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GABA_A receptor subunit 2 and subtypes confer unique kinetic properties on recombinant GABA_A receptor currents in mouse fibroblasts

Kevin F. Haas and Robert L. Macdonald *

Received 27 May 1998; accepted after revision 18 September 1998.

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

1. To determine their contributions to the rapid kinetic properties of GABA_A receptor (GABAR) currents, 1 and 3 subunit subtypes without or with or 2L subtypes were transiently coexpressed in mouse L929 fibroblasts to produce 13, 13, or 132L GABAR isoforms.

2. Brief (2-3 ms) applications of 1 mM GABA to outside-out membrane patches containing 13, 13, or 132L isoforms elicited currents that activated rapidly with monophasic time courses and deactivated rapidly with biphasic time courses. 132L currents exhibited a slower mean deactivation rate (76·1 ms) than 13 (34·1 ms) or 13 currents (42·8 ms).

3. During 1 mM GABA applications, 132L currents activated more rapidly (0·46 ms) than 13 currents (1·7 ms) or 13 currents (2·4 ms). During 4000 ms GABA applications, 13 and 132L currents desensitized with triphasic time courses to similar extents (13, 94·6 %; 132L, 92·4 %) and with similar mean rates (13, 352 ms; 132L, 462 ms). In contrast, 13 currents desensitized only 55·6 % with a biphasic time course and slower mean rate (1260 ms).

4. These experiments demonstrated that the 13 heterodimer formed a GABAR channel with rapid deactivation and rapid and nearly complete desensitization. Addition of the subunit did not alter the activation rate, but produced a receptor with slower and less complete desensitization. Addition of the 2L subtype increased activation rate, prolonged deactivation and changed the pattern of rapid desensitization.

5. Rapid kinetic and steady-state single-channel data were used to construct kinetic models that predicted the behaviour of the 132L and 13 currents. These models represent a reconciliation of macroscopic and steady-state single-channel data for GABARs and provide a framework for systematically assessing the functional significance of different GABAR isoforms.

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

GABA_A receptors (GABARs) mediate the majority of fast inhibitory neurotransmission in the mammalian brain. Functional GABARs are ligand-gated chloride ion channels composed of five individual subunits. These subunits derive from six identified families, many with multiple subtypes (1-6, 1-3, 1-3, , and ). When expressed in Xenopus oocytes or mammalian cells, different GABAR subunit combinations form receptors with unique pharmacological and biophysical properties (Macdonald & Olsen, 1994). These subunits do not assemble randomly, however, for while subunit combinations readily express in mammalian cells, addition of a subunit drives expression of isoforms (Angelotti & Macdonald, 1993). The majority of native receptors are believed to be formed by combinations of and subunits (McKernan & Whiting, 1996), although the recently characterized and subunits may substitute for or subunits in some instances (Hedblom & Kirkness, 1997; Davies et al. 1997). In the rat, the 2 subtype becomes the dominant subunit expressed at later developmental stages, and mRNA and membrane protein for this subtype are expressed in most brain regions. In contrast, the subunit is restricted only to a few cell populations in the postnatal rat that include thalamic relay neurons, cerebellar granule neurons and dentate granule neurons of the hippocampus (Laurie et al. 1992a,b; Wisden et al. 1992; Sperk et al. 1997). While the subunit has been shown to combine preferentially with the 6 subtype in cerebellar granule neurons (Jones et al. 1997), the GABAR subtypes that it combines with in dentate granule neurons remain unknown. The potential importance of hippocampal subunit-containing GABARs is underscored, however, by subunit knockout mice that exhibit spontaneous seizures (Olsen et al. 1997). For this investigation, we chose the 132L and 13 GABAR isoforms to determine the roles of and subunits in shaping GABAR currents.

132L and 13 GABAR whole-cell currents have been characterized previously in L929 fibroblasts, where incorporation of the subunit resulted in higher apparent GABA affinity, slower and less complete whole-cell current desensitization, and smaller whole-cell currents compared with receptors containing the 2 subtype (Saxena & Macdonald, 1994). 13 currents also desensitized more rapidly than 13 currents, although both faster (Fisher & Macdonald, 1997) and slower desensitization (Dominguez-Perrot et al. 1996) relative to 132L currents have been reported. In addition, 13 single channels had a smaller main channel conductance level (13 pS), while 132L and 13 channels had a similar larger main conductance level (27 pS). The 2L subtype, however, conferred a change on the open and closed properties of the receptor, leading to a tendency for longer duration openings and longer bursts of openings (Fisher & Macdonald, 1997). While suggesting major differences in channel gating and desensitization, these analyses did not resolve the rapid phases of activation, desensitization and deactivation of GABAR currents.

These rapid kinetic properties are critical to understanding the potential synaptic roles of the 132L and 13 GABAR isoforms. In previous studies of native receptors, rapid application of GABA to outside-out membrane patches containing many GABARs reproduced the rapid activation and deactivation of IPSCs (Maconochie et al. 1994; Jones & Westbrook, 1995; Tia et al. 1996; Galaretta & Hestrin, 1997; Mellor & Randall, 1997, 1998). Also, with this rapid application protocol, it was demonstrated that GABAR desensitization was an important factor in shaping the deactivation time course of macropatch responses (Jones & Westbrook, 1995, 1996). In addition, macropatch deactivation kinetics were altered by allosteric modulators of GABARs such as benzodiazepines (Lavoie & Twyman, 1996; Mellor & Randall, 1997) and the anaesthetic propofol (Zhu & Vicini, 1997), as well as by intracellular phosphatase activity (Jones & Westbrook, 1997). Moreover, different recombinant GABAR isoforms displayed unique rapid kinetic properties (Verdoorn, 1994; Tia et al. 1996; Lavoie et al. 1997) that probably contribute to the diversity in GABAergic synaptic responses.

In addition to predicting the synaptic behaviour of recombinant GABAR isoforms, rapid kinetic analysis of macroscopic currents may serve as a bridge between single-channel and whole-cell analysis, allowing for the development of more comprehensive kinetic models of GABAR behaviour that incorporate desensitization (Macdonald & Twyman, 1992). For this study, we implemented a GABA application system that allowed very rapid solution exchange (10-90 % rise time < 400 µs) during electrophysiological recordings from outside-out membrane patches containing multiple receptor channels. Using this technique, we determined the rapid activation, desensitization and deactivation kinetics of 132L and 13 GABAR currents and used these kinetic data in combination with steady-state single-channel analysis to develop comprehensive models of GABAR kinetic behaviour for these isoforms. In some instances, the 13 isoform was examined so that contributions of the and subunits could be more thoroughly assessed.

