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1
3
and
1
3
2L GABAA Receptor Currents
Departments of Neurology (H.-J.F., M.T.B., R.L.M.), Molecular Physiology and Biophysics (R.L.M.), and Pharmacology (R.L.M.), Vanderbilt University, Nashville, Tennessee
Received May 11, 2004; accepted July 8, 2004
| Abstract |
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isoforms, thought to dominate phasic (synaptic) GABAergic inhibition, have been extensively studied, the effects of pentobarbital on kinetic properties of 

GABAA receptors, thought to mediate tonic (extra- or perisynaptic) inhibition, are unknown. Using ultrafast drug delivery and single channel recording techniques, we demonstrate isoform-specific pentobarbital modulation of low-efficacy, minimally desensitizing
1
3 currents and high-efficacy, rapidly desensitizing
1
3
2L currents. Specifically, with saturating concentrations of GABA, pentobarbital substantially potentiated peak
1
3
receptor currents but failed to potentiate peak
1
3
2L receptor currents. Also, pentobarbital had opposite effects on the desensitization of
1
3
(increased) and
1
3
2L (decreased) receptor currents evoked by saturating GABA. Pentobarbital increased steady-state
1
3
receptor single channel open duration primarily by introducing a longer duration open state, whereas for
1
3
2L receptor channels, pentobarbital increased mean open duration by increasing the proportion and duration of the longest open state. The data support previous suggestions that GABA may be a partial agonist at 

isoforms, which may render them particularly sensitive to allosteric modulation. The remarkable increase in gating efficacy of
1
3
receptors suggests that 

isoforms, and by inference tonic forms of inhibition, may be important targets for barbiturates.
1 to
6,
1 to
4,
1 to
3,
,
,
, and
(Olsen and Macdonald, 2002
subunit, but these kinetic responses were not observed for receptors containing a
2L subunit (Wohlfarth et al., 2002
Pentobarbital is an anesthetic barbiturate and exerts its CNS effects by interacting with GABAA receptors (Olsen and Macdonald, 2002
). At low concentrations, pentobarbital potentiates GABAA receptor currents (Nicoll and Wojtowicz, 1980
; Schulz and Macdonald, 1981
) by increasing the proportion of longer single channel openings and thus increasing mean open duration. At higher concentrations, pentobarbital directly activates GABAA receptors (Nicoll and Wojtowicz, 1980
; Schulz and Macdonald, 1981
; Krampfl et al., 2002
). At millimolar concentrations, pentobarbital inhibits GABAA receptor function, probably through a low-affinity open channel block mechanism (Akaike et al., 1987
; Rho et al., 1996
; Akk and Steinbach, 2000
). These properties of pentobarbital (positive allosteric modulation, direct agonism, and open channel block) are similar to those reported for certain neurosteroids (Lambert et al., 1995
; Rupprecht and Holsboer, 1999
).
Although the effects of barbiturates on 

currents, thought to dominate phasic (synaptic) GABAergic inhibition, have been extensively studied, the modulatory effects of pentobarbital on the kinetic properties of 

GABAA receptors are still unknown. There is increasing evidence that 

receptors may be selectively targeted to extraor perisynaptic membranes, where they are thought to mediate tonic neuronal inhibition by responding to fluctuating concentrations of extracellular neurotransmitters such as GABA (Nusser et al., 1998
; Bai et al., 2001
; Stell et al., 2003
; Wei et al., 2003
). Recent studies have suggested that pentobarbital plays an important role in modulation of
subunit-containing GABAA receptors. Chronic pentobarbital treatment evoked an alteration of GABAA receptor
subunit mRNA in the CNS (Lin and Wang, 1996
), and pentobarbital potentiated the response of
subunit-containing GABAA receptors (Adkins et al., 2001
; Brown et al., 2002
). In the present study, we transfected cDNAs encoding rat
1,
3, and
or
1,
3, and
2L GABAA receptor subunits into human embryonic kidney (HEK 293T) cells and used ultrafast drug delivery and outside-out patch single channel recording techniques to characterize the distinct effects of pentobarbital on GABAA receptors containing
or
2L subunits.
