Published online January 28, 2008
doi:10.1085/jgp.200709871
The Journal of General Physiology, Vol. 131, No. 2, 163-181
The Rockefeller University Press, 0022-1295 $30.00
© 2008 Keramidas et al.
Agonist-dependent Single Channel Current and Gating in 
4β2
and 1β2
2S GABAA Receptors
Angelo Keramidas and
Neil L. Harrison
CV Starr Laboratory for Molecular Neuropharmacology, Department of Anesthesiology, Weill Medical College of Cornell University, New York, NY 10021
Correspondence to Angelo Keramidas: ank2013{at}cornell.med.edu
The family of 
-aminobutyric acid type A receptors (GABAARs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic GABAARs that are mainly comprised of 
1, β2, and 2 subunits, whereas tonic inhibition is mediated by extrasynaptic GABAARs comprised of 4/6, β2, and 
subunits. We investigated the activation properties of recombinant 4β2 and 1β22S GABAARs in response to GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at 4β2 GABAARs (β 
1600 s–1) than at 1β22S GABAARs (β 460 s–1), whereas GABA activated 1β22S GABAARs more rapidly (β 1800 s–1) than 4β2 GABAARs (β < 440 s–1). Single channel recordings of 1β22S and 4β2 GABAARs showed that both channels open to a main conductance state of 25 pS at –70 mV when activated by GABA and low concentrations of THIP, whereas saturating concentrations of THIP elicited 36 pS openings at both channels. Saturating concentrations of GABA elicited brief (<10 ms) openings with low intraburst open probability (PO 0.3) at 4β2 GABAARs and at least two "modes" of single channel bursting activity, lasting 100 ms at 1β22S GABAARs. The most prevalent bursting mode had a PO of 0.7 and was described by a reaction scheme with three open and three shut states, whereas the "high" PO mode (0.9) was characterized by two shut and three open states. Single channel activity elicited by THIP in 4β2 and 1β22S GABAARs occurred as a single population of bursts (PO 0.4–0.5) of moderate duration (33 ms) that could be described by schemes containing two shut and two open states for both GABAARs. Our data identify kinetic properties that are receptor-subtype specific and others that are agonist specific, including unitary conductance.
Abbreviations used in this paper: GABAAR, -aminobutyric acid type A receptor; LGIC, ligand-gated ion channel; RI, rectification index; THIP, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one.
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INTRODUCTION
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The -aminobutyric acid type A receptor (GABAAR) is a member of the Cys-loop superfamily of ligand-gated ion channels (LGICs), and the main inhibitory receptor in the brain. GABAARs are comprised of five homologous subunits that assemble to form a central, Cl–-selective pore. Cloning techniques have so far identified 16 subunits that occur in mammals, these include 1–6, β1–3, 1–3, , 
, 
, and 
subunits (Sieghart and Sperk, 2002
; Darlison et al., 2005).
At inhibitory synapses the most prevalent GABAAR is composed of 1, β2, and 2 subunits (McKernan and Whiting, 1996; Somogyi et al., 1996), which has been shown by covalent subunit cross-linking experiments to have a stoichiometry and arrangement of 2β21β21 (Chang et al., 1996; Baumann et al., 2002). This type of GABAAR can be found, for example, at synapses in the cerebellum (Vicini et al., 2001). GABA release at these fast inhibitory synapses is brief and results in a submillisecond, high concentration pulse of GABA, and this elicits a phasic or transient response that typically lasts <50 ms (Cobb et al., 1995; Hardie and Pearce, 2006). The inhibitory synaptic currents can be modeled mathematically using a pulse of 1 mM GABA for 300–500 µs (Jones and Westbrook, 1995; Lavoie et al., 1997; Schofield and Huguenard, 2007). The duration of the synaptic event is dependent on a variety of factors, of which one of the most important is the identity of the subunit, with 1 subunits mediating rapid decay kinetics (Vicini et al., 2001), whereas 2 and 3 subunits mediate slower events (Gingrich et al., 1995; Lavoie et al., 1997; Schofield and Huguenard, 2007).
In addition to the activation of conventional synaptic GABAARs by neurotransmitter release, a form of tonic inhibition occurs due to the sustained activation of GABAARs located extrasynaptically. These receptors are thought to be activated by low concentrations of agonists present in the extracellular space, such as GABA, as has been shown in the cerebellum (Brickley et al., 1996) and dentate gyrus (Mody, 2001), and taurine, as has been reported in the thalamus (Jia et al., 2008). Extrasynaptic GABAARs are believed to be composed of 4, β2/3, and subunits in the thalamus, dentate gyrus, and cortex (Sur et al., 1999; Sieghart and Sperk, 2002; Jia et al., 2005), with 6 subunits replacing 4 subunits in the cerebellum. As the and subunits have not been shown to coexist in the same pentamers, it is widely presumed that the subunit simply replaces the to give an arrangement of ββ in β GABAARs (Sigel et al., 2006). In rat thalamus, 60–70% of the 4 subunit protein coprecipitates with the subunit, which is thought to be exclusively extrasynaptic (Nusser et al., 1998), and all of the subunit is restricted to 4 subunit–containing receptors (Sur et al., 1999). The 4 and subunits confer pharmacological properties that differ from those of 1β22S GABAARs. GABAARs containing the 4 subunit are insensitive to conventional benzodiazepines (Knoflach et al., 1996; Whittemore et al., 1996) and show robust potentiation of GABA responses by neurosteroids (Whittemore et al., 1996). Concentration–response experiments on recombinant 4β2/3 GABAARs have demonstrated that these channels are activated with greater relative efficacy by the GABA analogue and hypnotic, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP; Gaboxadol) than GABA (Brown and Kerby, 2002; Jia et al., 2005), whereas the reverse applies to synaptic GABAARs.
