, however, receptor populations would be unable to achieve the same occupancy of long-lived agonist-bound states as would occur following longer pulses. With fewer receptors in these long-lived states, more rapidly deactivating postsynaptic responses occur. Furthermore, the response to saturating concentrations of GABA is thought to involve binding of two molecules of GABA by each receptor, with the longest lived states being achieved only when both GABA binding sites are occupied (Macdonald et al. 1989). When GABA is applied either very briefly or at subsaturating concentrations, not all receptors would be fully di-liganded, potentially also allowing for more rapidly deactivating currents (Mozrzymas et al. 2003).
To illustrate this idea, GABA (10 µM) was applied to 432L receptors for durations varying from 5 to 200 ms (Fig. 6B). For the cell shown in Fig. 6B, the 10–90% rise time in the presence of 10 µM GABA was 28 ms. When GABA was applied for brief periods, there was not only a truncation of the peak, but also more rapid deactivation. Although similar experiments were not performed with 132L receptors, the relatively rapid deactivation following applications of 1 mM GABA for 5 ms compared to 4000 ms durations suggests that a similar coupling of GABA application duration and deactivation time course occurs with these receptors as well. At synapses containing 432L receptors, very brief exposure to GABA would be predicted therefore to produce synaptic currents with small amplitudes and brief durations.
GABA sensitivities of 132L and 432L receptors were similar
In addition to the synaptic receptors mediating IPSCs, another population of GABAA receptors found outside the synaptic cleft respond to tonic, low levels of GABA ( 1 µM). Despite the small size of these tonic currents, their longevity allows a large overall charge transfer that can significantly alter neuronal excitability. Effective inhibition by extrasynaptic receptors would be best served by highly sensitive GABAA receptors that are able to respond during prolonged exposure to low GABA concentrations. Concentration–response curves were generated by applying varying GABA concentrations in random order with at least a 45 s wash between applications of each concentration (Fig. 7). Since there was substantial cell-to-cell variability in current size, a near-maximal (1 mM) GABA concentration was applied intermittently to allow normalization of peak currents and to assess current run-down during the experiments. The GABA EC50 values were similar for both 132L and 432L receptors (Fig. 7; GABA EC50 values were 14 µM (95% CI = 12–17 µM) and 15 µM (95% confidence interval (CI) = 14–17 µM) for pooled data from 132L and 432L peak currents, respectively).
Tonic GABA receptor currents measured in neurons are thought to be caused by low levels of extrasynaptic GABA that change much more slowly than GABA levels in the synaptic cleft. Therefore, we sought next to determine the pseudo-steady-state concentration-dependent response of 132L and 432L receptors to prolonged GABA application. Various concentrations of GABA were applied for 4 s, and the current at the end of the GABA application was measured (Fig. 8A, arrow). At GABA concentrations above 10 µM, the response to prolonged GABA was lower for 432L than for 132L receptors (Fig. 8B). It is worth noting that the concentration dependence of these pseudo-steady-state currents had remarkably limited dynamic ranges. A maximal pseudo-steady-state response was seen with 10 µM GABA, although higher concentrations were able to accelerate the rates of activation and desensitization (Fig. 8B). In the range of GABA concentrations likely to be present in the extrasynaptic spaces (1–2 µM), the responses of 432L and 132L receptors were similar.
The foregoing concentration dependence studies would suggest that despite increased desensitization to high GABA levels, both 132L and 432L receptors might respond reasonably well in an extrasynaptic environment. However, the previous studies all used a complete washout between GABA applications, which is probably different from the nearly constant low ambient levels of GABA thought to exist in the extrasynaptic space in some brain regions. To more realistically mimic extrasynaptic or perisynaptic GABAA receptor responses, we measured the GABAA receptor responses when pre-exposed to a prolonged low concentration of GABA. Cells were incubated for 45 s in 1 µM GABA (preapplication) followed by application of higher concentrations of GABA. Following a washout, the response to 1 mM GABA was measured and used to normalize all of the pre-exposed responses (Fig. 9A). It is possible that prolonged GABA application could produce a sufficient chloride ion flux that the normal electrochemical gradient was altered. Current–voltage relationships at the beginning and end of each sweep (Fig. 9A, arrows) confirmed that there was no change in the GABA reversal potential (data not shown). For both receptor isoforms, the maximal response to GABA following preapplication with low GABA concentrations was greatly reduced, compared to cells in which GABA was allowed to washout between applications (Fig. 9B). However, the suppressive effect of low tonic GABA levels was particularly prominent for 432L currents.
GABA receptor kinetic modelling
To make more quantitative predictions about the potential physiological roles of 1 and 4 subtype-containing GABAA receptors, a kinetic model was generated for these receptor isoforms that accounted for not only the observed macroscopic properties described above, but also for the previously published single channel properties (Haas & Macdonald, 1999; Akk et al. 2004) (Fig. 10). This model was based largely on the previous work of Haas & Macdonald (1999), but has been expanded to account for several additional microscopic and macroscopic kinetic observations. Specifically, single channel recordings from 1 GABAA receptors have consistently demonstrated three open states, with the relative contribution of the shortest open state (O1) being concentration dependent at GABA concentrations below 10 µM (Fisher & Macdonald, 1997). Although this suggests the presence of an additional GABA binding step distal to O1, a feature found in most of the existing models, it remains unclear why a residual component of O1 persists at higher concentrations. One possible explanation is that two open states with similar mean open times exist, one of which is mono-liganded and the other di-liganded. We have therefore added two additional states to the Haas and Macdonald kinetic model: a di-liganded closed (C3) and a di-liganded open state (O2). The addition of the closed state is intended to preserve the known burst characteristics of these receptors such that only one type of opening is observed per burst (Twyman et al. 1990). As with 1 receptors, single channel recordings from 4 receptors also demonstrated the presence of three open states that are concentration independent at GABA concentrations above 10 µM (Akk et al. 2004). We therefore applied the same kinetic model to those receptors.
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Figure 10. Kinetic model for the 132L and 432L receptor isoforms
A, a modified version of the kinetic model proposed for the 132L receptor isoform by Haas & Macdonald (1999) is shown (O, open; C, closed; D, desensitized). B, rate constants for the 132L and 432L receptor isoforms were determined by fitting the time course of macroscopic currents evoked by saturating and subsaturating GABA concentrations to the kinetic model in A after constraints were imposed based on published single-channel data (see Methods). Units for all rate constants are s–1 except for kon (M–1 s–1). Ca and Da, the optimized responses (black line) of 132L and 43 | |
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Abstract
Introduction