	METHODS

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Expression of recombinant GABARs

The cDNAs encoding rat 1, 3, 2L and GABAR subunit subtypes were individually subcloned into the plasmid expression vector pCMVNeo (Huggenvik et al. 1997). Mouse L929 fibroblasts (American Type Culture Collection, Rockville, MD, USA) were maintained in Dulbecco's Modified Eagle's medium supplemented with 10 % horse serum at 37°C in 5 % CO₂-95 % air and passaged prior to confluent growth. Cells were transfected with 4-8 µg of each subtype plasmid along with a plasmid (pEGFP; Clontech, Palo Alto, CA, USA) encoding for green fluorescent protein (GFP), in a 1:1:1:1 ratio using a modified calcium phosphate co-precipitation technique (Chen & Okayama, 1987) as previously described (Angelotti et al. 1993). The next day, cells were replated onto Mecanex gridded 35 mm culture dishes. Twenty-four hours after replating, electrophysiological recordings were performed on GFP-positive cells.

Electrophysiology

Patch-clamp recordings were performed on outside-out membrane patches pulled from L929 fibroblasts bathed in an external solution consisting of (mM): NaCl, 142; CsCl, 8; MgCl₂, 6; CaCl₂, 1; Hepes, 10; glucose, 10 (pH 7·4, 320 mosmol l^-1). Glass microelectrodes were formed from thick-walled borosilicate glass (World Precision Instruments, Pittsburgh, PA, USA) with a Flaming Brown electrode puller (Sutter Instrument Co., San Rafael, CA, USA), fire-polished to tip resistances of 10-20 M, then coated with Q-dope (GC Electronics, Rockford, IL, USA). Patch electrodes were filled with an internal solution consisting of (mM): CsCl, 153; MgCl₂, 1; MgATP, 2; Hepes, 10; EGTA, 5 (pH 7·3, 300 mosmol l^-1). This combination of internal and external solutions produced a chloride equilibrium potential of 0 mV. Outside-out membrane patches were pulled from positively transfected L929 cells and voltage-clamped at -75 mV using an EPC-7 amplifier (List). The intensity of GFP fluorescence was used to identify cells with relatively high or low expression to use for macropatch or single-channel recordings, respectively.

GABA was applied to outside-out membrane patches using a rapid application system (Franke et al. 1987) consisting of a double-barrelled theta tube (FHC, Brunswick, ME, USA) connected to a piezoelectric translator (Burleigh Instruments, Fishers, NY, USA). One barrel was perfused with the external recording solution and the other was perfused with a GABA-containing external solution. Activation of the translator drove the solution interface rapidly across the patch surface. The solution exchange time was monitored at the end of each recording by blowing out the patch and stepping a dilute (90 %) external solution across the open electrode tip to measure a liquid junction current. The 10-90 % rise times for solution exchange were consistently less than 400 µs. The recording chamber was continuously perfused with external solution to prevent accumulation of GABA in the bath. All experiments were performed at room temperature (22-23°C).

Rapid kinetic analysis

Outside-out patch data were low-pass filtered at 3 kHz, digitized at 10 kHz and analysed using the pCLAMP6 software suite (Axon Instruments) and Origin 4.1 (Microcal, Northampton, MA, USA). Multiple GABA-elicited responses (5-20) were acquired for each GABA concentration at 30 s intervals, then were averaged to form ensemble currents for analysis. Deactivation of ensemble currents was measured as the current decay after the removal of GABA. Activation was measured as the 10-90 % rise time to the peak current and desensitization as the decline from peak current in the continuing presence of GABA. The deactivation and desensitization time courses of ensemble GABAR currents were fitted using the Levenberg-Marquardt least squares method with one-, two-, or three-component exponential functions. The number of exponential components was incremented until the addition of another component did not significantly improve the fit (P < 0·01) as determined by an F test on the sum of squared residuals. For comparison of deactivation and desensitization time courses among currents from different isoforms, mean deactivation and desensitization rates were calculated using a weighted summation of the fitted exponential components. For a triphasic decay, this equation was (A₁₁ + A₂₂ + A₃₃), where ₁, ₂ and ₃ were the fitted time constants and A₁, A₂ and A₃ were the fitted component proportions. The extent of desensitization after 4000 ms GABA applications was measured as (peak current - fitted steady-state current)/(peak current). Numerical data were expressed as means ± S.E.M. Statistical significance was determined using Student's unpaired two-tailed t tests and ANOVAs (Student- Newman-Keuls test) where appropriate.

Single-channel analysis

Single-channel recordings were filtered at 2 kHz with an 8-pole Bessel filter (3 dB at 2 kHz), digitized at 20 kHz through a Digidata 2000 A/D converter (Axon Instruments) and acquired into Axoscope (Axon Instruments). Single-channel data were analysed using pCLAMP6 (Axon Instruments) and Interval5 (Dr Barry S. Pallotta, University of North Carolina, Chapel Hill, NC, USA). Single-channel events were identified with a 50 % threshold detection method. Subconductance levels were rarely observed (< 5 % of openings) but were included in the analysis if they reached the 50 % threshold. Recordings were only included in the kinetic analysis if overlaps of simultaneous openings occurred for less than 1 % of the openings. Overlapped openings and bursts were not included in the kinetic analysis. The presence of multiple channels would decrease the apparent duration of the longer closed components, but would have no effect on the open state properties. Duration histograms were constructed as described by Sigworth & Sine (1987) and fitted by a maximum likelihood method. The number of exponential functions required to fit the distribution was increased until additional components did not significantly improve the fit as determined by the log-likelihood ratio test (Horn, 1987; McManus & Magleby, 1988). Durations less than 1·5 times the system dead time (150 µs) including the 8-pole Bessel filter (3 dB cut-off at 2 kHz) were displayed in the histograms but were not included in the fit. For the definition of bursts, the two shortest closed components were considered as intraburst closures. A critical gap for each patch was calculated from the closed interval distribution to equalize the proportion of misclassfied events (Colquhoun & Sakmann, 1985).