In this study we used rapid agonist application techniques in conjunction with single channel recordings to develop kinetic schemes for recombinant 4β2 and 1β22S GABAARs, activated by GABA and THIP. Simple activation schemes derived from modeling fast agonist application experiments showed that THIP produces a greater opening rate constant at 4β2 GABAARs than GABA, whereas the converse was the case for 1β22S GABAARs. Saturating concentrations of THIP also evoked single channel openings with a larger conductance (36 pS) at both 4β2 and 1β22S GABAARs, whereas low concentrations of THIP and high and low concentrations of GABA elicited openings that were 25 pS at both channels. Kinetic analysis of single channel recordings obtained from 4β2 and 1β22S GABAARs corroborated the data from macropatch, ensemble experiments and revealed that channel bursting characteristics are both receptor and agonist specific. GABA induced up to three distinct modes in 1β22S GABAARs, identified by stable intraburst open probabilities; the mode with the highest intraburst open probability could be described by three open and two shut states. In contrast, only one gating mode was seen in 4β2 GABAARs, and the openings were brief. THIP elicited homogeneous bursting patterns in both channels, which consisted of brief bursts that could best be described by two open and two shut states.
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MATERIALS AND METHODS
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Expression of GABAARs in HEK293 Cells
HEK293 cells were plated onto poly-D-lysine–coated glass coverslips and transfected with cDNA encoding the human 1, mouse 4, rat β2, human 2S, and human subunits, using the calcium phosphate coprecipitation method. Mouse and rat subunits were combined with human subunits, first because of their availability, and second, because they show a high degree of identity and homology with their human counterparts. The cells were also transfected with the CD4 surface antigen, which acted as a transfection marker. Our preliminary cotransfections of 4, β2, and subunits were done at a transfection ratio of 1:1:2 (plasmid mass ratio), but we settled on a ratio of 1:1:5 for the present study as our data suggested that that ratio favored the inclusion of the subunit to produce ternary 4β2 GABAARs. We also cotransfected the 4 and β2 subunits at a ratio of 1:1. 1, β2, and 2S subunits were cotransfected at a ratio of 1:1:4. In addition, we mock transfected cells to test for the presence of endogenous THIP or GABA-gated currents. Transfected cells were washed after 24 h of exposure to the cDNA precipitate and used for patch-clamp recordings 48–72 h later.
Electrophysiology and Solutions
GABA and THIP-gated currents were recorded at room temperature (21 ± 1°C) using the excised outside-out patch configuration of the patch-clamp technique (Hamill et al., 1981). For a small number of experiments, whole-cell recordings were done as described in the results. Patches (and cells) were voltage clamped at –70 mV. Macropatch (ensemble) and single channel currents were recorded using an Axopatch-200A amplifier (Axon Instruments), filtered with a 4-pole Bessel filter that is built into the amplifier at a –3 dB value of 5 kHz (macropatch) or 10 kHz (single channels) and acquired at a sampling frequency of 20 or 50 kHz, respectively. The standard extracellular solution contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 D-glucose, and 10 HEPES, and titrated to a pH of 7.4 with NaOH. The standard intracellular solution contained (in mM) 60 NaCl, 60 CsCl, 3 KCl, 2 K2ATP, 4 MgCl2, 1 CaCl2, 5 EGTA, 10 D-glucose, 10 HEPES, 20 TEACl, and pH adjusted to 7.4 with NaOH. The patch pipettes used for macropatch experiments had resistances of 4–7 M
when filled with intracellular solution. GABA and THIP were dissolved at the desired concentrations in the extracellular solution. Solutions were delivered onto the patches via a double lumen glass tube, mounted on a piezoelectric transducer that was controlled by programmed, pClamp software. The time taken for the small solution interface (5 nm) between the adjacent streams to sweep across the patch was 100–150 µs (rising phase of open pipette response). Recorded patches were positioned adjacent to the solution interface to achieve fast and consistent solution exchange. Multiple (2–10) currents were recorded at each agonist concentration and averaged to produce a net current response for analysis. Patch pipettes used for single channel recordings were made of thick walled borosilicate glass and had resistances of 6–15 M when filled with intracellular solution. They were also coated with a silicon polymer (Sylgard 184) and fire polished to improve signal resolution. Recordings were made in the continuous presence of agonist. Saturating concentrations of THIP and GABA (10 mM) were used to determine the activation steps and associated rate constants for derived kinetic schemes.
