Skip to main content
PMC home page
The Journal of Physiology logoLink to The Journal of Physiology
. 2004 Oct 7;561(Pt 2):433–448. doi: 10.1113/jphysiol.2003.051839

The α1 and α6 subunit subtypes of the mammalian GABAA receptor confer distinct channel gating kinetics

Janet L Fisher 1
PMCID: PMC1665367  PMID: 15579538

Abstract

The GABAA receptors show a large degree of structural heterogeneity, with seven different subunit families, and 16 different subtypes in mammalian species. The α family is the largest, with six different subtypes. The α1 and α6 subtypes are among the most diverse within this family and confer distinct pharmacological properties to recombinant and neuronal receptors. To determine whether different single channel and macroscopic kinetic properties were also associated with these subtypes, the α1 or α6 subunit was expressed in mammalian cells along with β3 and γ2L subunits and the kinetic properties examined with outside-out patch recordings. The α1β3γ2L receptors responded to GABA with long-duration openings organized into multi-opening bursts. In contrast, channel openings of the α6β3γ2L receptors were predominately short in duration and occurred as isolated, single openings. The subunit subtype also affected the deactivation rate of the receptor, which was almost 2-fold slower for α6β3γ2L, compared with the α1β3γ2L isoform. Onset of fast desensitization did not differ between the isoforms. To determine the structural domains responsible for these differences in kinetic properties, we constructed six chimeric subunits, combining different regions of the α1 and α6 subunits. The properties of the chimeric subunits indicated that structures within the third transmembrane domain (TM3) and the TM3–TM4 intracellular loop conferred differences in single channel gating kinetics that subsequently affected the deactivation rate and GABA EC50. The effect of agonist concentration on the rise time of the current showed that the extracellular N-terminal domain was largely responsible for binding characteristics, while the transmembrane domains determined the activation rate at saturating GABA concentrations. This suggests that subunit structures outside of the agonist binding and pore-lining domains are responsible for the kinetic differences conferred by the α1 and α6 subtypes. Structural heterogeneity within these transmembrane and intracellular regions can therefore influence the characteristics of the postsynaptic response of GABAA receptors with different subunit composition.

The GABAA receptor (GABAR) mediates the majority of fast inhibitory synaptic transmission in the central nervous system (CNS). Mammalian GABARs are composed of pentameric combinations of α(1–6), β(1–3), γ(1–3), δ, ɛ, π or θ subunit subtypes. The pharmacological and physiological properties of GABARs are largely determined by their subunit and subtype composition (Mehta & Ticku, 1999; Korpi et al. 2002). Among the six different α subtypes, the α1 and α6 subtypes are the most divergent. The regional expression of α1 and α6 subunit mRNAs in the mammalian brain is different, suggesting distinct physiological roles for these subtypes. The α1 is the most widely expressed of all the α subtypes, and shows a high level of expression in most regions of the brain. Nearly half of all native GABARs in the adult brain are believed to contain an α1 subunit (McKernan & Whiting, 1996). The expression of the α1 subtype mRNA is developmentally regulated, increasing in virtually all brain regions from low levels in embryonic rat to higher levels in the adult (Laurie et al. 1992a). In contrast, the expression of the α6 subtype is restricted to the granule cells of the cerebellum and cochlea nuclei of the adult (Laurie et al. 1992b; Varecka et al. 1994) where it contributes to both synaptic and extrasynaptic populations (Nusser et al. 1998).

Substantial effort has focused on describing the pharmacological properties of recombinant GABARs composed of different subunit subtypes and determining the structures responsible for these properties (Korpi et al. 2002). However, much less is known about the effect of subtype composition on the single channel properties of these receptors. Among the α subtypes, only the properties of receptors containing α1 or α4 subunits have been described at the single channel level (Angelotti & Macdonald, 1993; Fisher & Macdonald, 1997; Haas & Macdonald, 1999; Akk et al. 2004). Gating characteristics of single channels influence the amplitude, shape and duration of the postsynaptic current. Much of the functional heterogeneity of native GABARs may be due to differences in the subunit composition of the receptors but because most neurones produce a number of different GABAR subunit subtypes, channel characteristics of native receptors cannot be readily assigned to a particular isoform. Previous studies from recombinant receptors have shown that the α subtype influences whole-cell desensitization and deactivation rates (Gingrich et al. 1995; Burgard et al. 1996; Tia et al. 1996a, 1996b; Lavoie et al. 1997; McClellan & Twyman, 1999; Bianchi et al. 2002). Additionally, results from transgenic animals suggest that the α1 subunit is necessary for a developmental shift in decay rate observed in many brain regions, supporting the idea that changes in the relative levels of α subtypes alter the kinetic properties of the receptors (Vicini et al. 2001; Goldstein et al. 2002; Koksma et al. 2003). Pathological conditions such as anxiety and epilepsy are also associated with changes both in α subtype expression and IPSC decay kinetics (Smith et al. 1998; Hsu et al. 2003; Cagetti et al. 2003).

The α1 and α6 subtypes share the least structural homology of the α family members (59% amino acid identity) (Lüddens et al. 1990; Tyndale et al. 1995). Which of the structural differences among the subtypes are functionally important in determining the unique channel characteristics associated with each receptor isoform? Determining the structures that regulate these properties will not only increase our understanding of the physical mechanisms that underlie channel function, but will also suggest potential targets for the development of drugs selective for these receptor isoforms. Most of the mutational analysis of GABAR subunits has examined agonist binding and transduction sites for GABA and allosteric modulators (Mehta & Ticku, 1999; Korpi et al. 2002), and little is known about the non-conserved sites that contribute to the unique characteristics associated with different subunits and subtypes.

The differences in the amino acid sequences between α1 and α6 subtypes occur primarily in the large extracellular N-terminal domain and in the intracellular loop between the third and fourth transmembrane domains (TMs), while the TMs themselves and the TM2–TM3 extracellular domain are highly conserved (83% amino acid identity). To determine the structural domains responsible for the different kinetic properties associated with the α1 and α6 subtypes, chimeric constructs of the rat α1 and α6 subunits were created with three different splice sites (Fig. 1). Plasmids containing the wild-type subunits or chimeric constructs were transiently transfected along with β3 and γ2L subunits into a human embryonic kidney cell line (HEK-293T) and the receptor properties were measured through whole-cell, single channel and rapid application recordings.

Figure 1. Locations of the splice sites for generation of the chimeric subunits.

Figure 1

Open in a new tab

Schematic representation of a GABAR subunit. The GABAR subunit structure includes four transmembrane domains (TM1–TM4), a large N-terminal extracellular domain, and a large intracellular domain between TM3 and TM4. The splice sites for the chimeric subunits were located within TM1 (site A), on the extracellular side of TM3 (site B), and on the intracellular side of TM4 (site C) as shown by the arrows.

Methods

Construction of chimeric subunits

Full-length cDNAs for rat α1, α6, β3 and γ2L GABAR subunits were obtained in the mammalian expression vectors pCMVneo (Robert Macdonald, Vanderbilt University). The A-site chimeras have already been characterized at the whole-cell level and the methods for construction of the chimera published (Fisher et al. 1997). To produce the other chimeric subunits, α1 and α6 cDNAs were subcloned into the pcDNA1.1/Amp plasmid (Invitrogen). Chimeras at splice sites B and C (Fig. 1) were created by taking advantage of unique restriction sites at these locations in the α1 and α6 subunits. In each case a single mutation was required in one subunit to either create or remove a restriction site. Mutations were made in the GABAR subunit cDNA using a commercial kit (QuikChange, Stratagene) and verified with sequencing of both strands. These mutations were silent, changing the nucleotide sequence but not the amino acid sequence. Once these restriction sites were in place, the plasmids were cut to release the section of the subunit from the splice site through a portion of the vector. The fragments were then gel-purified, swapped and re-ligated. Creation of the expected chimera was verified by sequencing. For the B-site chimera, a Nco I site was utilized at the start of TM3. This was a unique site in the α6 subunit. An additional Nco I site was removed from the α1 subunit with a CCT to CCC mutation, encoding a proline in both cases. For the C-site chimera, a unique Bsp106I site at the start of the TM4 was created in the α6 subunit with an ATA to ATC mutation, encoding an isoleucine residue in both cases. To produce this same restriction site in the α1, GAC was mutated to GAT, both encoding an aspartate residue.

Transfection of HEK-293T cells

Wild-type or chimeric α subunits were transiently transfected along with β3 and γ2L subunits into the human embryonic cell line HEK-293T (Genhunter, Nashville, TN, USA). The β3 and γ2L subtypes were selected for co-expression because they are found at high levels in most regions of the brain and may therefore potentially represent native isoforms. In addition, the kinetic properties of the α1β3γ2L isoform have been well described, and these previous reports provide a useful background for comparison (Fisher & Macdonald, 1997; Haas & Macdonald, 1999).

For selection of transfected cells, the plasmid pHook-1 (Invitrogen, San Diego, CA, USA) containing cDNA encoding the surface antibody sFv was also transfected into the cells. Cells were maintained in Dulbecco's modified Eagle medium (DMEM) plus 10% fetal bovine serum, 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin. Cells were passaged by a 5 min incubation with 0.05% trypsin−0.02% EDTA solution in phosphate-buffered saline (10 mm Na2HPO4, 150 mm NaCl, pH = 7.3).