Kinetic modelling

Modelling of the macroscopic currents was performed using SCoP (Berrien Springs, MI, USA). The models for 132L and 13 isoforms were based on a model developed to predict the steady-state single-channel kinetics of mouse spinal cord neurons (Macdonald et al. 1989; Twyman et al. 1990). This model incorporated two GABA binding steps of equal GABA affinity. The two shortest closed states were concentration-independent and fixed as the distal closed states emerging from the open states. To confirm the validity of this model for the application conditions of this study, we performed steady-state single-channel analysis at 1 mM GABA. For the 13 isoform, only two open states were identified. These were both modelled as doubly liganded open states. A single slow desensitized state was added to account for the longest closed duration. For the 132L isoform, we observed three open states and five closed states. Three desensitized states were included to explain the triphasic desensitization pattern. The sum of the closing rates from the open states was then fixed by the inverse of the mean open durations. Similarly, the inverses of the two briefest closed durations set the opening rates from the distal closed states (C₅-C₁₀). The remaining parameters were adjusted to fit best the macroscopic deactivation and desensitization rates and the single-channel open, closed and burst properties. Simulations of single-channel data using the models were performed with the Interval5 analysis software.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Macropatch 13, 13 and 132L currents at high GABA concentrations

Within the synaptic cleft, it has been predicted that GABA concentrations of 500 µM to 1 mM are reached within 100 µs, which decline rapidly over the course of a few milliseconds (Maconochie et al. 1994; Clements, 1996). To approximate a synaptic GABA time course, brief pulses of 1 mM GABA (2-3 ms) were applied to outside-out membrane patches pulled from L929 fibroblasts expressing 13, 13, or 132L GABAR channels. Changing application durations over this range did not measurably alter the deactivation kinetics of the currents. Longer (400-4000 ms) GABA applications were used to evaluate activation and desensitization kinetics. GABA application elicited currents that activated and deactivated rapidly, but each isoform exhibited distinct rapid kinetic properties.

Deactivation

Representative current traces for each isoform are shown in Fig. 1A with 2 ms applications of 1 mM GABA denoted by open-tip liquid junction currents. This example illustrates that currents from all three isoforms deactivated with similar fast components ranging from 11·1-18·5 ms. The slow deactivation component was variable, ranging from 73·1 ms for the 13 current to 182 ms for the 132L current. To compare the deactivation rates of the currents among isoforms, we utilized a weighted sum of the fitted deactivation time constants to calculate a mean deactivation rate (see Methods). The 13 currents deactivated most rapidly with a mean of 34·1 ms (n = 5 patches) followed by 13 currents at 42·8 ms (n = 4). The 132L current deactivated significantly more slowly at 76·1 ms (n = 6) (Fig. 1B). The slower deactivation of 132L currents was due to a significantly longer slow decay component (209 ms) than 13 (144 ms) and 13 currents (82·4 ms) (P < 0·05). In addition, for 132L currents, a significantly greater percentage of the decay was attributed to the slow component (32·0 %) than that for 13 currents (16·8 %) (P < 0·05) (Table 1).

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Figure 1. Current deactivation following brief applications of 1 mM GABA A, outside-out membrane patches were pulled from L929 fibroblasts expressing 13, 13 and 132L GABAR isoforms. In patches voltage-clamped at -75 mV, brief pulses (2-3 ms) of rapidly applied 1 mM GABA elicited inward GABAR currents that deactivated with biphasic time courses. Ensemble currents for 13 (n = 20 applications), 13 (n = 12) and 132L (n = 10) were shown. These deactivation time courses were fitted best with a two component exponential equation. The duration of GABA application was denoted by liquid junction currents above the traces. B, deactivation rates were compared by using a weighted sum of the fitted components ((proportion fast × f) + (proportion slow × s)) to obtain a mean deactivation rate. 13 currents deactivated with a mean of 34·1 ± 3·4 ms (n = 5 patches), similar to 13 currents at 42·8 ± 6·3 ms (n = 5). 132L currents deactivated significantly more slowly (*P < 0·05) at 76·1 ± 11·0 ms (n = 6).

Activation

Since 2-3 ms GABA applications were too short to assess current activation accurately, 400 ms applications were used to compare the activation rates of the three GABAR isoforms (Fig. 2, Table 1). The average rate of activation for each isoform was measured as the 10-90 % rise time of currents elicited by 1 mM GABA. The whole-cell GABA EC₅₀ for these isoforms was previously determined to be 2·1 µM for the 13 isoform, 2·8 µM for the 13 isoform, and 11·5 µM for the 132L isoform (Fisher & Macdonald, 1997). We predicted that activation rates would correlate with EC₅₀ values and that the 132L GABARs would activate relatively slowly compared with 13 and 13 GABARs. Yet, when currents from each isoform were normalized and overlaid (Fig. 2A), 13 and 13 currents activated relatively slowly compared with 132L currents. The mean 10-90 % rise time to peak current was 1·7 ms (n = 5) for the 13 isoform and 2·4 ms (n = 5) for the 13 isoform, but only 0·46 ms (n = 8) for the 132L isoform (Fig. 2B, Table 1). Thus, the 2L subtype conferred a more rapid rate of activation. A similar slower rise time of 13 currents in relation to 132L currents has also been demonstrated in whole-cell recordings with slower application of lower GABA concentrations (Dominguez-Perrot et al. 1996).

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Figure 2. Activation A, GABAR outside-out patch currents from 13, 13 and 132L isoforms were normalized and overlaid to compare the activation rates of currents evoked by rapid application of 1 mM GABA. B, 13 GABAR currents activated rapidly with a mean 10-90 % rise time to peak current of 1·7 ± 0·40 ms (n = 5), similar to the 13 activation rate (2·4 ± 0·27 ms, n = 5). 132L currents activated significantly more rapidly (*P < 0·05) with an mean 10-90 % rise time of 0·46 ± 0·04 ms (n = 8).

Table 1. Rapid kinetic properties of GABARs

	13	13	132L
Deactivation (2-3 ms)
n	5	4	6
f (ms)	14·5 ± 2·2	17·1 ± 4·2	12·4 ± 1·3
s (ms)	144 ± 18	82·4 ± 20	209 ± 21 *
Percentage fast	83·2 ± 3·3 *	57·3 ± 4·7	68·0 ± 2·9
Percentage slow	16·8 ± 3·3 *	42·7 ± 4·7	32·0 ± 2·9
Activation (400 ms)
n	5	5	8
10-90 % rise time (ms)	1·7 ± 0·40	2·4 ± 0·27	0·46 ± 0·04 *
Deactivation (400 ms)
n	-	5	7
f (ms)	-	14·7 ± 4·2	33·4 ± 2·9
s (ms)	-	79·7 ± 7·3	193 ± 31
Percentage fast	-	45·8 ± 15	10·5 ± 2·6
Percentage slow	-	54·1 ± 15	89·5 ± 2·6
Desensitization (4000 ms)
n	6	4	6
f (ms)	24·6 ± 3·2	75·7 ± 37·4	7·9 ± 1·5 *
i (ms)	200 ± 9·9	-	129 ± 26·4
s (ms)	1570 ± 149	2190 ± 311	1540 ± 135
Percentage fast	61·1 ± 7·0	48·0 ± 9·2	50·2 ± 4·6
Percentage intermediate	20·1 ± 6·8	-	21·8 ± 2·4
Percentage slow	18·8 ± 2·3	52·0 ± 9·2 *	28·1 ± 2·9
Extent (%)	94·5 ± 1·3	55·4 ± 3·6 *	92·4 ± 1·4