Data Analysis
To determine the activation rate constant in macropatch recordings, the onset phase of the current response was fitted to the following exponential equation:
 | (1) |
where, I(t) is the current at time t, Imax is the maximum current amplitude, and kobs is the observed pseudo-first order rate constant for current activation. The activation rate constant (kobs) was plotted against agonist concentration (log10 scale) for a range of concentrations and the data were fitted to a modified form of the Hill equation:
 | (2) |
where, [A] is the agonist concentration, EC50 is the concentration of agonist that produces half of the maximal response, n is the Hill coefficient (slope at EC50), kmaxobs is the maximal kobs, and yO is the lower activation rate constant as [A] approaches zero. From the fitted data we extracted the maximum (upper asymptote) and minimum (lower asymptote) rates of activation and the concentration that elicits the half-maximal kobs (kobsEC50).
We related our macropatch activation data to a modified del Castillo-Katz type scheme (Del Castillo and Katz, 1957) and assumed that the channels open from a diliganded state to single open state. Moreover, we treated the two putative agonist binding sites on each GABAAR as being identical and independent, so that the binding of one GABA molecule to its site does not affect the binding of the second GABA molecule:
| (SCHEME 1) |
Where R represents the receptor, A represents the agonist, AR and A2R are the mono- and diliganded receptor in the closed state, respectively, and A2R* is the diliganded receptor in the open state. k+1 and k–1 are the microscopic association and dissociation rate constants, respectively. β is the channel opening rate constant and is the shutting rate constant. We interpreted the difference between the upper and lower asymptote values in the kobs versus [agonist] plots as the opening rate constant in Scheme 1. The minimum rate of activation (lower asymptote) was taken as an estimate of the mean burst duration of the channels at low agonist concentration. In addition, reasonable estimates of agonist apparent affinity were obtained from the kobsEC50 values, after correcting for the independent binding site assumption (Maconochie and Steinbach, 1998; Keramidas et al., 2006). Estimates of the individual channel shutting, agonist binding, and unbinding rate constants were obtained by simulating macropatch currents in response to 1–2-ms exposure to agonist. Rapid application currents were simulated in conjunction with coupled differential equations that described the transitions between states. The simulations were done with Berkeley Madonna software (www.berkeleymadonna.com). Experimentally determined values, such as the opening rate constant and apparent affinity, were entered as fixed parameters in the simulations, whereas unknown rate constants, such as the shutting rate constant and the individual agonist binding and unbinding rate constants, were allowed to vary during the fitting.
Single channel current amplitude was determined by generating amplitude histograms for selected segments of record. Single channel conductance was calculated using the following expression:
 | (3) |
where is the single channel conductance, i is the single channel current, Vm is the transmembrane potential and Vrev is the potential at which current reverses direction. The values of Vrev were obtained from current–voltage measurements, covering a range of voltages between –60 mV to +60 mV (Keramidas et al., 2002). Liquid junction potential offsets were calculated and accounted for in the conductance calculation (Barry, 1994).
For single channel analysis, we opted to focus on the gating steps of fully (di-) liganded channels in our kinetic modeling. We used saturating concentrations of agonist to isolate the gating steps from agonist binding. By activating the channels with saturating concentrations of agonist, any agonist binding steps rapidly preequilibrate and the resulting activity can be safely assumed to arise from fully liganded channels. We also found that only saturating concentrations of THIP produces groups of openings that could be defined as clusters or bursts, especially at 4β2 channels. Subsaturating concentrations of THIP (e.g., 50 µM), mostly produced isolated openings at 4β2 channels, which precluded any useful analysis. Single channel kinetic analysis was done with the aid of QuB software (www.qub.buffalo.edu). Segments of activity that appeared to be due to multiple, overlapping openings (e.g., see Fig. 1 A and Fig. 4 A and Figs. S1 and S2) were excluded from analysis. Single channel currents were divided into segments or clusters of activity, initially by eye (see Fig. 4, C and D), and extracted to separate files within QuB for further analysis. Eye-selected clusters were separated from each other by quiescent periods lasting at least 100 ms (often much longer). Isolated openings that often occurred between clusters were ignored during cluster selection and not included in any subsequent analysis. Clusters were separated into discrete bursts by applying a critical shut time (tcrit) that marked the end of a burst (Colquhoun and Hawkes, 1995; Purohit and Grosman, 2006). tcrit values were determined of each patch by generating shut time histograms (MIL in QuB) for the idealized (SKM in QuB) eye-selected clusters of data (Lema and Auerbach, 2006; Plested et al., 2007). These preliminary fits, which were not used for further analysis or model derivation, were needed solely to determine tcrit values for the purpose of dividing eye-selected clusters into shorter segments of activity. The tcrit values were calculated by equalizing the area under the overlapping tails of the two longest shut distributions within eye-selected clusters. Clusters were divided into bursts because the longest shut "intracluster" duration generally ranged between 10 and 40 ms, making it possible that some clusters of openings arose from more than one channel, especially in patches where there was some evidence of more than one channel in the patch. To minimize the occurrence of this confound we opted to divide our clusters into burst, which increased the likelihood that the activity subjected to analysis arose from a single ion channel. The system dead time was 75 µs. Bursts were analyzed for duration, open probability (intraburst PO), and open and shut durations. Gating schemes were derived by generating shut and open dwell-time histograms to idealized burst of data by the method of maximum likelihood (MIL "Star" within QuB) and fitting mixtures of probability density functions to these histograms (Colquhoun and Hawkes, 1995; Lema and Auerbach, 2006). The number of individual exponential components in each dwell class (shut or open) resulting from the fit was taken as the minimum number of states in any underlying reaction scheme. Postulated schemes were then refitted to the idealized bursts by maximum likelihood, starting with initial rate constants of 250 s–1. Three criteria were used to rank schemes. First, the group of likelihood (log likelihood, LL) values obtained from three to five patches, for a particular experimental condition, was subjected to t test analysis. Second, we favored schemes that produced opening rate constants that were comparable to those obtained from macropatch experiments and, finally, schemes that reasonably simulated the macropatch data. Clusters of activity were separated into distinct modes based on intracluster open probability (PO), using the "Select" option in QuB.