| Materials and Methods |
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1,
3, and
or
1,
3, and
2L GABAA receptor subunits using a modified calcium phosphate precipitation method (Fisher and Macdonald, 1997a
Whole Cell and Single Channel Recordings. Whole cell macroscopic currents were obtained with the cell attached to the dish (whole cell recording) or after lifting of the cells (lifted cell recording). Single channel microscopic currents were recorded from excised outside-out patches. Recording electrodes were pulled on a P-87 Flaming Brown micropipette puller (Sutter Instrument Company, Rafael, CA). The whole cell and lifted cell recording electrodes were pulled from the thin-wall borosilicate glass tubing (i.d. 1.12 mm, o.d. 1.5 mm) (WPI, Sarasota, FL) with resistances between 0.8 to 2 M
, and the single channel recording electrodes were pulled from the thick-wall borosilicate glass tubing (i.d. 0.84 mm, o.d. 1.5 mm) (WPI) with resistances between 6 to 16 M
. All electrodes were fire polished on an MF-9 Microforge (Narishige, Tokyo, Japan). The single channel recording electrodes were coated with polystyrene Q-dope (GC Electronics, Rockford, IL) after fire polishing to minimize the noise during recording.
Currents were recorded with either an Axopatch 1D or 200A patch-clamp amplifier (Axon Instruments, Foster City, CA) and Digi-data 1200 series interface (Axon Instruments). The data were stored on a PC computer hard drive for offline analysis. Series resistance was not compensated; theoretically, this could lead to an underestimation of the extent of desensitization because voltage errors would be larger at the peak current than after receptor desensitization had occurred. However, we previously reported that desensitization rate and extent were not significantly affected by current size for a broad range of amplitudes, including very large current peaks (515 nA) that desensitized
90% over a 28-s application (Bianchi and Macdonald, 2002
), suggesting that series resistance errors did not significantly affect our interpretations.
Solutions, Drugs, and Drug Application. All the chemicals were purchased from Sigma-Aldrich (St. Louis, MO). External bath solution was composed of 142 mM NaCl, 1 mM CaCl2, 6 mM MgCl2, 8 mM KCl, 10 mM glucose, and 10 mM HEPES that was standardized to pH 7.4 with NaOH and osmolality to 323 to 329 mOsm. Recording electrodes were filled with an internal solution consisting of 153 mM KCl, 1 mM MgCl2, 10 mM HEPES, and 5 mM EGTA at pH 7.3 and osmolality between 301 and 309 mOsm. MgATP (2 mM) was added to the internal solution on recording days. These solutions produced an ECl near 0 mV and an EK at -75 mV.
GABA and pentobarbital sodium salt were prepared as stock solutions and were diluted to desired concentrations with external solution on the day of the experiment. Applications of drugs were performed using an ultrafast delivery device consisting of multibarrel tubes (two three-barrel square glass tubing glued together) connected to a perfusion fast-step system, a mechanical translation device (Warner Instrument, Hamden, CT). Each of the three-barrel square glasses was heated and manually pulled to the final barrel size (around 200 µm). The external bath solution and drug solutions were driven by gravity. This drug delivery device allowed rapid solution exchanges with 10 to 90% rise times consistently less than 2 ms (typically 0.41.0 ms) determined by switches between normal and dilute external solution at an open electrode. However, solution exchanges, even when cells were lifted from the recording dish, were likely to occur with a slower rate. Applications of drugs were separated by an interval of at least 45 s to minimize accumulation of desensitization. GABA-evoked single channel activity was recorded with and without pentobarbital application from excised outside-out patches during steady-state conditions (minutes of exposure to GABA). Voltage was clamped at -75 mV during single channel recordings. All the experiments were performed at room temperature.