The cells were transfected using a modified calcium phosphate method (Angelotti et al. 1993). Plasmids encoding GABAR subtype cDNAs were added to the cells in 1 : 1 ratios of 2 μg each plus 1 μg of the plasmid encoding sFv. Following a 4–6 h. incubation at 3% CO2, the cells were treated with a 15% glycerol solution in BBS buffer (50 mm Bes (N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid), 280 mm NaCl, 1.5 mm Na2HPO4) for 30 s. The selection procedure for sFv antibody expression was performed 20–28 h later as described in Greenfield et al. (1997). Briefly, the cells were passaged and mixed with 3 μl of magnetic beads coated with hapten (approximately 4.5 × 105 beads) (Chesnut et al. 1996). Following 30–60 min of incubation to allow the beads to bind to positively transfected cells, the beads and bead-coated cells were isolated using a magnetic stand. The selected cells were resuspended into DMEM, plated onto poly-lysine-treated glass coverslips coated with collagen and used for recording 18–28 h later.

Electrophysiological recording solutions and techniques

For all recordings the external solution consisted of (mm): 142 NaCl, 8.1 KCl, 6 MgCl2, 1 CaCl2, 10 glucose and 10 Hepes (4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid) with pH = 7.4 and osmolarity adjusted to 295–305 mosmol l−1. Recording electrodes were filled with an internal solution of (mm): 153 KCl, 1 MgCl2, 5 K-EGTA (ethylene glycol-bis (β-aminoethyl ether N,N,N′N′-tetraacetate), 2 MgATP and 10 Hepes with pH = 7.4 and osmolarity adjusted to 295–305 mosmol l−1. These solutions provided a chloride equilibrium potential near 0 mV. For single channel recordings the electrodes were coated with polystyrene Q-dope (GC Electronics). GABA was diluted into external solution from freshly made or frozen stocks in water. Patch pipettes were pulled from borosilicate glass with an internal filament (World Precision Instruments, Sarasota, FL, USA) on a two-stage puller (Narishige, Japan) to a resistance of 5–10 MΩ. For whole-cell recordings and single channel recordings GABA was applied to cells or excised outside out patches using a stepper solution exchanger with a complete exchange time of < 50 ms (open tip, SF-77B, Warner Instruments, Hamden, CT, USA). For macropatch rapid-application recordings the 3-barrel square glass was pulled to a final size near 200 μm. The 10–90% rise times of the junction potential at the open tip were consistently faster than 400 μs and were tested after each patch recording using a diluted external solution. There was a continuous flow of external solution through the chamber. Currents were recorded with an Axon 200B (Foster City, CA, USA) patch clamp amplifier and stored on hard drive for off-line analysis. All experiments were performed at room temperature (near 25°C).

Analysis of currents

Whole-cell currents were analysed using the programs Clampfit (pCLAMP8 suite, Axon Instruments, Foster City, CA, USA) and Prism (Graphpad, San Diego, CA, USA). Concentration–response data were fitted with a four-parameter logistic equation:

graphic file with name tjp0561-0433-m1.jpg

where n represents the Hill number. Fits were made to normalized data with current expressed as a percentage of the maximum response to GABA for each cell.

Macropatch currents were digitized at 10 kHz and analysed with the pCLAMP8 suite of programs (Axon Instruments). The desensitization or deactivation rate was determined by fitting the decay current with the Levenberg-Marquardt least squares method with one or two exponential functions, as determined by a significant improvement of the fit with additional components (F test of the sum of squared residuals). Weighted mean decay times were calculated by multiplying each time constant by its relative area.

Single channel currents were recorded on digital tape for offline analysis. Recordings were digitized at 20 kHz and filtered at 2 kHz. Open and closed intervals were detected with a 50% threshold method, with a transition recorded every time the current passed a threshold set at one-half of the amplitude of the main conductance level (Fetchan, pCLAMP8). Sub-conductance openings were rarely observed (< 0.1% of openings), and were not included in the analysis. Stability plots of open probability showed no time-dependent changes in channel activity. Analysis of the event dwell-times was performed with the Interval 5 program (Dr Barry Pallotta, University of North Carolina-Chapel Hill) using the maximum likelihood fitting method. The number of components required to fit the interval histograms was increased until an additional component did not significantly improve the fit (Horn, 1987). Confidence limits for the components of each fit were calculated by the method of m-unit likelihood intervals (Colquhoun & Sigworth, 1995). To define bursts the briefest one or two closed components were considered intraburst closures. The critical gap was calculated from the closed interval histogram for each patch using a method that equalized the proportions of misclassified events (Colquhoun & Sakmann, 1985).

Tukey-Kramer multiple comparisons tests were performed using the Instat program (Graphpad) with a significance level of P < 0.05. The logs of the GABA EC50 measurements were used for statistical comparison.

Results

The α1 and α6 subtypes confer different single channel kinetic properties

The α1 and α6 subtypes of the GABAR show distinct patterns of distribution throughout the brain and through development. These subtypes confer very different pharmacological properties to recombinant and native receptors. While some studies have examined the kinetic properties associated with these subunits at the whole-cell level, the single channel characteristics of receptors containing the α6 subtype have not been described. Channel gating properties influence the amplitude and duration of the postsynaptic response to GABA. Therefore, differences in these properties due to varying subunit composition could alter the effectiveness of GABAergic neurotransmission.

To compare the kinetic properties of α1β3γ2L and α6β3γ2L receptor channels, outside-out patches were exposed to submaximal (∼EC20–30) GABA concentrations for each isoform (10 μm for α1β3γ2L and 1 μm for α6β3γ2L). The durations of channel openings and closures were obtained from steady-state recordings (5–10 min). The α1β3γ2L receptors were characterized by relatively long-duration openings, which occurred in a pattern of bursts (Fig. 2) (Fisher & Macdonald, 1997). In contrast, the α6β3γ2L channel openings were relatively brief in duration, and tended to occur as isolated single openings rather than in bursts.

Figure 2. Single channel characteristics of receptors containing α1 or α6 subunit subtypes.

Figure 2

Open in a new tab

A, single channel GABAR currents were obtained from outside-out patches. Upper traces are continuous 10 s recordings from patches in response to 1 μm (α6β3γ2L) or 10 μm GABA (α1β3γ2L). The lower trace shows a 2 s segment extracted from the upper trace, as indicated by the bar. Patches were held at −100 mV. In this and all subsequent figures, channel openings are downward. For display the data were sampled at 1 ms per point and filtered at 100 Hz. B, duration histograms of open and closed intervals from individual patches were fitted with the sum of three to five exponential functions. For α1β3γ2L, the individual components for the open interval histogram shown had time constants (and relative areas) of 0.54 ms (0.485), 2.88 ms (0.304), and 5.64 ms (0.211) with 5377 intervals and a mean open time of 2.33 ms. The closed interval histogram was fitted with five components with time constants (and relative areas) of 0.28 ms (0.639), 1.48 ms (0.170), 7.8 ms (0.10), 123.3 ms (0.072) and 507.7 ms (0.019). For α6β3γ2L the open interval histogram shown was fitted with three components with time constants (and relative areas) of 0.40 ms (0.829), 1.21 ms (0.164) and 8.96 ms (0.007) with 2454 intervals and a mean open time of 0.59 ms. The closed interval histogram was fitted with four components with time constants (and relative areas) of 0.61 ms (0.107), 16.05 ms (0.396), 50.7 ms (0.446) and 283.0 ms (0.051).

The open duration histograms for the α1 subtype-containing channels were best fitted with the sum of three exponential distributions, and all three of the open states made a substantial contribution to the total area (Fig. 2B, Table 1, 1226–5377 open intervals per patch). However, the α6 subtype-containing receptors only rarely exhibited a third, long-duration open state. Although open interval histograms from some (3 of 7) of the α6β3γ2L patches were best fitted with three distributions, the longest open state contributed less than 1% of the area in all cases (Fig. 2B, Table 1, 2454–5278 open intervals per patch). Therefore, the mean open time of the α6β3γ2L receptors was significantly briefer than the α1β3γ2L receptors (Table 1). The relative areas of all the open states were significantly different between the receptor isoforms (Table 1). The time constant (τ) of the second longest open state was also significantly shorter for the α6β3γ2L receptor. This may reflect difficulty in accurately fitting components with low relative area, or may indicate a true difference in the stability of this open conformation. When examining only the histograms fitted best with three components, the 95% confidence limits for the time constant of the second open state showed a larger range (0.555–7.964 ms, mean of 1.15 ms) than the fits to the other states, which were typically less than 1.5 times the mean. For example, the 95% confidence limits for the longest open state in these histograms had a range of 4.05–6.68 ms (mean of 5.53 ms). When all open histograms from α6 subunit-containing receptors were fitted with only two exponential distributions, the average time constant for the longer open state was not significantly different from the 2nd longest open state of the α1β3γ2L channels.

Table 1.