Online Supplemental Material
This paper contains two supplemental figures, Figs. S1 and S2 (available at http://www.jgp.org/cgi/content/full/jgp.200709871/DC1). Fig. S1 shows single channel recordings of agonist competition experiments in 1β22S GABAARs using high and low concentrations of THIP (5 mM and 100 µM) and GABA (5 mM and 50 µM). Fig. S2 shows single channel recordings in 1β22S GABAARs at high and low concentrations of THIP (5 mM and 100 µM) and GABA (5 mM and 50 µM) at pipette potentials of –70 and +70 mV.
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RESULTS
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Identifying the 4β2 Pentameric GABAAR Using THIP
We recorded single channel currents in excised, outside-out membrane patches from cells transfected with 4, β2, and subunits, initially at a transfection ratio of 1:1:2. These patches exhibited two current amplitudes in response to saturating concentrations of THIP (10 mM), at a pipette potential of –70 mV. The most frequently observed openings had an amplitude of –2 pA, but we also observed –2.5 pA openings that were less frequent. These two current amplitudes could conceivably represent subconductance states of 4β2 GABAARs having the conventionally accepted stoichiometry of 24:2β2: (Sigel et al., 2006). However, we decided to transfect cells at other transfection ratios and subunit combinations to investigate the possibility that the observed heterogeneity in single channel amplitude arose from GABAARs of different composition. We first increased the proportion of subunit in our transfections to give a 4:β2: ratio of 1:1:5. These transfections yielded a predominant single channel, THIP-activated current amplitude of –2.46 ± 0.07 pA (n = 11 patches). An example of this activity is shown in Fig. 1 A along with the pooled amplitude histogram (Fig. 1 B, dark gray shade).
In our next set of transfections we omitted the subunit and cotransfected only the 4 and β2 subunits, at a ratio of 1:1. Fig. 1 A shows a typical example of single channel, THIP-activated currents from these transfections. The pooled amplitude histogram yielded a mean single channel current amplitude, calculated from 16 patches, of –1.96 ± 0.05 pA (Fig. 1 B, light gray shade). Similar experiments on patches excised from mock-transfected cells yielded no endogenous THIP (or GABA)-gated activity.
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Figure 1. Single channel recordings from GABAARs of different subunit compositions in response to GABA and THIP.(A) Two single channel records from patches expressing 4β2 GABAARs (above) and 4β2 GABAARs (below). The patches were excised from cells transfected with 4, β2, and subunits at a ratio of 1:1:5 (4:β2:) and 4 and β2 subunits at a ratio of 1:1 (4:β2), respectively. (B) Pooled amplitude histograms obtained from patches expressing 4β2 GABAARs (dark gray; peak = –2.46 ± 0.07 pA, n = 11 patches) and 4β2 GABAARs (light gray; peak = –1.96 ± 0.06 pA, n = 16 patches). (C) A single channel recording from a patch (above) expressing 4β2 GABAARs (transfected at 1:1:5) in response to 10 mM GABA and another recording (below) where 10 mM GABA and 10 mM THIP were applied sequentially to the same patch. (D) Pooled amplitude histograms for GABA-gated currents (light gray; peak = –1.72, n = 3 patches) and THIP-gated currents (dark gray, transferred from Fig. 1 D from 4β2 GABAARs). (E) Single channel activity elicited by THIP (above) and GABA (below) from two separate patches expressing 1β22S GABAARs, transfected at a ratio of 1:1:4. (F) Pooled amplitude histograms from patches expressing 1β22S GABAARs in response to GABA (light gray; peak = –1.68 pA, n = 9 patches) and THIP (dark gray; peak = –2.58 ± 0.08 pA, n = 10 patches). Currents were filtered to 2 kHz for display purposes. The peaks corresponding to zero current were omitted for clarity.