Desensitization

Previous whole-cell studies demonstrated that 1_x2L currents desensitized more rapidly and completely than 1_x currents (Saxena & Macdonald, 1994; Fisher & Macdonald, 1997). Long pulses (4000 ms) of rapidly applied GABA (1 mM) were used to examine the rapid desensitization of 13, 13 and 132L currents (Fig. 3). Peak currents varied with isoform, with the 132L isoform producing the largest currents (143·7 ± 44·6 pA, n = 12), followed by the 13 (48·7 ± 17·4 pA, n = 9) and 13 (11·9 ± 4·4 pA, n = 8) isoforms. During 4000 ms GABA applications, 13 and 132L current desensitization time courses were fitted best by the sum of three exponential functions, but 13 current desensitization was fitted best with only two exponential functions (Fig. 3A). 13 currents desensitized with a mean rate of 352 ± 43·7 ms, derived from fast, intermediate and slow time constants: _f = 24·6 ± 3·2 ms (61·1 ± 7·0 %), _i = 200 ± 9·9 ms (20·1 ± 6·8 %) and _s = 1570 ± 149 ms (18·8 ± 2·3 %) (n = 5). 132L currents desensitized with a similar mean rate of 461 ± 56·0 ms with _f = 7·9 ± 1·5 ms (50·2 ± 4·6 %), _i = 129 ± 26·4 ms (21·8 ± 2·4 %) and _s = 1540 ± 135 ms (28·1 ± 2·9 %) (n = 6). 13 currents desensitized with a significantly slower mean rate of 1260 ± 362 ms with _f = 75·7 ± 37·4 ms (48·0 ± 9·2 %) and _s = 2190 ± 311 ms (52·0 ± 9·2 %) (n = 5) (Fig. 3B, Table 1). The extent of desensitization was similar for 13 and 132L currents, being 94·5 ± 1·3 and 92·4 ± 1·4 %, respectively, but significantly less for 13 current at 55·4 ± 3·6 % (Table 1). While the desensitization time courses were similar for 13 and 132L currents, there were subtle differences. 132L currents showed a significantly more rapid fast component than 13 currents (P < 0·001), but a greater proportion of desensitization was concentrated in the slowest component (P < 0·05). Thus, addition of the subunit to 1 and 3 subtypes significantly reduced both the rate and the extent of desensitization, while addition of the 2L subtype to 1 and 3 subtypes changed the pattern of desensitization.

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Figure 3. Rapid phases of desensitization A, 1 mM GABA was rapidly applied for 4000 ms to outside-out membrane patches containing 13, 13 and 132L isoforms. For the representative traces shown, current desensitization was fitted with multicomponent exponential equations with time constants of 21·9 ms (f, inset), 218 ms and 1150 ms for the 13 current (n = 6), 36·8 ms (f, inset) and 2030 ms for the 13 current (n = 5), and 9·6 ms (f, inset), 153 ms and 1390 ms for the 132L current (n = 5). Time calibration for A and insets is 1000 ms and 150 ms, respectively. B, 13 and 132L currents desensitized with similar mean rates (352 ± 43·7 ms (n = 5) and 461 ± 56·0 ms (n = 6), respectively). 13 currents desensitized with a mean rate of 1260 ± 362 ms (n = 4) that was significantly slower (*P < 0·01) than 13 and 132L currents.

We examined the concentration dependence of desensitization by rapidly applying multiple concentrations of GABA for 4000 ms to outside-out patches containing the 13 or 132L isoforms (Fig. 4A). The 13 isoform desensitized minimally at 3 µM GABA and exhibited biphasic desensitization at 1 mM GABA. For 13 currents, due to the small current size and relatively low extent of desensitization, consistent fits of desensitization at GABA concentrations lower than 1 mM could not be obtained. For the 132L isoform, 1 µM GABA currents did not desensitize. At higher GABA concentrations, the desensitization time course was concentration dependent, with slower phases appearing at 10 µM GABA and the most rapid phase only appearing at high concentrations of GABA (Fig. 4A and B). At 10 µM GABA, a small-amplitude faster component of desensitization ( = 205 ± 47·7 ms, 16·9 ± 3·9 %; n = 6) was revealed, in addition to a predominant slow component ( = 1960 ± 289 ms, 83·1 ± 3·9 %; n = 6). At 100 µM GABA, a third fast component of desensitization was revealed (_f = 16·1 ± 5·3 ms, 23·7 ± 6·8 %; n = 5) in addition to the intermediate (_i = 147 ± 44·1 ms, 23·3 ± 4·6 %; n = 5) and slow (_s = 1710 ± 177 ms, 52·4 ± 5·5 %; n = 5) components. At 1 mM GABA desensitization was also triphasic (Table 1). There was not a significant change in the desensitization rates of these components as GABA concentration was increased (Fig. 4B), but instead there was a shift towards a greater proportion of fast desensitization at higher GABA concentrations (Fig. 4C). This concentration independence of desensitization rates was similar to the desensitization pattern of cultured hippocampal neuron GABAR outside-out patch currents (Celentano & Wong, 1994) and to 132L whole-cell currents (Dominguez-Perrot et al. 1997). This suggested that entry into desensitized conformations did not require additional GABA binding

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Figure 4. Concentration dependence of desensitization A, multiple GABA concentrations were applied to patches (13: 3 µM and 1 mM; 132L: 1, 10 and 100 µM and 1 mM) to examine the concentration dependence of desensitization. At 3 µM GABA, 13 ensemble currents (n = 15) exhibited minimal desensitization during a 4000 ms GABA application while ensemble currents elicited by 1 mM GABA (n = 12) exhibited more pronounced desensitization. For the 132L isoform, 1 µM GABA currents (n = 10) did not desensitize. At higher GABA concentrations, desensitization was concentration dependent, with slower phases of desensitization that appeared at 10 µM (n = 6) and a rapid phase of desensitization that appeared at higher GABA concentrations (1 mM shown; n = 5). B, for 132L currents, the desensitization rates ( values) for the slow (), intermediate () and fast phases () of desensitization were plotted as a function of GABA concentration, showing their nearly flat concentration dependence. C, the percentage contributions of the slow (), intermediate () and fast () desensitization phases to the total desensitization were plotted as a function of GABA concentration. The contribution of the slow phase decreased with increasing GABA concentration while the contribution of the fast phase increased after its appearance at 100 µM GABA.