Open interval properties

τ1 (ms) (% area) τ2 (ms) (% area) τ3 (ms) (% area) Mean open time (ms)
α1β3γ2L 0.53 ± 0.03 3.30 ± 0.44† 6.35 ± 0.45 2.41 ± 0.27†††
(n = 6) (50.5 ± 2.8%)††† (32.9 ± 2.5%)††† (16.6 ± 2.1%)†††
α6β3γ2L 0.36 ± 0.03 1.48 ± 0.29* 5.53 ± 1.8 0.46 ± 0.05***
(n = 7) (91.3 ± 2.2%)*** (8.0 ± 2.1%)*** (0.7 ± 0.5%)***
α1/α6-A 0.37 ± 0.03 1.42 ± 0.15* 5.22 ± 3.0 0.47 ± 0.04***
(n = 6) (91.9 ± 1.3%)*** (7.8 ± 1.3%)** (0.3 ± 0.2%)***
α1/α6-B 0.34 ± 0.05 1.37 ± 0.25* 6.17 ± 1.8 0.42 ± 0.08***
(n = 5) (93.0 ± 2.5%)*** (6.8 ± 2.5%)** (0.2 ± 0.1%)***
α1/α6-C 0.47 ± 0.06 2.88 ± 0.38† 5.86 ± 0.30 2.42 ± 0.30†††
(n = 6) (44.9 ± 4.1%)††† (35.7 ± 2.1%)††† (19.4 ± 2.5%)†††
α6/α1-A 0.36 ± 0.06 2.27 ± 0.16 5.84 ± 0.99 2.25 ± 0.36†††
(n = 5) (36.5 ± 5.0%)††† (46.6 ± 7.9%)††† (16.9 ± 5.1%)†††
α6/α1-B 0.52 ± 0.05 2.65 ± 0.64 6.04 ± 2.0 1.82 ± 0.26†††
(n = 5) (50.1 ± 8.3%)††† (37.6 ± 8.8%)††† (12.3 ± 1.9%)††
α6/α1-C 0.39 ± 0.02 1.57 ± 0.17* 5.23 ± 0.82 0.45 ± 0.27***
(n = 6) (95.2 ± 0.8%)*** (4.3 ± 0.9%)*** (0.5 ± 0.3%)***
Open in a new tab

Significantly different from α1β3γ2L:

*

P < 0.05

**

P < 0.01

***

P < 0.001.

Significantly different from α6β3γ2L:

†

P < 0.05,

††

P < 0.01

†††

P < 0.001.

The closed interval histograms for the α1β3γ2L channels were best fitted with the sum of five exponential distributions (Fig. 2B). Because the durations and relative contributions of longer closed states are subject to greater error with multi-channel patches, only the briefer closures and burst properties were examined in detail. The two briefest components, generally considered intraburst closures, made a large contribution (65.3 ± 3.9%, n = 6) to the total area. The histograms for the α6β3γ2L isoform were fitted with four or five components, and often lacked one of the two briefest components compared with α1β3γ2L (Fig. 2B). In contrast to receptors containing the α1 subtype, the shorter closed components (τ < 10 ms) made a minor contribution to the total area, averaging 19.8 ± 5.2% (n = 7, P < 0.001 compared with α1β3γ2L), consistent with the isolated appearance of these channel openings. As with the longest open state, the rare relative occurrence of these brief closures led to difficulty in discriminating these components and accurately fitting their time constants.

To examine the burst properties of these isoforms, a critical gap (τcrit) representing the termination of bursts was defined between the brief closed components. For the α1β3γ2L receptor and for α6β3γ2L patches fitted with five exponential distributions, the two shortest components were assigned as intraburst closures. For histograms from α6β3γ2L receptors fitted with four components, only the briefest was considered intraburst since the next shortest component had a τ greater than 10 ms, and was therefore more comparable to the third-longest closed component of the α1β3γ2L receptor. Using these techniques, the τcrit was not significantly different between the isoforms, with an average of 1.96 ± 0.17 ms for α1β3γ2L and 2.10 ± 0.28 ms for α6β3γ2L (P > 0.05). Distributions of burst durations were fitted with the sum of three exponential distributions, and distributions of openings/burst were fitted with the sum of two to three geometric functions. The α1β3γ2L receptors showed significantly longer average burst durations (6.91 ± 1.05 ms, n = 6) compared with the α6β3γ2L receptors (0.96 ± 0.19 ms, n = 7). The average number of openings per burst was also higher for α1β3γ2L receptors (2.49 ± 0.25 openings, n = 6) compared with α6β3γ2L (1.41 ± 0.09 openings, n = 7).

At the submaximal GABA concentration used here, the α6β3γ2L receptors rarely enter long open states. For the α1β3γ2L isoform, higher GABA concentrations lead to a greater proportion of longer open states (Fisher & Macdonald, 1997). The longer open states are also associated with longer bursts, both in mean duration and in the number of openings per burst (Twyman et al. 1990). Therefore, higher GABA concentrations also lead to a greater contribution from the briefer closed states, which are best modelled as terminal states connected to each of the open states (Twyman et al. 1990; Haas & Macdonald, 1999). If the burst structure for the α6β3γ2L receptor channels is similar to that for the α1β3γ2L isoform, higher GABA concentrations might lead to an increased contribution from longer open states, leading to greater openings per bursts and more brief closures. This could clarify the time constants for these components of the closed and open interval histograms.

Design of α1/α6 chimeric subunits

These results show that the α1 and α6 subtypes confer distinct channel gating kinetics to recombinant receptors. To examine the structural differences between the subunits responsible for these characteristics, chimeric subunits of the α1 and α6 subunits were designed to isolate the contributions of the transmembrane domains. The chimeric subunits contained the structure of one of the α subtypes up to the splice site, with the remainder of the subunit structure from the other subtype. The splice sites were located within TM1 (site A), at the N-terminal side of TM3 (site B) and at the N-terminal side of the TM4 (site C) (Fig. 1). The region of the subunit between splice sites A and B included the TM2 and a short extracellular domain between TM2 and TM3. The region between splice sites B and C spanned TM3 and the large intracellular loop between TM3 and TM4. For the C-site chimeras only the TM4 and the short extracellular C-terminal domain was contributed from the other subtype.

Single channel properties of receptors containing chimeric subunits

Steady-state recordings were made from outside-out patches at a holding potential of −100 mV in response to continuous application of an EC20–30 GABA concentration for each isoform. The channel openings produced by the α6/α1-Aβ3γ2L receptor in response to 0.3 μm GABA were similar to the α1β3γ2L isoform, occurring in a series of bursts, with a significant contribution from long-duration openings (Fig. 3). Fits of open interval histograms showed that these channels produced three open states, with average durations and relative contributions comparable to those seen with the α1β3γ2L isoform (Table 1). The burst characteristics were also similar to the α1 subtype-containing receptors (Fig. 3B). None of these properties were significantly different from the α1β3γ2L receptor (P > 0.1). In contrast, the α1/α6-A chimeric subunit, which contained the N-terminal domain of the α1 subtype, produced channel openings that were predominantly brief in duration (Fig. 3). The open state and burst characteristics were similar to receptors containing the wild-type α6 subunit (Table 1, Fig. 3B) and none of the kinetic properties were significantly different from α6β3γ2L (P > 0.1). The large extracellular domain is responsible for much of the structural heterogeneity between the α subtypes; however, it does not appear to make a significant contribution to the differences in single channel kinetics. Therefore, structural differences within the agonist binding sites are not likely to be responsible for the differences between the α1 and α6 subunits in channel gating characteristics.

Figure 3. Single channel and burst properties of receptors containing chimeric subunits.

Figure 3

Open in a new tab

A, traces shown are continuous 1 s recordings from outside-out patches in response to steady-state applications of 0.3 μm (α6/α1-A, α6/α1-B), 1 μm (α6, α6/α1-C), 10 μm (α1, α1/α6-C) or 30 μm GABA (α1/α6-A, α1/α6-B). Patches were voltage clamped at −100 mV. For display the data were sampled at 500 μs per point and filtered at 100 Hz. Schematics of the chimeric constructs are shown to the left, with boxes indicating the transmembrane domains. B, mean burst durations were calculated by weighting the time constants from the fit of the histograms by their relative areas. Histograms were typically fitted with three components. The average number of openings per burst were calculated by a weighted mean of the fit of the openings/burst distributions, which were typically fitted with the sum of two to three geometric components. Bars represent means ± s.e.m. and the numbers in parentheses indicate the number of patches. Dotted lines represent the mean of the wild-type receptors. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate a significant difference from α1β3γ2L. †P < 0.05, ††P < 0.01 and †††P < 0.001 indicate a significant difference from α6β3γ2L.

The role of the TM2 and its flanking domains was examined with the B-site chimeras, which exchanged the N-terminal domain of the subunits through the start of TM3. Like the A-site chimeric subunits, the α6/α1-Bβ3γ2L receptors were similar to α1β3γ2L receptors in their channel properties, with the longest of three open states making a significant contribution to the area of the open interval histogram (Fig. 3, Table 1). The burst characteristics were also indistinguishable from receptors containing the α1 subtype (Fig. 3B). None of these kinetic properties were significantly different from the α1β3γ2L receptor (P > 0.1). Again, as with the A-site chimera, the α1/α6-B chimeric subunit conferred single channel characteristics similar to the α6β3γ2L receptor, with predominantly brief-duration, isolated openings (Fig. 3, Table 1). The open state and burst properties were not significantly different from the α6β3γ2L isoform.