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Our single channel measurements suggest that transfecting 4, β2, and subunits at a ratio of 1:1:2 likely results in the expression of GABAARs of different subunit compositions, including not only the desired ternary 4β2 GABAARs, but possibly binary 4β2 channels. This inference is supported by our pooled amplitude histograms and statistical treatment of data obtained at different subunit transfection ratios. We performed a t test (unpaired) on the measurements of single channel current amplitude between the two groups of transfections (4 and β2 at 1:1 and 4, β2, and at 1:1:5). This analysis produced a significant difference in current amplitude between the two groups, with a P value of <0.0001, suggesting that the latter transfection ratio results in the expression of a channel that opens to a distinct amplitude. We infer that this channel is likely the ternary 4β2 GABAAR.
For subsequent sections of this study, including macropatch measurements, we transfected 4, β2, and subunits at a 1:1:5 ratio, as our data suggest that this ratio favorably biases the expression of ternary 4β2 GABAARs. Moreover, in our single channel analysis we will disregard the –2 pA currents in the same transfections as we cannot rule out the possibility that these openings are due to 4β2 binary GABAARs.
Single Channel Amplitude in 4β2 and 1β22S GABAARs Is Agonist Dependent
In this set of experiments we measured and compared single channel current amplitude (and conductance) in 4β2 and 1β22S GABAARs in response to GABA and THIP at a pipette potential of –70 mV. Saturating concentrations of GABA usually failed to evoke any currents in 4β2 GABAARs in most patches that exhibited THIP-activated currents. Generally, the single channel openings in 4β2 GABAARs that were observed in response to GABA were very short in duration, consisted mostly of single open–shut events, and disappeared within a few seconds of recording. GABA-gated single channel activity of 4β2 GABAARs has been previously reported (Akk et al., 2004). That study also noted the absence of discrete clusters of openings, even though saturating concentrations (1 mM) of GABA were used. A notable feature of the GABA-gated currents that we observed was that they had a smaller current amplitude than when activated by THIP. As shown in Fig. 1 C and the accompanying pooled amplitude histogram in Fig. 1 D, GABA produced a single channel amplitude of –1.72 ± 0.07 pA (n = 3 patches). An unpaired, two-tailed t test revealed that the THIP-activated current amplitude was significantly greater than GABA-gated current (P < 0.0001).
We also examined single channel activity in cells transfected with 1, β2, and 2S subunits, at a ratio of 1:1:4 in response to THIP. These patches yielded a single current amplitude of –2.58 ± 0.08 pA (n = 10 patches; Fig. 1 E) at a pipette potential of –70 mV. In contrast, when we exposed 1β22S GABAARs to GABA we observed single channel currents of two distinct amplitudes. One of these was –1.1 pA (unpublished data), but this was relatively rare, accounting for 30% of openings in only 3 out of 14 patches exposed to 10 mM GABA. The majority of the activity had an amplitude of –1.68 ± 0.06 pA (n = 9 patches; Fig. 1 E) and occurred in clusters, separated by quiescent periods that generally lasted several seconds (see also Fig. 4 C). This GABA-gated activity was similar to that previously reported for 1β12S channels (Lema and Auerbach, 2006) and the closely related 1β22L channels (Steinbach and Akk, 2001), also expressed in HEK293 cells. Fig. 1 F shows pooled amplitude histograms for THIP and GABA-gated single channel currents in 1β22S GABAARs. A statistical treatment (unpaired, two-tailed t test) of the single channel currents induced by THIP and GABA revealed that each agonist opened the 1β22S GABAAR to a distinct and significantly different amplitude (P < 0.0001). To explore the observation of differential, agonist-specific single channel current amplitude, we also performed agonist competition experiments on 1β22S GABAARs using GABA and THIP. The agonist application protocol involved coapplying the two agonists, in addition to applying each agonist separately, to the same patch. The experiments were done with the following pairs of agonist concentrations: 5 mM GABA and 5 mM THIP, 5 mM GABA and 100 µM THIP, and, finally, 50 µM GABA and 5 mM THIP (all at –70 mV). As shown in Fig. S1 (available at http://www.jgp.org/cgi/content/full/jgp.200709871/DC1), when 5 mM GABA and 5 mM THIP were coapplied the resulting current amplitude (–2.24 ± 0.05 pA) was not distinguishable from that of THIP alone (–2.22 ± 0.06 pA, P = 0.8431, paired t test). In contrast, the amplitude elicited by 5 mM GABA alone was –1.62 ± 0.02 pA (n = 4 patches, Fig. S1 A and Table S1), which an ANOVA test (one-way with repeated measures) revealed as significantly smaller (P < 0.0001). An example of the second type of competition experiment is shown in Fig. S1 B. Here, when 5 mM THIP was applied alone or in combination with 50 µM GABA, the current amplitudes were indistinguishable, being –2.44 ± 0.04 pA for THIP and –2.47 ± 0.03 pA for THIP plus GABA (P = 0.5559, paired t test). 