Data Analysis and Simulations. Whole cell currents were analyzed offline using Clampfit 8.1 (Axon Instruments) and GraphPad Prism 2.01 (GraphPad Software Inc., San Diego, CA). Peak currents were measured directly (manually) relative to the baseline, and residual currents at "steady state" were measured from the end of 28-s application to baseline after recovery. The extent of potentiation of GABA current by pentobarbital was measured by dividing the peak current of coapplication of a given concentration of GABA and pentobarbital by the peak current evoked by GABA alone. The resulting ratio was multiplied by 100 and expressed as percentage of GABA control. The concentration-response data were normalized and fitted using a four-parameter logistic equation with a variable slope: I = Imax/(1 + 10(LogEC50-Logdrug) · Hill slope). I represented the current evoked by a given concentration of GABA with or without pentobarbital coapplication. Imax denoted the maximal GABA peak current. The extent of current desensitization was measured as a percentage of current reduction, calculated by dividing the amount of desensitized current (amplitude of peak current - amplitude of current at the end of the 4- or 28-s GABA application) by peak current and multiplying by 100. The rate of deactivation was analyzed using standard exponential Levenberg-Marquardt methods. Deactivation currents were fitted with one or two exponential components in the form of a1
1 + a2
2, where a1 and a2 represented the relative amplitudes of the exponential components, and
1 and
2 denoted the time constant. A weighted
was calculated to compare the rates of deactivation using the formula a1 ·
1/(a1 + a2) + a2 ·
2/(a1 + a2). Single channel data were acquired at 50-µs intervals, filtered at 2 kHz, and analyzed offline with Fetchan 6.0 (Axon Instruments) using 50% threshold detection. Although most patches contained multiple channels based on overlapped openings, only single amplitude openings were included in the analysis. Kinetic analysis was performed using Interval 5 (Dr. Barry S. Pallotta, University of North Carolina, Chapel Hill, NC). Open duration histograms were generated and fitted by the maximum likelihood method. The number of exponential components was increased until an additional exponential component did not significantly improve the fit as determined by a log-likelihood ratio test automatically performed by the software. Events with intervals less than 1.5 times of the estimated system dead time (100 µs) were plotted but not included in the fitting.
Simulations were carried out with the Berkeley Madonna 8.0 software package (www.berkeleymadonna.com), using the fourth order Runge-Kutta algorithm to solve model differential equations with a 100-µs step size. Simulated currents were imported into GraphPad Prism 2.01 (GraphPad Software Inc.) where noise was added before display.
Data were reported as mean ± S.E.M. Paired Student's t test was used to compare current features before and after pentobarbital treatment. Unpaired Student's t test was used to analyze the alterations between different treatment groups. The difference was considered to be statistically significant for p < 0.05.
| Results |
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2L and
Subunit-Containing GABAA Receptors to GABA. Whole cell currents were recorded by rapid application of different concentrations of GABA to HEK cells transfected with cDNAs encoding rat
1,
3, and
2L or
1,
3, and
GABAA receptor subunits (Fig. 1A). Cells were voltage clamped at -20 mV for cells transfected with
1,
3, and
2L subunits and at -50 mV for those transfected with
1,
3, and
subunits due to the smaller amplitude of currents recorded in these receptors (Wohlfarth et al., 2002
G) were greater for
1
3
2L receptors than for
1
3
receptors (Fig. 1B). Mean maximal peak
G for
1
3
2L receptors (233.4 ± 41.1 nS; n = 7) was significantly higher than that for
1
3
receptors (21.0 ± 4.0 nS; n = 6) (p < 0.001). The mean EC50 values for
1
3
2L receptors (8.5 ± 6.4 µM) and
1
3
receptors (5.8 ± 0.7 µM) were not significantly different. The Hill slope for both
1
3
2L and
1
3
receptors was 1.4.
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Differences in Direct Activation and Open-Channel Block of
2L or
Subunit-Containing GABAA Receptors by Pentobarbital. As reported previously (Schulz and Macdonald, 1981
; Rho et al., 1996
; Thompson et al., 1996
; Krampfl et al., 2002
), pentobarbital directly activated GABAA receptors at high concentrations. In the present study, pentobarbital-evoked whole cell currents were recorded from
1
3
2L and
1
3
receptors, followed by application of saturating GABA (0.3 mM) for each cell (Fig. 2, A and C). Similar to GABA, pentobarbital evoked smaller mean peak
G for
1
3
receptors than for
1
3
2L receptors (Fig. 2, B and D, squares). At 1000 µM pentobarbital,
1
3
receptor mean peak
G (7.0 ± 2.4 nS; n = 7) was significantly smaller than
1
3
2L receptor
G (109.4 ± 19.0 nS; n = 8) (p < 0.001). However, at higher pentobarbital concentrations, the mean peak
G for
1
3
2L receptors declined. This effect of high pentobarbital concentration was not observed for
1
3
receptors up to 3000 µM pentobarbital (Fig. 2, B and D, squares). At 3000 µM pentobarbital, the mean peak
G was still significantly smaller for
1
3
receptors than for
1
3
2L receptors (p < 0.05).