The C-site chimeras examined the contribution from TM4 to the single channel characteristics. Receptors containing the α6/α1-C chimeric subunit had single channel properties similar to those with the α6 subunit, with isolated, brief-duration openings (Fig. 3, Table 1). The open state and burst properties were not significantly different from the α6β3γ2L receptor (P > 0.1). Channel openings from α1/α6-Cβ3γ2L receptors were similar to those containing the α1 subtype, with long-duration openings occurring in a pattern of bursts (Fig. 3, Table 1). None of the single channel kinetic properties differed significantly from the α1β3γ2L receptor (P > 0.1).

These findings suggest that the region of the subunit that includes TM3 and the TM3–TM4 intracellular domain contains the structural differences responsible for the unique kinetic properties associated with the α1 and α6 subtypes. The N-terminal extracellular domain and the 1st, 2nd and 4th transmembrane domains do not appear to contribute to the differences in open state or burst properties between the α1 and α6 subtypes. Additional chimeric constructs exchanging only the TM3 and TM3–TM4 domains would be necessary to confirm that interactions with other regions do not contribute to these single channel properties.

GABA sensitivity of receptors containing α1/α6 chimeric subunits

Consistent with previous reports (Ducic et al. 1995; Saxena & Macdonald, 1996; Fisher et al. 1997), the α1β3γ2L and α6β3γ2L isoforms differed in their GABA sensitivity. GABA was applied for 5 s to cells voltage-clamped at −50 mV in the whole-cell recording configuration. The GABA EC50 was about 10 times higher for receptors containing the α1 subtype compared with those with the α6 subtype (Fig. 4). The average GABA EC50 for the α1β3γ2L receptors was 18.5 ± 1.7 μm (n = 5), while that of the α6β3γ2L receptors was 1.8 ± 0.22 μm (n = 7). To examine the structural differences between the subunits that contribute to this pharmacological difference, we examined the GABA sensitivity of receptors containing each of the chimeric subunits.

Figure 4. GABA sensitivity of receptors containing wild-type and chimeric subunits.

Figure 4

Open in a new tab

A, representative whole-cell traces from transfected HEK-293T cells. The peak current in response to varying concentrations of GABA was measured. GABA was applied for 5 s, as indicated by the bar, to cells voltage clamped at −50 mV. The same time scale applies to all traces. B, concentration–response relationships were constructed by normalizing the peak response to each concentration of GABA as a percentage of the maximum current response for each cell. Points shown are the means ± s.e.m. (n = 4–7 cells). Averaged data were fitted with a four-parameter logistic equation represented by the dashed (wild-type) or continuous (chimeras) line.

The measured GABA EC50 is influenced by downstream signal transduction events as well as by the binding affinity of the receptor. Since the GABA binding sites are formed by the extracellular N-terminal domains, it was initially expected that the GABA EC50 of the chimeric subunits would be similar to the subunit that contributed this region or, alternatively, intermediate to the wild-type isoforms. Instead, receptors containing the α1/α6-A and -B site chimeras were less sensitive to GABA than either wild-type isoform (Fig. 4), with average EC50 values of 94.8 ± 10.2 μm (α1/α6-A, n = 5) and 117.2 ± 4.4 μm (α1/α6-B, n = 6) (P > 0.2 compared with one another, P < 0.05 compared with α1β3γ2L). Conversely, receptors with the α6/α1-A or -B chimeric subunits were more sensitive to GABA than the wild-type receptors, with average EC50 values of 0.45 ± 0.14 μm (α6/α1- A, n = 4) and 0.48 ± 0.06 μm (α6/α1-B, n = 6) (P > 0.2 compared with one another, P < 0.05 compared with α6β3γ2L). The GABA sensitivity of receptors containing the C-site chimeras was similar to the wild-type subunit that contributed their N-terminal structure, with average EC50 values of 19.1 ± 2.9 μm (α1/α6-C, n = 7, P > 0.2 compared with α1β3γ2L) and 1.7 ± 0.3 μm (α6/α1-C, n = 5, P > 0.2 compared with α6β3γ2L). These shifts in GABA sensitivity are consistent with a previous report examining only the A-site chimeric subunits (Fisher et al. 1997).

These results suggest that the regions between splice sites B and C influence GABA sensitivity for both the α1 and α6 subunits. This portion of the subunit includes TM3 and the TM3–TM4 intracellular loop. When this region is contributed from the α1 subunit, the EC50 is decreased, whereas when it is contributed from the α6 subunit, EC50 is increased. Structures in transmembrane or intracellular domains have not been suggested to form part of the agonist-binding pockets or directly alter GABA affinity. However, they could affect GABA EC50 by altering the signal transduction or gating transitions downstream of agonist binding (Colquhoun, 1998). The observed shifts in GABA sensitivity are consistent with the single channel properties conferred by the chimeric subunits. Channels with longer open time and burst duration should produce an apparent higher agonist affinity, even with no changes in the binding steps, if the agonist is less able to unbind from the open conformations. Conversely, simply reducing open time and burst duration can shift the equilibrium toward receptor conformations from which agonist unbinds, causing an apparent reduction in GABA sensitivity.

Effect of GABA concentration on receptor activation

Because of the potential influence of downstream gating transitions, concentration–response relationships and measurements of EC50 values often provide a poor estimate of agonist binding characteristics for ligand-gated channels (Colquhoun, 1998). The dependence of the channel activation rates on agonist concentration has been used to differentiate binding rates, or affinity, from the signal transduction processes that gate the channel, or efficacy (Clements & Westbrook, 1991; Lewis et al. 2003). The current rise time in response to agonist concentrations below EC50 values is related to binding affinity, while the maximum rise time, seen with saturating concentrations, is determined in large part by channel gating properties. The relationship between the activation rate and the GABA concentration was examined for receptors containing wild-type or chimeric subunits to clarify the structural determinants underlying the differences between the α1 and α6 subtypes in agonist binding and channel gating.

GABA concentrations from 1 μm to 10 mm were rapidly applied for 400 ms to excised outside-out patches held at −50 to −70 mV and the time for the current to increase from 10 to 90% of the peak response measured. At higher, saturating concentrations (1–10 mm), receptors containing the α1 subtype had rise times about 2 times faster than those containing the α6 subtype (Fig. 5A). This is consistent with greater gating efficacy for the α1β3γ2L isoform. Increasing GABA concentration from 1 to 10 mm did not substantially alter the rise time, suggesting that the receptor binding sites are saturated by 1 mm GABA for both receptor isoforms. As anticipated from the higher GABA sensitivity of the α6β3γ2L receptors, the rise times at GABA concentrations below 100 μm were significantly faster than those seen for the α1β3γ2L receptors (Fig. 5B).

Figure 5. Rise time and desensitization properties.

Figure 5

Open in a new tab

A, outside out macropatches were pulled from transfected cells and 1 mm GABA applied for 400 ms. Traces show current rise of α1β3γ2L (black trace) and α6β3γ2L (grey trace). Current amplitude was normalized to allow comparison of rise time. 1 mm GABA produced the maximum 10–90% rise time for receptor isoforms containing wild-type or chimeric subunits. Symbols and bars represent the average ± s.e.m. and the number in parentheses indicates number of patches. **P < 0.01 and ***P < 0.001 indicate a significant difference from α1β3γ2L while ††P < 0.01 and †††P < 0.001 indicate a significant difference from α6β3γ2L. Dashed lines show the average response for the α1β3γ2L and α6β3γ2L isoforms. B, GABA concentrations ranging from 1 μm to 10 mm were applied for 400 ms to excised patches voltage clamped at −50 to −70 mV. Symbols and bars represent the average time ± s.e.m. for the current to increase from 10 to 90% of the maximal current (n = 3–13 patches). No data were obtained at 1 μm GABA for the α1/α6-A or α1/α6-B chimeric subunits because of low current amplitude. C, 1 mm GABA was applied for 400 ms to excised patches held at −50 to −70 mV to examine onset of desensitization. The current decay was fitted with the sum of two exponential components with similar kinetic properties for both α1β3γ2L and α6β3γ2L isoforms.

Receptors containing the chimeric subunits showed a mixed pattern of dependence on GABA concentration (Fig. 5A and B). The α1/α6-C and α6/α1-C chimeric subunits produced rise times similar to the wild-type subunit that contributed the N-terminal domain. Receptors with the α1/α6-A or α1/α6-B chimeric subunits had rise times like the α1β3γ2L receptor at concentrations below 100 μm (Fig. 5B). Although the whole-cell concentration–response relationships indicated that these subunits reduced GABA sensitivity, the similarity in rise times at low GABA concentrations suggests that the binding rate was not different from the receptors containing the α1 subunit. At higher GABA concentrations, receptors containing the α1/α6-A or α1/α6-B chimeric subunits had rise times similar to α6β3γ2L receptors, with maximum rates significantly slower than the α1β3γ2L isoform (Fig. 5A and B). This is consistent with the channel gating properties of these receptors, which are similar to receptors containing the α6 subunit.