We used 400 ms GABA applications to evaluate the effect of desensitization on current deactivation for 132L and 13 currents. Due to the relatively small size of 13 currents and their high extent of desensitization after 400 ms, we were unable to assess their deactivation following desensitization. Desensitization should prolong deactivation as more of the receptors equilibrate into desensitized states (Jones & Westbrook, 1995). This occurred with 132L currents, which deactivated predominantly with the slow component (193 ± 31 ms, 89·5 ± 2·6 %) following 400 ms applications of 1 mM GABA rather than predominantly with the fast component (12·4 ± 1·3 ms, 68·0 ± 2·9 %) following brief GABA applications (Table 1). Moreover, a receptor that minimally desensitized should deactivate at nearly the same rate following short and long GABA applications. This was true of the 13 currents, which deactivated with nearly identical rate constants and proportions following brief and prolonged GABA applications (Table 1).

Single-channel properties

We were interested in reconciling previous models of receptor function based on single-channel or whole-cell data for these isoforms. While previous studies examined single-channel properties of these isoforms at lower GABA concentrations, we were interested in the pattern of activity at predicted synaptic GABA concentrations. Thus, steady-state single-channel data at 1 mM GABA, the same concentration used to examine rapid macroscopic current properties, were obtained for the 132L and 13 isoforms. Representative single-channel traces illustrated that 13 single channels had a main conductance level (23·8 ± 0·37 pS; n = 4) similar to 132L channels (25·9 ± 0·60 pS; n = 4) (Table 2), but opened less frequently and for shorter durations (Fig. 5A). The open probability (NP_o) was significantly lower for 13 (2·27 ± 0·57 %) channels than for 132L (10·5 ± 1·5 %) channels (P < 0·05). Open interval analysis demonstrated at least three open states for 132L receptors and at least two open states for 13 receptors. Closed interval histograms were fitted best by five closed states for each isoform (Fig. 5B, Table 2). The number of open and closed states and their durations corroborated previous findings from these isoforms obtained at lower GABA concentrations (Fisher & Macdonald, 1997).

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Figure 5. Single-channel characteristics A, single-channel GABAR currents from outside-out membrane patches were voltage-clamped at -75 mV. Traces shown were continuous 700 ms recordings in response to 1 mM GABA. Channel openings are downwards. B, duration histograms of open and closed intervals were formed from steady-state single-channel data. Open duration histograms for 132L GABARs were fitted best with three components, while the 13 open duration histogram was fitted best with only two components. Closed interval histograms were fitted best by five components. For a summary of steady-state single-channel properties see Table 2.

GABAR kinetic modelling

Previous investigations of the steady-state single-channel properties of mouse spinal cord neuron GABARs led to the development of a working kinetic model (Macdonald et al. 1989; Twyman et al. 1990). This model incorporated two GABA binding steps and three open states (O) with interconnected concentration-dependent and concentration-independent (distal) closed states (C). Subsequent investigations of recombinant 112L and 132L GABARs demonstrated similar single-channel main conductances and open and closed properties (Angelotti & Macdonald, 1993; Fisher & Macdonald, 1997). This model was incomplete, however, as it did not adequately explain GABAR desensitization. Also, this model did not address the single-channel properties of the 13 isoform, which exhibited only two resolvable open states with brief durations, leading to a low open probability. Taking this model as a framework, we used macroscopic rapid kinetic data and steady-state single-channel data to construct more comprehensive models to describe the kinetic behaviour of the 13 (Fig. 6A) and 132L isoforms (Fig. 7A). For the 13 isoform (Fig. 6A), only two open states were identified. These were both modelled as doubly liganded open states. A single slow desensitized state (D_s) was added to account for the longest closed duration. For the 132L isoform (Fig. 7A), we observed three open states. Three desensitized states were needed to explain the triphasic desensitization pattern. Desensitized states were entered only from doubly liganded states, as no concentration dependence of desensitization rates was found. The sum of the closing rates from the open states was then fixed by the inverse of the mean open durations. Similarly, the inverses of the two briefest closed durations set the opening rates from the distal closed states (C₅ - C₁₀). Another important constraint was to have the model generate a P_o lower than the measured NP_o for single channels. The remaining model parameters were optimized to fit the macroscopic activation, desensitization and deactivation rates of outside-out patch currents (Figs 6B and 7B). These optimized parameters were then used to generate simulated single-channel data, and the optimization process was continued until the models best predicted both the macroscopic rapid kinetic and steady-state single-channel open, closed and burst properties.

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Figure 6. Kinetic model for the 13 isoform A, a kinetic model for the 13 isoform was derived from steady-state single-channel analysis and rapid kinetic analysis of currents from outside-out membrane patches. B, the rate constants in the model were optimized to best fit the time course of the 13 macroscopic currents and the single-channel open, closed and burst properties (see Methods). O, open state; C, closed state; D, desensitized state. Units for all rate constants were s-1 except for kon (m-1 s-1). C, the optimized model currents were superimposed on averaged 13 data traces for 2 ms (n = 4), 400 ms (n = 5) and 4000 ms (n = 4) applications of 1 mM GABA (application bars above traces). The same currents are depicted on an expanded time scale in the insets. Time calibrations for 2 and 400 ms applications and insets are 150 and 10 ms, respectively. Time calibrations for 4000 ms and inset are 1000 and 150 ms, respectively.

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Figure 7. Kinetic model for the 132L isoform A, a kinetic model for the 132L isoform was derived from steady-state single-channel analysis and rapid kinetic analysis of currents from outside-out membrane patches. B, the rate constants in the model were optimized to best fit the time course of the 13 macroscopic currents and the single-channel open, closed and burst properties (see Methods). Units for all rate constants were s-1 except for kon (m-1 s-1). C, the optimized model currents were superimposed on averaged 132L data traces for 2 ms (n = 6), 400 ms (n = 8), and 4000 ms (n = 6) applications of 1 mM GABA (application bars above traces). The same currents are depicted on an expanded time scale in the insets. Time calibrations for 2 and 400 ms applications and insets are 150 and 10 ms, respectively. Time calibrations for 4000 ms application and inset are 1000 and 150 ms, respectively.

The desensitization seen in macroscopic currents could have resulted from a redistribution of chloride ions during the course of the recordings, a redistribution of receptor conformations among relatively less stable short bound closed states, or the entrance of the receptor into more stable long-lived bound closed (desensitized) states. We examined the possibility of chloride ion redistribution by rapidly stepping a desensitizing current between -75 and 75 mV (reversal potental for Cl^-, E_Cl = 0 mV) and found symmetrical currents even after several seconds of GABA application (K. F. Haas & R. L. Macdonald, unpublished observations), ruling out chloride ion redistribution as an explanation.