50 µM GABA applied alone produced an amplitude of –1.70 ± 0.03 (n = 8 patches), which was again significantly smaller than when the patches were exposed to 5 mM THIP alone or in combination with GABA (ANOVA, P < 0.0001, Table S1). Fig. S1 C shows recordings from a patch where 5 mM GABA and 100 µM THIP were either applied separately or coapplied. In contrast to saturating concentrations of THIP, 100 µM THIP elicited an amplitude of –1.80 ± 0.03 pA, which was not statistically different from that elicited by 5 mM GABA alone (–1.73 ± 0.05 pA) or 5 mM GABA with 100 µM THIP (–1.78 ± 0.04, n = 8 patches, AVOVA, P = 0.2429, Table S1). It is also noteworthy that the current amplitude evoked by 5 mM and 50 µM GABA and 100 µM THIP were not significantly different from each other (ANOVA, P = 0.2125). In three patches we were also able to obtain enough spontaneous (agonist-free) single channel openings to construct reliable amplitude histograms (unpublished data). These measurements yielded an amplitude of –1.60 ± 0.06 pA, which was not significantly different from GABA-gated openings (paired t test, P = 0.3781). Fig. S1 D shows single channel activity from a patch that exhibited some spontaneous openings and was subsequently and sequentially exposed to GABA (50 µM), THIP (100 µM), and THIP (5 mM).
To further characterize single channel current amplitude elicited by the two agonists, we recorded activity at pipette potentials of –70 and +70 mV, from the same patch, using either saturating concentrations of GABA or THIP (5 mM) or subsaturating concentrations of GABA (50 µM) or THIP (100 µM). Examples of these experiments are provided in Fig. S2. For reference, the amplitude measurements and statistical comparisons are tabulated in Table S2, along with a measure of current rectification (rectification index, RI), defined as the ratio of single channel current amplitude measured at pipette potentials of –70 and +70 mV.
Saturating concentrations of THIP (5 mM) applied to 1β22S GABAARs produced similar single channel current amplitude at –70 mV (–2.32 ± 0.07 pA) and +70 mV (2.30 ± 0.07 pA, paired t test, P = 0.839, n = 8 patches, Fig. S2 A), resulting in an RI of 1. 100 µM THIP elicited a significantly greater current amplitude at –70 mV (–1.76 ± 0.11 pA) than at +70 mV (1.46 ± 0.03 pA, paired t test, P = 0.038, n = 5 patches, Fig. S2 B), producing slight inward rectification (RI = 1.10). This was also the case when using GABA as the agonist at both saturating and subsaturating concentrations. 5 mM GABA elicited a current amplitude of –1.66 ± 0.06 pA at –70 mV and 1.45 ± 0.04 pA at +70 mV (n = 8 patches, paired t test, P = 0.025, RI = 1.14, Fig. S2 C). Similarly, 50 µM GABA elicited a current amplitude of –1.74 ± 0.06 pA at –70 mV and 1.40 ± 0.10 pA at +70 mV (n = 3 patches, paired t test, P = 0.003, RI = 1.24; Fig. S2 D, Table S2).
Using Eq. 3, single channel currents of –2.46 and –1.70 pA activated by THIP and GABA, respectively, and corresponding reversal potentials (unpublished data) for the two agonists, we calculated the unitary conductance of 4β2 GABAARs. The calculations yielded conductances of 35.5 and 24.6 pS for THIP and GABA, respectively, at –70 mV. Similar calculations for 1β12S GABAARs produced conductance values of 37.2 pS for THIP and 25.0 pS for GABA (Table I).
For subsequent single channel analysis of 4β2 GABAARs we focused on the THIP-activated –2.46 pA current, and for 1β22S GABAARs, the THIP-activated –2.58 pA and GABA-activated –1.68 pA activity. Due to its rarity, and the possibility that the lower conductance, GABA-gated activity in 1β22S GABAARs could be mediated by binary 1β2 channels (Wagner et al., 2004; Li et al., 2006), which were –1.1 pA, we decided not to analyze these particular currents any further.
Current Activation Rates in 4β2 and 1β22S GABAARs are Agonist Dependent
Current activation was examined over a range of concentrations of GABA and THIP for 4β2 and 1β22S GABAARs, using ultrafast agonist application on excised outside-out patches. For 4β2 GABAARs in response to GABA, data from small cells were also included, as these generally gave larger currents with improved signal–noise ratios. However, in spite of our efforts, it was only possible to obtain reliable activation rate data for the higher concentrations or GABA (10, 20, and 30 mM) for these channels.
The rate of current activation was measured by fitting Eq. 1 to the onset phase of the current (Fig. 2, A and B), from which the observed activation rate constant (kobs) was obtained. kobs was then plotted as a function of agonist concentration over the entire range of concentrations tested (Fig. 2, C and D).