In a comparable pattern, receptors containing the α6/α1-A or α6/α1-B chimeric subunits had rise times like the α6β3γ2L receptor at concentrations below 100 μm (Fig. 5B). Again, the whole-cell studies showed that these subunits increased GABA sensitivity, but these results suggest that the binding affinity was not different from α6β3γ2L receptors. At higher GABA concentrations, receptors containing these chimeric subunits had rise times similar to α1β3γ2L receptors, with maximum rates significantly faster than the α6β3γ2L isoform (Fig. 5A and B). This is consistent with higher gating efficacy, like that conferred by the α1 subunit and as seen in the single channel recordings.

Desensitization of receptors containing α1 or α6 subunit subtypes

Most recombinant and native GABARs show desensitization, a decrease in current amplitude with continued agonist application. Entry into fast desensitized states can influence current rise times as well as deactivation rates following removal of agonist. The desensitization properties of receptors containing α1 subunits have been well characterized, and depending upon the duration of application, are typically fitted with the sum of two to four exponential distributions (Haas & Macdonald, 1999). Conflicting findings have been reported for recombinant α6βxγ2 receptors, with an early study showing little desensitization (Tia et al. 1996b) while more recent work found substantial fast desensitization (Bianchi et al. 2002).

When 1 mm GABA was applied for 400 ms, no significant differences were observed between the α1β3γ2L and α6β3γ2L receptor isoforms (Fig. 5C). The current decay during the application period was fitted with the sum of two exponential components for both isoforms. For receptors containing the α1 subunit, the weighted average time constant for the current decay was 50.2 ± 11.2 ms. The two components had averages of 8.4 ± 0.9 ms (63.6 ± 3.8%) and 128.9 ± 31.8 ms, with an average residual current of 33.1 ± 2.3%. The α6 subunit-containing receptors showed similar characteristics, with averages of 10.9 ± 1.8 ms (61.2 ± 3.6%) and 128.9 ± 11.8 ms and a weighted average of 55.9 ± 5.8 ms. The average current remaining at the end of the application was 27.8 ± 3.1% of the peak current.

Deactivation kinetics of receptors containing wild-type and chimeric subunits

Differences in single channel kinetic properties and/or agonist binding affinity would be expected to alter the characteristics of the synaptic currents mediated by receptors containing the α1 or α6 subunits. Stable, agonist-bound conformations, such as long-duration open or closed (desensitized) states, serve to ‘trap’ the agonist in a high-affinity binding state (Bianchi & Macdonald, 2001). The previous results suggest that entry into fast desensitized states is similar for receptors containing either the α1 or α6 subtype. Therefore, in the absence of other effects, increasing open time and/or burst durations is predicted to slow deactivation in addition to decreasing GABA EC50 (Colquhoun, 1998; Bianchi & Macdonald, 2001). To estimate the kinetic properties of postsynaptic currents, a saturating GABA concentration (1 mm) was applied for < 8 ms to outside-out macropatches held at −50 to −70 mV. The deactivation rate was determined for receptors containing either the wild-type α1 or α6 subunits or the chimeric subunits. Decay currents were fitted with the sum of two exponential distributions and weighted means were calculated by multiplying each time constant by its relative area.

The average decay time constant of the α6β3γ2L receptors was significantly slower than that of the α1β3γ2L receptors (Fig. 6). This is consistent with the higher GABA sensitivity (lower GABA EC50) of the α6-containing receptor, but not with its single channel characteristics. A slower deactivation rate with incorporation of the α6 subunit compared with an α1 subunit was also observed with receptors containing the δ subunit (Bianchi et al. 2002). In that study, the properties of chimeric α1/α6 subunits suggested that the deactivation rate was determined primarily by structural differences within the extracellular N-terminal domain, possibly due to effects on ligand binding. Although no single channel kinetics were reported, the data suggested that a difference in binding affinity was the predominant determinant of the difference in deactivation.

Figure 6. Current deactivation of receptors containing wild-type or chimeric subunits.

Figure 6

Open in a new tab

A, outside out patches were pulled from cells transfected with the subunits indicated. GABA (1 mm) was applied for < 8 ms to cells voltage clamped at −50 to −70 mV. Traces shown are 3 s in duration. B, the current decay was fitted with the sum of 1–2 exponential components. Symbols and bars represent the weighted average ± s.e.m. and the number in parentheses indicates number of patches. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate a significant difference from α1β3γ2L while †P < 0.05, ††P < 0.01 and †††P < 0.001 indicate a significant difference from α6β3γ2L.

Consistent with the relationship between GABA affinity and channel deactivation, the chimeric constructs with high sensitivity to GABA (α6/α1-A and -B) had the slowest deactivation rates while those with low sensitivity to GABA (α1/α6-A and -B), had very rapid deactivation rates (Fig. 6). The C-site chimeric subunits had deactivation time constants similar to the wild-type subunit that contributed their N-terminal domains.

The low gating efficacy (brief open time and burst durations) of the α6β3γ2L channels would tend to speed deactivation. Therefore, it is most likely that its slower deactivation rate is due to a higher binding affinity for GABA and that this characteristic is associated with the extracellular N-terminal domain of the subunit. This is consistent with the concentration dependence of the current rise times. The even slower deactivation rates of the α6/α1-A and -B chimeric subunits would be predicted from their longer channel open time compared with the α6β3γ2L receptor. These receptors would be expected to have high binding affinity similar to the α6 subunit (conferred by the N-terminal extracellular domain) and high efficacy gating (conferred by the TM3 and TM3–TM4 loop of the α1 subunit), both of which should slow deactivation. In contrast, receptors containing the α1/α6-A and -B chimeric subunits have shorter channel open times in addition to binding characteristics conferred by the N-terminal domain of the α1 subunit, leading to their very rapid deactivation rates.

Discussion

The GABAR α1 and α6 subunit subtypes have very different expression patterns in the mammalian brain and have been shown to confer distinct pharmacological and functional properties to recombinant and native receptors. These results show that these subtypes also produce channels with different single channel gating kinetics. Channel openings from the α1β3γ2L isoform were relatively long in duration, and were organized into multi-opening bursts. The α6β3γ2L isoform, in contrast, had brief-duration, isolated openings, suggesting that GABA acts as a low efficacy agonist at this receptor. While there are no previous reports of the single channel kinetic properties of receptors containing an α6 subunit, these findings are consistent with the single channel traces published for an allelic variant of α6 subunit shown in Angelotti et al. (1992), for which no kinetic analysis was reported. These characteristics are also similar to GABA-activated channels from recombinant α4β2γ2 receptors (Akk et al. 2004) as well as receptors containing the δ subunit (Fisher & Macdonald, 1997). The α4 and α6 subtypes are structurally related, and share many pharmacological characteristics. Therefore, it may not be surprising that they confer similar kinetic properties. Interestingly, it has been argued that GABA acts as a lower efficacy or partial agonist at δ subunit-containing receptors (Bianchi & Macdonald, 2003). Pentobarbital and some neurosteroids are more effective agonists than GABA at δ subunit-containing receptors, and single channel recordings have shown that they can produce long-duration openings and bursts, unlike GABA (Bianchi & Macdonald, 2003; Feng et al. 2004). These agonists also produce greater whole-cell current amplitudes than GABA at α6βγ receptors (Thompson et al. 1996; Belelli et al. 2002). Examination of the effect of pentobarbital or neurosteroid agonists at the single channel level may confirm that receptors containing the α6 subunit are capable of producing long-duration openings in response to agonists that are more effective than GABA.

Alterations or abnormalities in α1 and α6 subunit expression may lead to the development of neurological and behavioural disorders. A shift from slow to fast decay kinetics has been correlated in several neurone populations to an increase in α1 subunit expression and a corresponding decrease in the α2 and α3 subunits (Brussaard et al. 1997; Robello et al. 1999; Okada et al. 2000; Hutcheon et al. 2000; Mellor et al. 2000; Vicini et al. 2001; Brussaard & Herbison, 2000; Bosman et al. 2002). The cerebellar localization of the α6 subunit suggests that it may be involved in motor control, particularly in the motor effects of ethanol and other sedatives (Korpi et al. 1993). Chronic treatment with benzodiazepines increases α6 subunit expression and decreases α1 subunit expression (O'Donovan et al. 1992). Since α6 subunit-containing receptors are insensitive to benzodiazepines, this change may lead to tolerance, a commonly observed clinical problem. A similar pattern of change is seen with alcohol dependence, as chronic ethanol administration increases α6 mRNA and decreases α1 mRNA (Mhatre & Ticku, 1992). Reducing the α6 subunit expression in cerebellar granule cells decreased agonist affinity, consistent with the higher GABA sensitivity of α6 subunit-containing recombinant receptors (Zhu et al. 1996; Makela et al. 1997). Extrasynaptic receptors containing the α6 subunit are believed to contribute the tonic current observed in cerebellar granule cells (Brickley et al. 1996; Rossi & Hamann, 1998; Nusser et al. 1998).