In the 13 isoform model, the initial peak current was due primarily to initial synchronous brief O₁ openings, while the rapid phase of desensitization was caused by a rapid redistribution of receptors into the remaining doubly liganded states. To accurately reflect the slow macroscopic desensitization and the long closed periods seen in single-channel recordings, a desensitized state with very slow entry and exit rates was needed (D_s, Fig. 6A). Kinetic rate constants were optimized to best fit averaged current traces for the brief (2 ms) and prolonged (400 or 4000 ms) 1 mM GABA applications and the steady-state single-channel properties (Table 2). Traces simulated from the optimized model parameters (Fig. 6B) were overlaid on averaged current traces for the 13 isoform (Fig. 6C).

For the 132L isoform, the initial current peak was primarily due to synchronous intermediate O₂ openings. Three desensitized states (D_f, D_i and D_s; Fig. 7A) were needed to account for the triphasic desensitization during 4000 ms applications. Kinetic rate constants were optimized to fit best averaged current traces for the brief and prolonged 1 mM GABA applications and the steady-state single-channel properties (Table 2). Traces simulated from the optimized model parameters (Fig. 7B) were overlaid on averaged current traces for the 132L isoform (Fig. 7C). When the desensitized states were altered in placement, other configurations such as entry into desensitization from an open state, or alternating the positions of fast and slow desensitized states did not reproduce the measured pattern of desensitization.

Table 2. Single-channel properties of GABARs

	13		132L
	Measured	Simulated	Measured	Simulated
Number of patches	4	-	4	-
Number of openings	7565	10000	10535	10000
Conductance (pS)	23·8 ± 0·37	-	25·9 ± 0·60	-
NPo × 100 (%)	2·27 ± 0·57 *	1·40	10·5 ± 1·5	3·68
Mean open time (ms)	0·74 ± 0·11*	-	2·14 ± 0·07	-
Mean shut time (ms)	35·6 ± 8·1	-	21·0 ± 3·4	-
Open intervals
1 (ms)	0·33	0·34	0·30	-
	(0·31-0·34)	(0·31-0·36)	(0·22-0·35)	-
Area1 (%)	79·2	86·2	23·6	-
	(78·0-79·9)	(79·8-87·5)	(18·0-25·8)	-
2 (ms)	0·98	1·01	1·92	1·87
	(0·95-0·98)	(0·83-1·25)	(1·03-2·20)	(1·29-2·16)
Area2 (%)	20·8	13·9	48·0	67·1
	(20·6-21·0)	(13·5-14·3)	(45·5-51·0)	(63·5-68·5)
3 (ms)	-	-	3·47	3·62
	-	-	(2·86-4·49)	(2·90-4·84)
Area3 (%)	-	-	28·4	32·9
	-	-	(22·9-33·2)	(32·3-34·1)
Closed intervals
1 (ms)	0·27	0·27	0·20	0·21
	(0·22-0·30)	(0·23-0·32)	(0·17-0·22)	(0·19-0·23)
Area1 (%)	12·2	14·0	38·0	48·1
	(10·6-13·4)	(12·7-16·0)	(35·8-39·3)	(44·4-50·0)
2 (ms)	3·50	2·24	1·64	1·35
	(2·20-5·66)	(1·23-4·00)	(1·44-1·80)	(0·97-1·83)
Area2 (%)	20·9	9·0	35·7	15·9
	(18·2-22·2)	(8·4-9·8)	(33·1-37·0)	(13·9-17·6)
3 (ms)	15·2	10·5	8·89	8·21
	(12·3-37·9)	(9·3-12·2)	(7·81-9·89)	(6·89-9·85)
Area3 (%)	50·1	54·6	21·3	21·9
	(45·4-55·4)	(50·2-58·7)	(19·6-23·8)	(20·0-23·8)
4 (ms)	68·8	55·8	95·5	72·6
	(57·6-78·3)	(50·9-62·1)	(86·2-103)	(64·5-81·8)
Area4 (%)	15·8	22·2	4·0	12·7
	(13·6-19·2)	(19·1-25·2)	(3·8-4·4)	(11·5-14·2)
5 (ms)	1630	3990	990	4200
	(1470-1760)	(3720-4250)	(949-1030)	(3890-4530)
Area5 (%)	1·0	0·56	1·0	1·2
	(0·8-1·1)	-	(0·95-1·4)	(1·15-1·25)
Mean burst duration (ms)	1·51*	0·94	6·35	6·39
	(1·31-1·72)	(0·80-1·07)	(5·95-6·79)	(6·08-6·71)

Based on these kinetic models for the 13 and 132L isoforms, simulated single-channel data were generated (Table 2). For the 13 isoform, the model predicted similar open, closed and burst properties to those measured for 13 channels. The simulated P_o of 1·40 % was less than the measured NP_o of 2·27 %. The low P_o was achieved by relatively slow opening rates ( values), meaning that a large number of channels (210) were needed to produce a small macroscopic current (14 pA) (Fig. 6B and C). The single desensitized state (D_s) accounted for the slow macroscopic desensitization and for the infrequent long duration closings present in single-channel recordings. The longest closed duration for measured single-channel data would have been shortened by the presence of multiple channels, which probably explained the shorter measured (1630 ms) than simulated (3990 ms) mean duration for this long closed state.

For the 132L isoform, in most cases, the model predicted similar single-channel properties to those measured from 132L channels. The model accounted for the intermediate (O₂) and long duration (O₃) open states and their relative proportions (Table 2). Similar to the 13 isoform model, the simulated P_o (3·68 %) was lower than the measured NP_o (10·5 %). While the 132L model accounted for the durations and proportions of longer duration (O₂ and O₃) openings at 1 mM GABA, it could not account well for the number of brief (O₁) openings recorded at high GABA concentration. The simulated closed interval data were also fitted best by five closed states, for at high GABA concentrations, relatively minimal time was spent in the unbound or monoliganded states (C₁ and C₂), which could not be resolved from the brief distal closed states (C₅-C₁₀). The predicted mean burst duration (6·39 ms) was nearly identical to the measured mean burst duration (6·35 ms).

To illustrate the similarity in measured and simulated single-channel properties, several seconds of measured and simulated single-channel traces were juxtaposed (Fig. 8). These traces illustrated the relatively low open probability at high GABA concentrations for both measured and simulated single-channel currents. At these high GABA concentrations, unbound closed states would probably have been very brief, so that any long closed states most probably represented entry into a desensitized conformation (Fig. 8). The several second duration closures (**) most probably included a visit into D_i and D_s, while other more frequent intraburst long closures (*) most probably included entry into D_f (mean duration = 45·4 ms). Thus, desensitization probably produced the characteristic pattern of clusters of openings separated by long closed periods.