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With higher pentobarbital concentrations, a "rebound" current occurred upon washout of pentobarbital (Fig. 2, A and C). This is consistent with rapid unbinding of pentobarbital from a low-affinity, open channel block site as suggested previously (Rho et al., 1996
; Thompson et al., 1996
; Wooltorton et al., 1997
; Dalziel et al., 1999
; Akk and Steinbach, 2000
; Krampfl et al., 2002
). For both
1
3
2L and
1
3
receptors, the mean peak
G of rebound current (measured from the current amplitude at the onset of the rebound current) increased as the pentobarbital concentrations increased from 200 to 3000 µM (Fig. 2, B and D, circles). The mean peak
G of rebound current was significantly less for
1
3
receptors than for
1
3
2L receptors at both 1 and 3 mM pentobarbital (p < 0.05).
For
1
3
2L receptors, maximal mean peak
G evoked by pentobarbital was significantly smaller than mean peak
G evoked by a saturating GABA concentration. Maximal mean peak
G evoked by pentobarbital averaged 58.5 ± 8.0% (p < 0.01) of GABA-evoked mean peak
G. For
1
3
receptors, maximal mean peak
G evoked by pentobarbital was 127.1 ± 18.6% of that evoked by a saturating GABA concentration, but this difference did not reach statistical significance. For
1
3
2L receptors, maximal mean rebound
G evoked by pentobarbital was significantly smaller than that evoked by a saturating GABA concentration (80.2 ± 9.1%; p < 0.05). However, for
1
3
receptors, maximal mean rebound
G evoked by pentobarbital was 1479.0 ± 590.4% (p < 0.01) of that evoked by a saturating GABA concentration.
For
1
3
2L receptors, the pentobarbital-evoked direct current declined during the pentobarbital application (Fig. 2A). This decline likely represented receptor desensitization, although a contribution from open channel block could not be ruled out. This apparent current desensitization was concentration-dependent (3003000 µM pentobarbital) and was manifested differently in
1
3
and
1
3
2L receptors (Fig. 3A). Whereas only minimal desensitization was observed for
1
3
receptor currents up to 3000 µM pentobarbital (Fig. 3A), more extensive desensitization was observed for
1
3
2L receptors at pentobarbital concentrations greater than 300 µM (Fig. 3A). It is possible, however, that a rapidly equilibrating open channel block mechanism was "masking" macroscopic manifestations of entry into desensitized states, and thus the measurements of apparent desensitization may be underestimated for both isoforms. Furthermore, the activation time with pentobarbital tended to be slower than that observed with GABA, and slow macroscopic activation would preclude observation of fast desensitization (Bianchi and Macdonald, 2002
).
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For
1
3
receptors, the deactivation rates of GABA-evoked currents were not concentration-dependent for concentrations up to 1 mM (data not shown), but significant slowing of deactivation was observed at 3000 µM pentobarbital (Figs. 2C and 3B). For
1
3
2L receptors, the deactivation rates of GABA-evoked current were not concentration-dependent at concentrations greater than 10 µM (data not shown). However, the deactivation rates of pentobarbital-evoked current were concentration-dependent at concentrations greater than 300 µM (Figs. 2A and 3B).
Pentobarbital Enhanced Currents Evoked by a High Concentration of GABA from
More than
2L Subunit-Containing GABAA Receptors. Because low concentrations of pentobarbital (3050 µM) caused little or no direct activation of current, they were considered modulatory concentrations. Occasionally, pentobarbital at 50 µM alone produced small direct currents for
1
3
2L receptors, but the direct currents were minimal (<2%) compared with maximal GABA-evoked currents. Pentobarbital concentrations in this range potentiated GABA-evoked currents for both
1
3
and
1
3
2L receptors. Coapplication of a range of GABA concentrations with 50 µM pentobarbital slightly potentiated
1
3
2L receptor currents (Fig. 4B). The currents evoked by coapplication of GABA and pentobarbital as well as GABA alone were normalized to 300 µM GABA. The normalized GABA concentration-response curve was shifted upward (Fig. 4B), with a maximal extent of enhancement of 133.9 ± 19.9% (n = 8). The mean EC50 for GABA + 50 µM pentobarbital (3.8 ± 1.9 µM) was not significantly different from that of GABA alone (8.5 ± 6.4 µM). Pentobarbital at 30 µM also potentiated
1
3
receptor currents (Fig. 4C). The concentration-response curve was shifted upward (Fig. 4D), and the amplitude of this shift (225.9 ± 15.1%; n = 7) was significantly greater than that observed with
1
3
2L receptors (p < 0.001). The mean EC50 for GABA + 30 µM pentobarbital (5.3 ± 1.0 µM) was not significantly different from that of GABA alone (5.8 ± 0.7 µM).