The properties of receptors containing α1/α6 chimeric subunits suggest that differences in GABA sensitivity and deactivation rate are predominately determined by the extracellular N-terminal domain. However, these characteristics are also influenced by channel gating properties, which appear to be conferred by the TM3 and/or the TM3–TM4 intracellular loop. We still lack a full understanding of physical changes in channel structure that underlie gating of the GABAR. Combined with studies of the structurally related glycine and nAChRs, hydrophobic residues within TM2 have been suggested to serve as the channel gate (Unwin, 2003). The conformational changes that result in channel opening may involve rotating or tilting of the TM2 helices (Tang et al. 2002; Unwin, 2003; Lester et al. 2004). Therefore it was somewhat surprising that the regions surrounding and including TM2 did not appear to be responsible for the kinetic differences associated with the α1 and α6 subtypes. Residues within this domain line the channel pore and are important for regulation of many channel properties, including gating, conductance and permeability and mediate the effects of some allosteric modulators (Karlin & Akabas, 1995; Mehta & Ticku, 1999). In addition, recent evidence has shown that electrostatic interactions between charged residues in the N-terminal and TM2–TM3 extracellular domains of the α subunits are important in signal transduction of agonist binding (Kash et al. 2003). However, the structure of the subunits in this region is highly conserved, with only five amino acids different between the α1 and α6 subtypes from splice site A to B. While this domain may be important for many general GABAR channel properties, structural differences within it do not appear responsible for the distinct single channel properties associated with the α1 and α6 subtypes.

The possibility that the structures within TM3 may control gating characteristics is interesting. Electron microscopy suggests that this domain interacts with the TM2 domain, possibly regulating its movement during activation (Miyazawa et al. 2003). The TM2 and TM3 domains have been suggested to contribute to a water-filled cavity, which forms the binding pocket for volatile anaesthetics and ethanol (Mihic et al. 1997; Krasowski et al. 1998, 2001; Ueno et al. 1999; Krasowski & Harrison, 2000). It has been reported that several residues within TM3 are accessible to the aqueous solution, and that changes in its secondary structure occur with GABA binding (Williams & Akabas, 1999). Recent studies examining cross-linking of engineered cysteine residues also provided evidence for a close relationship between residues within TM2 and TM3 of the glycine receptor subunits (Lobo et al. 2004). The TM3 domain is highly conserved among the GABAR subunits, and there are only two residues that differ between the α1 and α6 subtypes. Mutations of residues within TM3 increase spontaneous activity of the GABAR (Ueno et al. 1999) and have been shown to affect gating kinetics of the nAChR (Campos-Caro et al. 1997; Wang et al. 1999). Recently, an inherited form of epilepsy was reported in which the alanine 294 residue within the TM3 of the α1 subunit is mutated to aspartate (Cossette et al. 2002). Kinetic analysis of recombinant receptors showed that the mutation accelerated deactivation, slowed desensitization and reduced channel open time, consistent with a role for the TM3 in regulating channel gating efficacy (Fisher, 2004).

Regulation of GABAR kinetic properties by structures within the TM3–TM4 intracellular loop is also a strong possibility, especially as this region exhibits substantial heterogeneity among subunit subtypes. Since phosphorylation of residues within the intracellular loop of several subunits alters receptor function, structural changes within this domain are clearly able to influence channel behaviour (Mehta & Ticku, 1999). Genetic studies have linked a mutation (P385S) in this domain of the human α6 subunit to alcoholism and reduced sensitivity to the motor and anxiolytic effects of benzodiazepine agonists (Iwata et al. 1999, 2000; Hoffman et al. 2002). In structurally related receptors, a mutation that encoded a six amino acid duplication within the intracellular loop of the nAChR altered its gating kinetics (Milone et al. 1998) while this region of the 5HT3 serotonin receptor was found to influence its single channel conductance (Kelley et al. 2003). Interactions between the M3–M4 intracellular domain and M4 have been proposed to control channel gating of the nAChR (Akk & Steinbach, 2000).

Further studies examining the role of individual residues within these domains of the α subunit subtypes as well as other GABAR subunit family members may shed more light on the structures that regulate the kinetic properties of ligand-gated channels.

Acknowledgments

Thanks to Annette Smith and Dr Steve Wilson for help with construction of the chimeric subunits and to Dr Paul Housley for preparation of the antigen-coated magnetic beads. This work was supported by the University of South Carolina School of Medicine Research Development Fund, the South Carolina Commission on Higher Education, the PhRMA foundation, the National Science Foundation/EPSCoR (EPS-1032573) and NIH/BRIN (8-P0RR16461A).