The resulting data were fitted to a Hill type equation (Eq. 2) to obtain the upper and lower asymptotic levels and the concentration that elicits the half-maximal kobs (kobs EC50; Table I). For both agonists the kobs increased with increasing agonist concentration, plateauing to a maximum at 10–20 mM for both agonists. For 4β2 GABAARs, the kobs was greater for THIP than GABA, reaching a maximum for the two agonists of 2058 s–1 and 442 s–1, respectively (Fig. 2 C). The converse was the case for 1β22S GABAARs, with GABA producing a higher activation rate constant than THIP. The maximum kobs value for GABA was 1986 s–1 and for THIP was 660 s–1 (Fig. 2 D). The maximum activation rate constant for GABA-gated 1β22S GABAARs, estimated from medium-sized cells (Keramidas et al., 2006), is comparable, although slightly lower than that obtained here from excised patches using the same ultrafast perfusion system.
With decreasing agonist concentration, current activation became increasingly slower, plateauing to distinct, nonzero asymptotic levels. For 4β2 channels activated by THIP, the lower asymptote was 434 s–1. For 1β22S channels the lower asymptote kobs values were 202 s–1 and 186 s–1 for THIP and GABA, respectively. These values were not significantly different (Table I).
The agonist concentration that produced the half maximal kobs values (kobs EC50) varied with channel type. 4β2 channels had a kobs EC50 for THIP of 1.64 mM. 1β22S GABAARs had statistically indistinguishable kobs EC50s, which were 696 µM for THIP and 704 µM for GABA (Table I). These results indicate that 4β2 GABAARs have a lower apparent affinity for THIP than 1β22S GABAARs.
Simulating Macroscopic Currents and Simple Kinetic Schemes
To obtain estimates of rate constants in Scheme 1 for both channels and agonist we fitted simulated currents to real macropatch currents in response to 1–2-ms applications of 30 mM agonist, as previously described (Keramidas et al., 2006). To improve the accuracy of the fit and reduce the number of free parameters in the fitting procedure, we imposed the following constraints on our simulations. These constraints were based on experimentally derived parameters obtained from the kobs versus concentration plots (Table I). First, we set the opening rate constant (β) in Scheme 1 to equal the estimated value for β* from the kobs plot, after subtracting the lower asymptotic value (Maconochie et al., 1994; Keramidas et al., 2006) and fixed this parameter in the fitting procedure. Second, we took our experimental kobs EC50 values to be reasonable estimates of the equilibrium dissociation constant for the second binding step in Scheme 1 (Kd = 2k–1/k+1), after making a minor correction for equivalent and independent binding sites (Maconochie and Steinbach, 1998), which we assumed. This gave us a ratio of dissociation rate constant to association rate constant (k–1/k+1), which we fixed during the simulations, allowing effectively only the k+1 value itself to vary in the simulations of 1β22S GABAAR currents and THIP-activated 4β2 GABAAR currents. When simulating GABA-activated 4β2 GABAAR currents we set β to the upper asymptote (although this is likely an overestimate) and allowed k–1 and k+1 to vary freely as our GABA-gated kobs plot did not extend far enough to obtain a lower asymptote or kobs EC50, respectively. In addition, we estimated the channel open probability (POmacropatch) in response to 1–2-ms exposure to agonist using nonstationary fluctuation analysis. The POmacropatch values we obtained were, for 4β2 GABAARs, GABA–0.25 and THIP–0.36, and for 1β22S GABAARs, GABA–0.56 and THIP–0.42 (unpublished data). The currents were then scaled to the POmacropatch values before generating the simulations. Fig. 3 shows examples of real currents and the superimposed simulated currents generated with the above fitting constraints.
Accompanying each real and simulated current pair is the corresponding kinetic scheme and averaged rate constants (from three to four patches each) for both GABAARs and agonists. The rate constants estimated in Scheme 1 for GABA-activated 4β2 GABAARs were (Fig. 3 A; β was set to 440 s–1) = 86 ± 6 s–1, k+1 = 0.14 ± 0.06 x 106 M–1s–1, and k–1 = 44 ± 3 s–1, and for THIP-activated 4β2 GABAARs (Fig. 3 B; β was set to 1600 s–1), = 268 ± 17 s–1, k+1 = 2.1 ± 0.3 M–1s–1, and k–1 = 1434 ± 26 s–1. For THIP-activated 1β22S GABAARs, the rate constants were (Fig. 3 C; β was set to 460 s–1) = 283 ± 5 s–1, k+1 = 3.3 ± 0.2 106 M–1s–1, and k–1 = 957 ± 12 s–1, and for GABA-activated 1β22S GABAARs (Fig. 3 D; β was set to 1800 s–1), = 310 ± 14 s–1, k+1 = 1.4 ± 0.2 x 106 M–1s–1, and k–1 = 410 ± 22 s–1.
Single Channel Burst Characteristics Are Determined by Agonist Type
To obtain information about gating kinetics of fully liganded channels, we used saturating concentrations of THIP and GABA (10 mM) to elicit single channel currents in 4β2 and 1β22S GABAARs. THIP activated 4β2 GABAARs more consistently and produced more complex bursts than GABA (Fig. 4 A and Fig. 6 A), so we focused most of our efforts on the THIP-activated activity.
Segments (clusters) of THIP-activated activity were divided into bursts by applying a critical shut time (tcrit), which ranged between 17 and 37 ms. Table II summarizes the mean burst durations and intraburst open probability (PO) for 4β2 GABAARs activated by THIP and GABA. As can be seen from Table II and Fig. 4 A and Fig. 6 A, THIP induced bursts of activity in 4β2 GABAARs that were of relatively short duration (32.6 ± 4.4 ms) and had an intraburst PO of 0.40 ± 0.02.
GABA-activated bursts were much briefer (7.17 ± 0.58 ms) and had an intraburst PO of 0.25 ± 0.05. Similar analysis was applied to THIP- and GABA-activated single channel activity in 1β22S GABAARs. In response to 10 mM THIP, single channel openings in 1β22S GABAARs occurred as short bursts, defined by tcrit values that ranged between 17 and 25 ms. These bursts were similar in duration and structure to the THIP-activated activity in 4β2 GABAARs (Fig. 4, B and D, and Fig. 7 A). The mean burst duration and intraburst PO for this activity was 31.6 ± 3.9 ms and 0.47 ± 0.03, respectively (Table II). Notably, THIP induced shorter inactive periods, normally associated with channel desensitization, than GABA at 1β22S GABAARs. The range of the longest mean shut duration, measured from three patches in the presence of THIP was 0.5–10 s (Fig. 7), whereas, in the presence of GABA the channels shut for periods ranging from 2 to 200 s (n = 4 patches, Fig. 4 C and Fig. 8). These observations suggest that THIP elicits similar bursting patterns at both channels, whereas GABA elicits single channel activity that is substantially different from that elicited by THIP at the same channel.
In addition to longer-lived desensitization, GABA induced long clusters of activity that could be divided into distinct bursting patterns on the basis intraburst PO. This kinetic phenomenon has recently been reported for 1β12S channels recorded in the intact-patch configuration (Lema and Auerbach, 2006). To our knowledge, the present study is the first to report distinct bursting patterns, or modes, in excised outside-out patches for GABAARs. Moreover, our data suggest that bursting modes are agonist specific, with THIP eliciting only one discernable type of bursting mode and GABA eliciting at least two distinct modes. Clusters of GABA-gated activity were separated into modes using "Select" within QuB, as previously described (Lema and Auerbach, 2006). These were extracted to separate files for division into bursts by applying a tcrit. tcrit values were generally shorter for the high PO bursts. They ranged between 8 and 12 ms and 9 and 17 ms for high (H-Mode) and medium (M-Mode) PO bursts, respectively. Instances where modal switching occurred within the same cluster were not included in the analysis. Fig. 5 A shows a channel (presumably the same channel) that enters an M-Mode cluster then switches to an H-Mode cluster.
Most patches exhibited two gating modes, with the M-Mode PO being the most prevalent. The lowest PO mode (L-Mode) was the least prevalent, occurring only in 3 of 15 patches and was not analyzed any further (Fig. 5 A). The mean burst durations and corresponding intraburst POs for the H-Mode and M-Mode were as follows: H-Mode, 122.6 ± 19.7 ms and 0.87 ± 0.02 (n = 5 patches); M-Mode, 101.0 ± 8.3 ms and 0.69 ± 0.02 (n = 11 patches, Table II).
Single Channel Burst Analysis
We used saturating agonist concentrations (10 mM) to model gating kinetics in 4β2 GABAARs in response to THIP and 1β22S GABAARs in response to THIP and GABA. Fig. 6 shows an example of THIP-activated activity in 4β2 GABAARs and the accompanying dwell histograms for bursts.
As can be seen in the figure, the data were best fit with two shut and two open components. The corresponding mean time constants (and fractions) for each exponential component, from five patches, are shown in Table III.
THIP-activated bursts in 1β22S GABAARs were also described best with a mixture of two open and two shut components (Fig. 7, n = 3 patches), strongly suggesting that THIP elicits similar activation kinetics in both channels.
The main difference in the time constants between the two channels was that the first shut time constant (
C1) was nearly threefold greater and over twofold more prevalent for 1β22S GABAARs. GABA-gated bursts required three shut and three open components to adequately describe M-Mode activation (Fig. 8, n = 10 patches).
This is the same number of components reported for 1β12S GABAARs (Lema and Auerbach, 2006). For the H-Mode activity, only two shut and three open components were required (Fig. 9, n = 5 patches), in contrast to that reported by Lema and Auerbach (2006).
Notably, the longest open time constant in H-Mode activity was over 2.5–fold longer than the corresponding time constant in the M-Mode activity.
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