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Pentobarbital Produced Similar Alterations in Peak Current, Desensitization, and Deactivation of Currents Evoked by a Submaximal Concentration of GABA for
and
2L Subunit-Containing GABAA Receptors. To demonstrate better the fast component of desensitization and to determine the effect of pentobarbital on GABAA receptor currents evoked by low (1 µM) GABA concentrations during coapplication, cells were lifted from the recording dishes, and pentobarbital was preapplied for 1.5 s before a 4-s coapplication of GABA and pentobarbital. Pentobarbital (100 µM, a concentration that evoked very small currents from both
1
3
2L and
1
3
receptors) substantially enhanced both
1
3
2L (660.5 ± 111.9%; n = 6) and
1
3
(872.0 ± 105.9%; n = 8) receptor currents, but no significant difference in enhancement was observed between these two isoforms (Fig. 5, AC). When activated by 1 µM GABA,
1
3
2L and
1
3
receptor currents showed minimal apparent desensitization (<3%; Fig. 5, A, B, and D). Coapplication of 1 µM GABA with 100 µM pentobarbital significantly increased the extent of desensitization similarly for both
1
3
2L and
1
3
receptors (
10%; Fig. 5, A, B, and D) (p < 0.01). For these experiments, the extent of current loss during the 4-s applications was used to describe desensitization, rather than fitted desensitization time constants, because the rate of decay was slow relative to the application length, precluding accurate fitting of the time constants.
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The currents evoked by 1 µM GABA deactivated significantly faster for
1
3
receptors than for
1
3
2L receptors (Fig. 5, A, B, and E) (p < 0.001). For
1
3
receptors, the mean current deactivation time constants were significantly increased by pentobarbital from 86.8 ± 12.4 to 271.8 ± 45.9 ms (p < 0.01). In the presence of pentobarbital, the mean current deactivation time constants were significantly increased from 321.2 ± 29.4 to 1257.4 ± 237.9 ms for
1
3
2L receptors (p < 0.05). The mean current deactivation time constants in the presence of pentobarbital were significantly smaller for
1
3
receptors than for
1
3
2L receptors (p < 0.001) (Fig. 5E).
Pentobarbital Evoked a Greater Enhancement of Peak Amplitude and Desensitization with a Saturating Concentration of GABA for
than for
2L Subunit-Containing GABAA Receptors. Although pentobarbital (100 µM) modulation of currents evoked by a 4-s application of low concentration of GABA (1 µM) was not significantly different between
1
3
and
1
3
2L receptors, modulation of currents evoked by a 4-s application of a saturating concentration of GABA (1 mM) by pentobarbital (100 µM) produced a much larger effect on
1
3
than
1
3
2L currents (we used the same preapplication protocol as that for application of 1 µM GABA). Pentobarbital did not enhance the peak
1
3
2L receptor current evoked by 1 mM GABA (99.4 ± 5.8%; n = 7) (Fig. 6, A and C). However, pentobarbital evoked a substantial enhancement in peak
1
3
receptor current (526.4 ± 98.3%; Fig. 6, B and C). Note that in the coapplication condition under which
1
3
2L receptor GABA concentration-response curves were generated, 50 µM pentobarbital potentiated currents evoked by 300 µM GABA. The basis for the difference between this enhancement compared with results with 100 µM pentobarbital preapplied for 1.5 s before a jump into 1 mM GABA is unclear but may be related to increased direct gating of the receptors (visible during the preapplication period) that may have resulted in a small amount of "predesensitization", similar to what has been described for preincubation in low concentrations of GABA (Overstreet et al., 2000
).