References

  1. Akk G, Bracamontes J, Steinbach JH. Activation of GABAA receptors containing the α4 subunit by GABA and pentobarbital. J Physiol. 2004;566:387–399. doi: 10.1113/jphysiol.2003.058230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akk G, Steinbach JH. Structural elements near the C-terminus are responsible for changes in nicotinic gating kinetics following patch excision. J Physiol. 2000;527:405–417. doi: 10.1111/j.1469-7793.2000.t01-2-00405.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Angelotti TP, Macdonald RL. Assembly of GABAA receptor subunits: α1β1 and α1β1γ2S subunits produce unique ion channels with dissimilar single-channel properties. J Neurosci. 1993;13:1429–1440. doi: 10.1523/JNEUROSCI.13-04-01429.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Angelotti TP, Tan F, Chahine KG, Macdonald RL. Molecular and electrophysiological characterization of a allelic variant of the rat α6 GABAA receptor subunit. Mol Brain Res. 1992;16:173–178. doi: 10.1016/0169-328x(92)90209-t. [DOI] [PubMed] [Google Scholar]
  5. Angelotti TP, Uhler MD, Macdonald RL. Assembly of GABAA receptor subunits: analysis of transient single-cell expression utilizing a fluorescent substrate/marker gene technique. J Neurosci. 1993;13:1418–1428. doi: 10.1523/JNEUROSCI.13-04-01418.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Belelli D, Casula A, Ling A, Lambert JJ. The influence of subunit composition on the interaction of neurosteroids with GABAA receptors. Neuropharm. 2002;43:651–661. doi: 10.1016/s0028-3908(02)00172-7. [DOI] [PubMed] [Google Scholar]
  7. Bianchi MT, Haas KF, Macdonald RL. α1 and α6 subunits specify distinct desensitization, deactivation and neurosteroid modulation of GABAA receptors containing the δ subunit. Neuropharmacology. 2002;43:492–502. doi: 10.1016/s0028-3908(02)00163-6. [DOI] [PubMed] [Google Scholar]
  8. Bianchi MT, Macdonald RL. Agonist trapping by GABAA receptor channels. J Neurosci. 2001;21:9083–9091. doi: 10.1523/JNEUROSCI.21-23-09083.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bianchi MT, Macdonald RL. Neurosteroids shift partial agonist activation of GABAA receptor channels from low- to high-efficacy gating patterns. J Neurosci. 2003;23:10934–10943. doi: 10.1523/JNEUROSCI.23-34-10934.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bosman LWJ, Rosahl TW, Brussaard AB. Neonatal development of the rat visual cortex: synaptic function of GABAA receptor α subunits. J Physiol. 2002;545:169–181. doi: 10.1113/jphysiol.2002.026534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Brickley SG, Cull-2 SG, Farrant M. Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors. J Physiol. 1996;497:753–759. doi: 10.1113/jphysiol.1996.sp021806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Brussaard AB, Herbison AE. Long-term plasticity of postsynaptic GABAA-receptor function in the adult brain: insights from the oxytocin neurone. Trends Neurosci. 2000;23:190–195. doi: 10.1016/s0166-2236(99)01540-4. [DOI] [PubMed] [Google Scholar]
  13. Brussaard AB, Kits KS, Baker RE, Willems WPA, Leyting-Vermeulen JW, Voorn P, Smit AB, Bicknell RJ, Herbison AE. Plasticity in fast synaptic inhibition of adult oxytocin neurons caused by switch in GABAA receptor subunit expression. Neuron. 1997;19:1103–1114. doi: 10.1016/s0896-6273(00)80401-8. [DOI] [PubMed] [Google Scholar]
  14. Burgard EC, Tietz EI, Neelands TR, Macdonald RL. Properties of recombinant γ-aminobutyric acidA receptor isoforms containing the α5 subunit subtype. Mol Pharm. 1996;50:119–127. [PubMed] [Google Scholar]
  15. Cagetti E, Liang J, Spigelman I, Olsen RW. Withdrawal from chronic intermittent ethanol treatment changes subunit composition, reduces synaptic function, and decreases behavioral responses to positive allosteric modulators of GABAA receptors. Mol Pharm. 2003;63:53–64. doi: 10.1124/mol.63.1.53. [DOI] [PubMed] [Google Scholar]
  16. Campos Caco A, Rovira JC, Vicente Agullo F, Ballesta JJ, Sala S, Criado M, Sala F. Role of the putative transmembrane segment M3 in gating of neuronal nicotinic receptors. Biochemistry. 1997;36:2709–2715. doi: 10.1021/bi9623486. [DOI] [PubMed] [Google Scholar]
  17. Chesnut JD, Baytan AR, Russell M, Chang MP, Bernard A, Maxwell IH, Hoeffler JP. Selective isolation of transiently transfected cells from a mammalian cell population with vectors expressing a membrane anchored single-chain antibody. J Immunol Meth. 1996;193:17–27. doi: 10.1016/0022-1759(96)00032-4. [DOI] [PubMed] [Google Scholar]
  18. Clements JD, Westbrook GL. Activation kinetics reveal the number of glutamate and glycine binding sites on the N-methyl-D-aspartate receptor. Neuron. 1991;7:605–613. doi: 10.1016/0896-6273(91)90373-8. [DOI] [PubMed] [Google Scholar]
  19. Colquhoun D. Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. Br J Pharm. 1998;125:924–947. doi: 10.1038/sj.bjp.0702164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Colquhoun D, Sakmann B. Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J Physiol. 1985;369:501–557. doi: 10.1113/jphysiol.1985.sp015912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Colquhoun D, Sigworth FJ. Fitting and statistical analysis of single-channel records. In: Sackmann B, Neher E, editors. Single-Channel Recording. New York: Plenum Press; 1995. pp. 483–587. [Google Scholar]
  22. Cossette P, Liu L, Brisebois K, Dong H, Lortie A, Vanasse M, Saint-Hilaire J-M, Carmant L, Verner A, Lu W-Y, Wang YT, Rouleau GA. Mutation of GABRA1 in an autosomal dominant form of juvenile myoclonic epilepsy. Nature Genet. 2002;31:184–189. doi: 10.1038/ng885. [DOI] [PubMed] [Google Scholar]
  23. Ducic I, Caruncho HJ, Zhu WJ, Vicini S, Costa E. γ-aminobutyric acid gating of Cl− channels in recombinant GABAA receptors. J Pharmacol Exp Ther. 1995;272:438–445. [PubMed] [Google Scholar]
  24. Feng HJ, Bianchi MT, Macdonald RL. Mol Pharmacol. Vol. 66. 2004. Pentobarbital differentially modulates α1β3δ and α1β3γ2L GABAA receptor currents; pp. 981–1003. [DOI] [PubMed] [Google Scholar]
  25. Fisher JL. A mutation in the GABAA receptor α1 subunit linked to human epilepsy affects channel gating properties. Neuropharm. 2004;46:629–637. doi: 10.1016/j.neuropharm.2003.11.015. [DOI] [PubMed] [Google Scholar]
  26. Fisher JL, Macdonald RL. Single-channel properties of GABAA receptors containing γ2 or δ subtypes expressed with α1 and β3 subtypes in L929 cells. J Physiol. 1997;505:283–297. doi: 10.1111/j.1469-7793.1997.283bb.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Fisher JL, Zhang J, Macdonald RL. The role of α1 and α6 subtype amino-terminal domains in allosteric regulation of γ-aminobutyric acida receptors. Mol Pharmacol. 1997;52:714–724. doi: 10.1124/mol.52.4.714. [DOI] [PubMed] [Google Scholar]
  28. Gingrich KJ, Roberts WA, Kass RS. Dependence of the GABAA receptor gating kinetics on the α-subunit isoform: implications for structure–function relations and synaptic transmission. J Physiol. 1995;489:529–543. doi: 10.1113/jphysiol.1995.sp021070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Goldstein PA, Elsen FP, Ying S-W, Ferguson C, Homanics GE, Harrison NL. Prolongation of hippocampal miniature inhibitory postsynaptic currents in mice lacking the GABAA receptor α1 subunit. J Neurophysiol. 2002;88:3208–3217. doi: 10.1152/jn.00885.2001. [DOI] [PubMed] [Google Scholar]
  30. Greenfield LJ, Jr, Sun F, Neelands TR, Burgard EC, Donnelly JL, Macdonald RL. Expression of functional GABAA receptors in transfected L929 cells isolated by immunomagnetic bead separation. Neuropharmacology. 1997;36:63–73. doi: 10.1016/s0028-3908(96)00150-5. [DOI] [PubMed] [Google Scholar]
  31. Haas KF, Macdonald RL. GABAA receptor subunit γ2 and δ subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts. J Physiol. 1999;514:27–45. doi: 10.1111/j.1469-7793.1999.027af.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hoffman WE, Balyasnikova IV, Mahay H, Danilov SM, Baughman VL. GABA α6 receptors mediate midazolam-induced anxiolysis. J Clin Anesth. 2002;14:206–209. doi: 10.1016/s0952-8180(02)00343-4. [DOI] [PubMed] [Google Scholar]
  33. Horn R. Statistical methods for model discrimination: applications to gating kinetics and permeation of the acetylcholine receptor channel. Biophys J. 1987;51:255–263. doi: 10.1016/S0006-3495(87)83331-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Hsu F-C, Waldeck R, Faber DS, Smith SS. Neurosteroid effects on GABAergic synaptic plasticity in hippocampus. J Neurophys. 2003;89:1929–1940. doi: 10.1152/jn.00780.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Hutcheon B, Morley P, Poulter MO. Developmental change in GABAA receptor desensitization kinetics and its role in synapse function in rat cortical neurons. J Physiol. 2000;522:3–17. doi: 10.1111/j.1469-7793.2000.t01-5-00003.xm. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Iwata N, Cowley DS, Radel M, Roy-Byrne PP, Goldman D. Relationship between a GABAAα6 Pro385Ser substitution and benzodiazepine sensitivity. Am J Psych. 1999;156:1447–1449. doi: 10.1176/ajp.156.9.1447. [DOI] [PubMed] [Google Scholar]
  37. Iwata N, Virkkunen M, Goldman D. Identification of a naturally occurring Pro385-Ser385 substitution in the GABAA receptor α6 subunit gene in alcoholics and healthy volunteers. Mol Psych. 2000;5:316–319. doi: 10.1038/sj.mp.4000706. [DOI] [PubMed] [Google Scholar]
  38. Karlin A, Akabas MH. Toward a structural basis for the function of the nicotinic acetylcholine receptors and their cousins. Neuron. 1995;15:1231–1244. doi: 10.1016/0896-6273(95)90004-7. [DOI] [PubMed] [Google Scholar]
  39. Kash TL, Jenkins A, Kelley JC, Trudell JR, Harrison NL. Coupling of agonist binding to channel gating in the GABAA receptor. Nature. 2003;421:272–275. doi: 10.1038/nature01280. [DOI] [PubMed] [Google Scholar]
  40. Kelley SP, Dunlop JI, Kirkness EF, Lambert JJ, Peters JA. A cytoplasmic region determines single-channel conductance in 5-HT3 receptors. Nature. 2003;424:321–324. doi: 10.1038/nature01788. [DOI] [PubMed] [Google Scholar]
  41. Koksma J-J, van Kesteren RE, Rosahl TW, Swart R, Smit AB, Lüddens H, Brussaard AB. Oxytocin regulates neurosteroid modulation of GABAA receptors in supraoptic nucleus around parturition. J Neurosci. 2003;23:788–797. doi: 10.1523/JNEUROSCI.23-03-00788.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Korpi ER, Gründer G, Lüddens H. Drug interactions at GABAA receptors. Prog Neurobiol. 2002;67:113–159. doi: 10.1016/s0301-0082(02)00013-8. [DOI] [PubMed] [Google Scholar]