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Figure 8. Comparison of desensitization in measured and simulated single-channel currents A, single-channel GABAR currents during application of 1 mM GABA to an outside membrane patch containing 132L GABARs. The trace shown was a continuous 4·5 s recording filtered at 1 kHz for display. B, simulated single-channel currents at 1 mM GABA from the 132L isoform model. The trace shown was a continuous 4·5 s simulation. For both A and B, single asterisks denoted some of the closed states that probably involved entry into Df and the double asterisks denoted a long closed state that probably involved entry into Di and Ds.

These models should also explain the concentration-dependent changes in the current time course (Fig. 3). Simulated currents were generated at 3 µM and 1 mM GABA from the 13 kinetic model, and at 1 µM, 10 µM and 1 mM GABA from the 132L kinetic model (Fig. 9). These simulated currents were overlaid on normalized averaged currents from 13 (n = 4) and 132L (n = 6) isoforms, where multiple GABA concentrations were applied to the same patch. While developed and optimized for macroscopic kinetic and steady-state single-channel data evoked by 1 mM GABA, the model predicted the time courses of GABAR currents evoked by lower GABA concentrations. For the 13 currents, the simulated activation rate was somewhat slower than that measured at 3 µM GABA, but the comparison was difficult to make as only small currents were obtained at this concentration. For the 132L currents, the measured and simulated activation rates were nearly identical (Fig. 9, insets). Also, the nearly flat concentration dependence of desensitization rates for 132L currents was predicted by the model.

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Figure 9. Model predictions at different GABA concentrations A, the kinetic model developed for the 13 isoform was used to generate simulated currents for 4000 ms applications of 3 µM and 1 mM GABA. The simulated currents were overlaid on measured current responses at these same GABA concentrations. The measured current traces shown were averaged responses from three patches (3 µM) and four patches (1 mM) normalized to the 1 mM peak current values. B, the kinetic model developed for the 132L isoform was used to generate simulated currents for 4000 ms applications of 1 µM, 10 µM and 1 mM GABA. The simulated currents were overlaid on measured current responses at these same GABA concentrations. The current traces shown were normalized averaged responses from six patches where multiple GABA concentrations were applied to the same patch. A small increase (<10 %) in the peak amplitude of simulated current at lower GABA concentrations (3 µM for the 13 model; 1 and 10 µM for the 132L model) was needed to reproduce the amplitude of the measured currents. This was probably due to rundown during recordings. Insets show the same currents on an expanded time scale for comparison of activation rates at low GABA concentrations. Time calibrations for normal and expanded current traces are 1000 and 150 ms, respectively.

Model simulations

One important issue was whether the brief GABA applications used in this study could accurately mimic synaptic conditions. While the time course of GABA in the synaptic cleft has not been measured, predictions were made based on models of the time course of the glutamate at some excitatory central synapses (Clements, 1996). In a simple model, this transmitter time course has been predicted to rise to a peak concentration around 1 mM within 100 µs and decay with a monoexponential time course over several milliseconds. We generated simulated currents elicited by GABA with this predicted synaptic time course and compared the deactivation time course for 13 and 132L currents (Fig. 10). This comparison demonstrated only small differences between the deactivation with the synaptic time course and that with a 2 ms square pulse, although the difference was greater for the 13 currents. Nonetheless, the fitted deactivation rates and proportions were maintained, suggesting that the square pulse application protocol provided a good model for predicting the synaptic time course of recombinant GABAR currents.

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Figure 10. Simulated synaptic currents A simulated exponentially rising and decaying GABA time course (inset) was used to elicit simulated currents in the 13 and 132L kinetic models. The GABA time course was based on the predicted time course of GABA in the synaptic cleft with a peak concentration of 1 mM reached within 100 µs and a monoexponential decay with a time constant of 2 ms. These currents (dotted lines) were compared with currents elicited by a 2 ms square pulse 1 mM GABA application (continuous lines), illustrating the nearly identical current time courses with these modelled and simulated currents.

Another important issue was the role that desensitization played in shaping the deactivation of GABAR currents. For the 132L model, we compared the time course of deactivation with and without both fast and slow desensitization and also with varying rates of desensitization (Fig. 11). Even without desensitization, the decay of current following a brief pulse of GABA was biexponential (Fig. 11B). Fast desensitization (D_f) acted to blunt the peak current achieved and had an important role in prolonging the current decay, similar to the model presented by Jones & Westbrook (1995) for GABARs from cultured hippocampal neurons (Fig. 11C). Slow desensitization (D_i and D_s) acted as a current sink over this time course, blunting the degree of the slow component of decay (Fig. 11D). Varying the rate of entry into or exit from D_f also dramatically altered the rate of deactivation (Fig. 11E and F).

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Figure 11. Simulations addressing the role of desensitization For the 132L isoform, the role of fast and slow desensitization in shaping the current time course was assessed. A, using the optimized 132L kinetic model, a simulated current was generated by a 2 ms application of 1 mM GABA. B, a non-desensitizing current exhibited a larger peak amplitude and deactivated with a biexponential time course. C, the removal of fast desensitization (Df) increased the peak current amplitude and shortened the slow phase of deactivation. D, the removal of slow phases of desensitization (Di and Ds) increased the magnitude and duration of the slow phase of deactivation. E and F, varying the rate of entry into and exit from Df by 5-fold altered the shape of the deactivation time course. The original current time course is also shown (dotted line).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

We used rapid GABA application to outside-out membrane patches containing 13, 13 and 132L GABAR isoforms to evaluate the contributions of the 2L and subunits to the rapid activation, deactivation and desensitization of recombinant GABARs. The 13 currents activated relatively slowly, but exhibited rapid and nearly complete desensitization. Addition of the subunit substantially decreased the rate and extent of desensitization. In contrast, addition of the 2L subtype increased activation rate and changed the pattern of desensitization. For the 13 and 132L isoforms, steady-state single-channel and rapid kinetic data were used to develop more comprehensive models of GABAR kinetic behaviour that began to reconcile microscopic and macroscopic kinetics.

Effect of receptor composition on deactivation

The net rate of deactivation was isoform dependent, with both 13 and 13 currents deactivating more rapidly than 132L currents. This result was primarily due to a longer, more pronounced slow deactivation component to 132L currents. The deactivation kinetics of 13 currents were very similar to those of recombinant 632L currents that did not show any rapid desensitization in HEK 293 cells (Tia et al. 1996). This corroborated the finding that a homogeneous population of non-desensitizing receptors still deactivated with a biphasic time course. Moreover, the more rapid deactivation of 13 relative to 132L currents was similar to that reported for recombinant 12 vs. 122L receptors expressed in HEK 293 cells (Tia et al. 1996). Our findings demonstrated that the 2L and subunit subtypes conferred unique biophysical properties on GABARs which were evident over a synaptically relevant time course.

Effect of receptor composition on activation rate

The more rapid activation of the 132L currents initially seemed paradoxical as this isoform had a higher whole-cell GABA EC₅₀ than the 13 and 13 isoforms. However, at the microscopic kinetic level, models of the 13 and 132L currents readily explained this activation rate difference on the basis of GABA binding and gating rates. The predominant determinant of activation rate differences was a slower opening rate for 13 receptors since the modelled binding (k_on) rates were nearly identical.

As neurons integrate many inputs over time, the rapidity with which maximum inhibitory drive is achieved following GABA release could potentially be an important receptor property (Maconochie et al. 1994). If the GABA time course at the synapse was prolonged, the more than 3-fold faster activation rate conferred by the introduction of the 2L subtype could be important in determining the extent and type of inhibition achieved.

Effect of receptor composition on desensitization

The rate and extent of rapid desensitization showed isoform dependency. Our finding of minimal desensitization of 13 currents was similar to that described for recombinant 632L GABAR currents in HEK 293 cells (Tia et al. 1996). While we have been unable to duplicate these findings with 632L receptors in our cell system (K. F. Haas & R. L. Macdonald, unpublished observations), the implications of a non-desensitizing receptor remain the same. Desensitization could play a critical role in the repetitive activation of postsynaptic GABARs, where repetitive high frequency inhibitory responses would be attenuated for desensitizing receptor combinations, but not for non-desensitizing receptors.

Recent evidence suggested, however, that subunit-containing receptors may be primarily extrasynaptic on the soma of cerebellar granule neurons (Nusser et al. 1998). While the subunit-containing isoform present in hippocampal dentate granule neurons remains unknown, it most probably is not an 13 combination (Jones et al. 1997). It remains to be demonstrated whether all subunit-containing combinations exhibit similar rapid desensitization kinetics, but a non-desensitizing extrasynaptic receptor with high GABA affinity would be ideally suited for providing tonic inhibition.

The more rapid fast phase of desensitization in 132L currents than in 13 currents was qualitatively similar to that seen when the same isoforms were studied in HEK 293 cells (Dominguez-Perrot et al. 1997). The more prominent slow phase of desensitization in 132L currents, however, contributed to the trend towards a slower mean desensitization rate for 132L currents and may explain their slower desensitization rate in other whole-cell studies (Fisher & Macdonald, 1997).

Many studies have demonstrated that desensitization plays a critical role in shaping the deactivation time course of native GABAR responses in outside-out patch currents and neuronal IPSCs (Jones & Westbrook, 1995; Galarreta & Hestrin, 1997; Mellor & Randall, 1997). Moreover, a greater degree of fast desensitization has been linked to a prolonged slow phase of deactivation in cultured hippocampal neurons (Jones & Westbrook, 1995). This finding is supported by our results showing slower deactivation of the more rapidly desensitizing 132L currents relative to the less desensitizing 13 currents. However, this did not explain the more rapidly deactivating, yet highly desensitizing 13 currents. 13 currents may recover more rapidly from desensitization, leading to a more rapid deactivation rate, but this would need to be confirmed by paired-pulse experiments to examine recovery from desensitization.

2L and subunit structural determinants of rapid kinetic properties

The more rapid activation rate conferred by the 2L subtype could have been due to more rapid binding of GABA to the receptor, more rapid coupling of binding to gating, and/or to a more rapid gating rate. The GABA binding pocket has been suggested to be on the subunit, but EC₅₀ differences between isoforms have demonstrated that binding, coupling, and/or gating are influenced by other subunit families. Our models predicted similar binding rates (k_on) for 13 and 132L GABARs, and the activation rates were relatively insensitive to the unbinding (k_off) rates. The opening rate to the predominant open state for the 132L isoform (1800 s^-1), however, was much faster than that for the 13 isoform (80 s^-1). Thus, the models we developed for the 13 and 132L isoforms predicted that gating played the definitive role in the activation rate differences.

The structural determinants of activation and desensitization gating seem likely to be found in the subunit region spanning the first and second transmembrane domains (TM1 and TM2), which includes a short cytoplasmic loop linking TM1 and TM2. Previous studies have identified residues at M2 positions 5', 9' and 12' that influence macroscopic desensitization rates of recombinant 11 GABAR currents (Tierney et al. 1996; Birnir et al. 1997a,b), but amino acids at these positions are conserved between and 2L subtypes. Sequence alignments of and 2L subtypes revealed differences in multiple amino acids toward the extracellular end of the M1 domain and differences in one neutral and two charged residues in the TM1-TM2 linker (Tyndale et al. 1995). The M2 domains were relatively similar except for substitution of a threonine for a valine at the 1' position and a region of variability towards the extracellular end of the M2 domain. While speculation about the role of these differences would be premature, the domains of the and subunits that confer the binding and gating differences should be further tested with / subunit chimeras and site-specific mutagenesis.

Reconciling whole-cell and outside-out patch macroscopic data

For meaningful interpretation of both whole-cell and macroscopic outside-out patch data, the correlation of the two must be understood. For both 13 and 132L currents, the slow phases of desensitization revealed by 4000 ms GABA applications were similar in duration to the most prominent phase of desensitization in whole-cell studies, lending credence to the comparison between macropatch and whole-cell macroscopic kinetics for these recombinant GABARs. The rapid desensitization of 13 and 132L currents predicted that desensitization attenuated up to 60 % of the true peak current during the 50-100 ms needed for GABA to reach its peak concentration in whole-cell studies of the same isoforms (Fisher & Macdonald, 1997). Even more rapid whole-cell applications (Gingrich et al. 1995; Dominguez-Perrot et al. 1996) probably were not fast enough to resolve the most rapid phase of desensitization found here in 132L currents. Rapid application studies, however, predicted that this rapid phase of desensitization was probably the most important factor in shaping the synaptic time course of synaptic GABAR currents (Jones & Westbrook, 1995; Tia et al. 1996; Mellor & Randall, 1998; current study). Thus, the outside-out patch desensitization kinetics correlated with whole-cell desensitization kinetics, but, with the more rapid application, synaptically relevant faster phases of desensitization were resolved.

Reconciling macroscopic and single-channel data

Since any macroscopic current was simply the result of the underlying activity of man