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Currents evoked from
1
3
2L receptors by 1 mM GABA exhibited substantial desensitization (78.5 ± 3.5%; Fig. 6, A and D), consistent with previous studies on recombinant GABAA receptors containing a
subunit (Haas and Macdonald, 1999
; Burkat et al., 2001
; Bianchi and Macdonald, 2002
). However, only minimal desensitization was observed for
1
3
receptors with this concentration of GABA (14.1 ± 3.9%; Fig. 6, B and D), similar to our previous reports (Haas and Macdonald, 1999
). Pentobarbital differentially altered desensitization for
1
3
and
1
3
2L receptor currents evoked by 1 mM GABA. The mean percentage of current reduction was significantly increased to 39.1 ± 6.3% (p < 0.01) for
1
3
receptors. In contrast, the mean percentage of current reduction was significantly decreased by pentobarbital by
10% to 69.5 ± 5.5% for
1
3
2L receptors (p < 0.05) (Fig. 6D).
To evaluate more accurately the effects of pentobarbital on the extent of desensitization, longer duration GABA applications were required for the residual currents to approach a quasi steady state. Thus, both
1
3
and
1
3
2L currents were evoked by long-duration (28-s) pulses of GABA (1 mM). The enhancement of peak currents for long-duration GABA application was within the range of that for 4-s applications for both isoforms. However, the extent of desensitization, compared with 4-s applications, was increased for both isoforms. Specifically, pentobarbital significantly increased the mean residual currents from 868.0 ± 338.1 to 1196.0 ± 392.0 pA for
1
3
2L receptors (n = 5) (p < 0.05) as a consequence of a significant reduction in the extent of desensitization from 83.8 ± 2.9 to 78.1 ± 2.2% (p < 0.05) after 28-s GABA application (Fig. 7, AC). For
1
3
receptors (n = 4), pentobarbital induced a significant increase in apparent desensitization from 30.0 ± 9.6 to 59.6 ± 10.6% (p < 0.05) after 28-s GABA application, but the mean residual current amplitudes were significantly increased after pentobarbital treatment from 252.5 ± 60.1 to 512.5 ± 121.5 pA (p < 0.05) (Fig. 7, DF).
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Although pentobarbital produced differential changes in enhancement of peak current and desensitization for
1
3
and
1
3
2L receptor currents evoked by a saturating concentration of GABA, the rates of deactivation were prolonged in the presence of pentobarbital in both of these receptors after 4-s GABA application (Fig. 6, A and B). The mean current deactivation time constants after activation with 1 mM GABA alone were significantly smaller for
1
3
receptors than for
1
3
2L receptors (p < 0.001) (Fig. 6E). Pentobarbital significantly increased the mean current deactivation time constants from 118.2 ± 32.4 to 471.9 ± 127.8 ms (p < 0.05) for
1
3
receptors and also significantly increased the mean current deactivation time constants from 388.8 ± 44.6 to 1168.9 ± 272.1 ms for
1
3
2L receptors (p < 0.05) (Fig. 6E).
Pentobarbital Introduced a Long-Duration Open State for
Subunit-Containing GABAA Receptor Single Channel Currents. To explore possible bases for the different effects of pentobarbital in modulating macroscopic GABA-evoked currents from
and
2L subunit-containing GABAA receptors, single channel currents were recorded without and with pentobarbital during steady-state application of GABA to outside-out membrane patches containing
1
3
or
1
3
2L receptors. Data were analyzed from 40 to 480 s after the patch was excised.
1
3
2L receptor single channel currents exhibited bursting openings in the presence of 1 mM GABA (Fig. 8A13). The distribution of open states was fitted best with three exponential functions (Fig. 8A4), similar to our previous reports (Fisher and Macdonald, 1997b
; Haas and Macdonald, 1999
). Coapplication of 1 mM GABA with 100 µM pentobarbital increased the mean duration of channel openings (Fig. 8B13), although the distribution of open states was still best described by three exponential functions (Fig. 8B4). Compared with GABA alone, pentobarbital significantly increased the mean channel open duration from 1.45 ± 0.26 ms (n = 5) to 4.86 ± 1.06 ms (n = 5) for
1
3
2L receptors (p < 0.05) (Table 1). The time constant of the shortest exponential function (
1) was not significantly altered by pentobarbital. However, pentobarbital significantly decreased the relative area of the shortest exponential function (A1) (p < 0.05) (Table 1). Neither the time constant (
2) nor the relative area (A2) of the second exponential function was significantly altered for
1
3
2L receptors (Table 1). Interestingly, the third exponential function time constant (
3) was significantly increased from 4.63 ± 0.81 to 9.37 ± 1.11 ms (p < 0.01), and the relative area (A3) was significantly increased from 18.2 ± 3.04 to 41.4 ± 7.21% (p < 0.05) (Table 1). Pentobarbital at 100 µM alone directly activated single channel currents (Fig. 8C13). The open-duration histogram of channel activity evoked by 100 µM pentobarbital (n = 4) was similar to that evoked by 1 mM GABA (Fig. 8A1 and C1), except that
1 was significantly smaller with pentobarbital alone treatment (p < 0.05). Compared with pentobarbital alone, the mean open duration and
3 were significantly increased with coapplication of GABA and pentobarbital (p < 0.05) (Table 1).
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In contrast to the "high-efficacy" bursting behavior of
1
3
2L receptor single channel currents,
1
3
receptor single channel currents displayed only brief openings (Fig. 9A13). The distribution of open states was fitted best with two exponential functions (Fig. 9A4), similar to our previous reports (Fisher and Macdonald, 1997b
; Haas and Macdonald, 1999
). Coapplication of 1 mM GABA with 100 µM pentobarbital resulted in an increased mean open duration (Fig. 9B13). The distribution of open states in the presence of pentobarbital, however, required three exponential functions (Fig. 9B4). Compared with GABA alone, coapplication of GABA and pentobarbital significantly increased the mean open duration from 0.53 ± 0.04 ms (n = 6) to 1.08 ± 0.17 ms (n = 7) for
1
3
receptors (p < 0.05) (Table 1). A1 was significantly decreased by pentobarbital (p < 0.05), although
1 was not significantly altered.
2 was significantly increased from 0.90 ± 0.10 to 1.27 ± 0.10 ms in the presence of pentobarbital (p < 0.05). Coapplication of GABA and pentobarbital introduced a third longer duration open state for
1
3
receptors with a time constant of 4.03 ± 0.59 ms and relative area of 10.3 ± 2.15% (Table 1). The single channel activity evoked by 100 µM pentobarbital alone (n = 5) was different from that evoked by 1 mM GABA alone because open-state distributions required a third longer-duration open state (
3 = 2.95 ± 0.62 ms, A3 = 3.50 ± 0.57%) (Fig. 9C14).
1 was significantly smaller for pentobarbital alone treatment than for GABA alone treatment (p < 0.001) (Table 1). Compared with pentobarbital alone, the mean open duration (p < 0.05),
1 (p < 0.001),
2 (p < 0.01), and A3 (p < 0.05) were significantly greater with coapplication of GABA and pentobarbital (Table 1).
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Although it was possible that pentobarbital altered closed times or opening frequency in these experiments, we could not accurately analyze these properties because most patches in the current study exhibited overlapping openings, which indicated the presence of multiple active channels. The presence of more than one channel will result in spuriously high measures of open probability and open frequency, as well as low apparent closed duration measurements. Analysis was performed only on individual open durations; burst analysis was not performed due to the presence of multichannel patches that precluded accurate closed state analysis (required to define intraburst closed durations).
Simulation of
1
3
and
1
3
2L Receptor Currents. Although a detailed mechanism of action for pentobarbital could not be proposed based on the current results, we felt that simulations may shed light on two interesting features of the macroscopic data. First, pentobarbital potentiated the peak amplitude of
1
3
currents but did not potentiate that of
1
3
2L currents evoked by a saturating GABA concentration, despite the clear increase in efficacy by prolonging steady-state mean open time. Although this might have been related to limitations of resolving the "true" peak of a rapidly activating and desensitizing current using whole cell methods, could this observation be related to underlying receptor kinetics? Second, pentobarbital exhibited opposite effects on the macroscopic desensitization of
1
3
and
1
3
2L currents. Although this could be related to isoform-specific pentobarbital modulation of rate constants of desensitized states, was it possible that an increase in single channel open time could partly contribute to both of these apparently opposite effects? To explore these issues, we generated simulated currents using our previously established models for
1
3
and
1
3
2L receptors (Haas and Macdonald, 1999
) (Fig. 10, A and B). For the purposes of this analysis, we were limited to qualitative comparisons, particularly because the models were generated using data obtained with excised patches, a condition of substantially higher temporal resolution than the whole cell configuration (Bianchi and Macdonald, 2002
).
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