  43. Korpi ER, Kleingoor C, Kettenmann H, Seeburg PH. Benzodiazepine-induced motor impairment linked to point mutation in cerebellar GABAA receptor. Nature. 1993;361:356–359. doi: 10.1038/361356a0. [DOI] [PubMed] [Google Scholar]
  44. Krasowski MD, Harrison NL. The actions of ether, alcohol and alkane general anaesthetics on GABAA and glycine receptors and the effects of TM2 and TM3 mutations. Br J Pharm. 2000;129:731–743. doi: 10.1038/sj.bjp.0703087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Krasowski MD, Koltchine VV, Rick CE, Ye Q, Finn SE, Harrison NL. Propofol and other intravenous anesthetics have sites of action on the γ-aminobutyric acid type A receptor distinct from that for isoflurane. Mol Pharmacol. 1998;53:530–538. doi: 10.1124/mol.53.3.530. [DOI] [PubMed] [Google Scholar]
  46. Krasowski MD, Nishikawa K, Nikolaeva N, Lin A, Harrison NL. Methionine 286 in transmembrane domain 3 of the GABAA receptor β subunit controls a binding cavity for propofol and other alkylphenol general anesthetics. Neuropharm. 2001;41:952–964. doi: 10.1016/s0028-3908(01)00141-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Laurie DJ, Seeburg PH, Wisden W. The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. III. Olfactory bulb and cerebellum. J Neurosci. 1992b;12:1063–1076. doi: 10.1523/JNEUROSCI.12-03-01063.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Laurie DJ, Wisden W, Seeburg PH. The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development. J Neurosci. 1992a;12:4151–4172. doi: 10.1523/JNEUROSCI.12-11-04151.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Lavoie AM, Tingey JJ, Harrison NL, Pritchett DB, Twyman RE. Activation and deactivation rates of recombinant GABAA receptor channels are dependent on α-subunit isoform. Biophys J. 1997;73:2518–2526. doi: 10.1016/S0006-3495(97)78280-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Lester HA, Dibas MI, Dahan DS, Leite JF, Dougherty DA. Cys-loop receptors: new twists and turns. Trends Neurosci. 2004;27:329–336. doi: 10.1016/j.tins.2004.04.002. [DOI] [PubMed] [Google Scholar]
  51. Lewis TM, Schofield PR, McClellan AML. Kinetic determinants of agonist action at the recombinant human glycine receptor. J Physiol. 2003;549:361–374. doi: 10.1113/jphysiol.2002.037796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Lobo IA, Trudell JR, Harris RA. Cross-linking of glycine receptor transmembrane segments two and three alters coupling of ligand binding with channel opening. J Neurochem. 2004;90:962–969. doi: 10.1111/j.1471-4159.2004.02561.x. [DOI] [PubMed] [Google Scholar]
  53. Lüddens H, Pritchett DB, Köhler M, Killisch I, Keinänen Monyer H, Sprengel R, Seeburg PH. Cerebellar GABAA receptor selective for a behavioural alcohol antagonist. Nature. 1990;346:648–651. doi: 10.1038/346648a0. [DOI] [PubMed] [Google Scholar]
  54. McClellan AML, Twyman RE. Receptor system response kinetics reveal functional subtypes of native murine and recombinant human GABAA receptors. J Physiol. 1999;515:711–727. doi: 10.1111/j.1469-7793.1999.711ab.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. McKernan RM, Whiting PJ. Which GABAA-receptor subtypes really occur in the brain? Trends Neurosci. 1996;19:139–143. doi: 10.1016/s0166-2236(96)80023-3. [DOI] [PubMed] [Google Scholar]
  56. Makela R, Uusi-Oukari M, Homanics GE, Quinlan JJ, Firestone LL, Wisden W, Korpi ER. Cerebellar γ-aminobutyric acid type A receptors: pharmacological subtypes revealed by mutant mouse lines. Mol Pharm. 1997;52:380–388. doi: 10.1124/mol.52.3.380. [DOI] [PubMed] [Google Scholar]
  57. Mehta AK, Ticku MK. An update on GABAA receptors. Brain Res Rev. 1999;29:196–217. doi: 10.1016/s0165-0173(98)00052-6. [DOI] [PubMed] [Google Scholar]
  58. Mellor JR, Wisden W, Randall AD. Somato-synaptic variation of GABAA receptors in cultured murine cerebellar granule cells: investigation of the role of the α6 subunit. Neuropharm. 2000;39:1495–1513. doi: 10.1016/s0028-3908(00)00007-1. [DOI] [PubMed] [Google Scholar]
  59. Mhatre MC, Ticku MK. Chronic ethanol administration alters γ-aminobutyric acidA receptor gene expression. Mol Pharmacol. 1992;42:415–422. [PubMed] [Google Scholar]
  60. Mihic SJ, Ye Q, Wick MJ, Koltchine VV, Krasowski MD, Finn SE, Mascia MP, Valenzuela CF, Hanson KK, Greenblatt EP, Harris RA, Harrison NL. Sites of alcohol and volatile anaesthetic action on GABAA and glycine receptors. Nature. 1997;389:385–389. doi: 10.1038/38738. [DOI] [PubMed] [Google Scholar]
  61. Milone M, Wang H-L, Ohno K, Prince R, Fukudome T, Schen X-M, Brengman JM, Griggs RC, Sine SM, Engel AG. Mode switching kinetics produced by a naturally occurring mutation in the cytoplasmic loop of the human acetylcholine receptor ɛ subunit. Neuron. 1998;20:575–588. doi: 10.1016/s0896-6273(00)80996-4. [DOI] [PubMed] [Google Scholar]
  62. Miyazawa A, Fujiyoshi Y, Unwin N. Structure and gating mechanism of the acetylcholine receptor pore. Nature. 2003;423:949–955. doi: 10.1038/nature01748. [DOI] [PubMed] [Google Scholar]
  63. Nusser Z, Sieghart W, Somogyi P. Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. J Neurosci. 1998;18:1693–1703. doi: 10.1523/JNEUROSCI.18-05-01693.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. O'Donovan MC, Buckland PR, Spurlock G, McGuffin P. Bidirectional changes in the levels of messenger RNA encoding γ-aminobutyric acidA receptor α subunits after flurazepam treatment. Eur J Pharm. 1992;226:335–341. doi: 10.1016/0922-4106(92)90051-v. [DOI] [PubMed] [Google Scholar]
  65. Okada M, Onodera K, Van Renterghem C, Sieghart W, Takahashi T. Functional correlation of GABAA receptor α subunits expression with the properties of IPSCs in the developing thalamus. J Neurosci. 2000;20:2202–2208. doi: 10.1523/JNEUROSCI.20-06-02202.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Robello M, Amico C, Cupello A. Evidence of two populations of GABAA receptors in cerebellar granule cells in culture: different desensitization kinetics, pharmacology, serine/threonine kinase sensitivity, and localization. Biochem Biophys Res Comm. 1999;266:603–608. doi: 10.1006/bbrc.1999.1861. [DOI] [PubMed] [Google Scholar]
  67. Rossi DJ, Hamann M. Spillover-mediated transmission at inhibitory synapses promoted by high affinity α6 subunit GABAA receptors and glomerular geometry. Neuron. 1998;20:783–795. doi: 10.1016/s0896-6273(00)81016-8. [DOI] [PubMed] [Google Scholar]
  68. Saxena NC, Macdonald RL. Properties of putative cerebellar γ-aminobutyric acidA receptor isoforms. Mol Pharmacol. 1996;49:567–579. [PubMed] [Google Scholar]
  69. Smith SS, Gong QH, Li X, Moran MH, Bitran D, Frye CA, Hsu F-C. Withdrawal from 3α-OH-5α-pregnan-20-one using a pseudopregnancy model alters the kinetics of hippocampal GABAA-gated current and increases the GABAA receptor α4 subunit in association with increased anxiety. J Neurosci. 1998;18:5275–5284. doi: 10.1523/JNEUROSCI.18-14-05275.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Tang P, Mandal PK, Xu Y. NMR structures of the second transmembrane domain of the human glycine receptor α1 subunit: model of pore architecture and channel gating. Biophys J. 2002;83:252–262. doi: 10.1016/S0006-3495(02)75166-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Thompson SA, Whiting PJ, Wafford KA. Barbiturate interactions at the human GABAA receptor: dependence on receptor subunit combination. Br J Pharm. 1996;117:521–527. doi: 10.1111/j.1476-5381.1996.tb15221.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Tia S, Wang JF, Kotchabhakdi N, Vicini S. Developmental changes of inhibitory synaptic currents in cerebellar granule neurons: role of GABAA receptor α6 subunit. J Neurosci. 1996a;16:3630–3640. doi: 10.1523/JNEUROSCI.16-11-03630.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Tia S, Wang JF, Kotchabhakdi N, Vicini S. Distinct deactivation and desensitization kinetics of recombinant GABAA receptors. Neuropharm. 1996b;35:1375–1382. doi: 10.1016/s0028-3908(96)00018-4. [DOI] [PubMed] [Google Scholar]
  74. Twyman RE, Rogers CJ, Macdonald RL. Intra-burst kinetic properties of the GABAA receptor main conductance state of mouse spinal cord neurones in culture. J Physiol. 1990;423:193–220. doi: 10.1113/jphysiol.1990.sp018018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Tyndale RF, Olsen RW, Tobin AJ. GABAA receptors. In: North RA, editor. Ligand- and Voltage-Gated Ion Channels. Boca Raton: CRC Press; 1995. pp. 265–290. [Google Scholar]
  76. Ueno S, Wick MJ, Ye Q, Harrison NL, Harris RA. Subunit mutations affect ethanol actions on GABAA receptors expressed in Xenopus oocytes. Br J Pharmacol. 1999;127:377–382. doi: 10.1038/sj.bjp.0702563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Unwin N. Structure and action of the nicotinic acetylcholine receptor explored by electron microscopy. FEBS Lett. 2003;555:91–95. doi: 10.1016/s0014-5793(03)01084-6. [DOI] [PubMed] [Google Scholar]
  78. Varecka L, Wu CH, Rotter A, Frostholm A. GABAA/benzodiazepine receptor α6 subunit mRNA in granule cells of the cerebellar cortex and cochlear nuclei: expression in developing and mutant mice. J Comp Neurol. 1994;339:341–352. doi: 10.1002/cne.903390304. [DOI] [PubMed] [Google Scholar]
  79. Vicini S, Ferguson C, Prybylowski K, Kralic J, Morrow AL, Homanics GE. GABAA receptor α1 subunit deletion prevents developmental changes of inhibitory synaptic currents in cerebellar neurons. J Neurosci. 2001;21:3009–3016. doi: 10.1523/JNEUROSCI.21-09-03009.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Wang HL, Ohno K, Milone M, Brengman J, Evoli A, Batocchi AP, Middleton LT, Christodoulou K, Engel AG, Sine SM. Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel gating. Nat Neurosci. 1999;2:226–233. doi: 10.1038/6326. [DOI] [PubMed] [Google Scholar]
  81. Williams DB, Akabas MH. γ-aminobutyric acid increases the water accessibility of M3 membrane spanning segment residues in γ-aminobutyric acid type A receptors. Biophys J. 1999;77:2563–2574. doi: 10.1016/s0006-3495(99)77091-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Zhu WJ, Wang JF, Vicini S, Grayson DR. α6 and γ2 subunit antisense oligonucleotides alter γ-amino butyric acid receptor pharmacology in cerebellar granule neurons. Mol Pharmacol. 1996;50:23–33. [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES