Introduction

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GABA_A receptors are the predominant inhibitory neurotransmitter receptors in the vertebrate central nervous system. When activated, the Cl channel of the receptor opens, leading to an influx of Cl and neuronal hyperpolarization. GABA_A receptors are pentameric hetero-oligomers composed of assemblies of different subunits. Based upon sequence homology, these subunits have been grouped into different classes designated (1-6), (1-4), (1-3), , ,  (Hevers and Luddens, 1998), and the recently discovered  (Bonnert et al., 1999). Hydropathy analysis has revealed that each subunit spans the membrane four times (TM1-TM4), with the second transmembrane domain (TM2) lining the channel pore (Hevers and Luddens, 1998).

GABA_A receptors possess a variety of allosteric binding sites through which different drugs can modulate the GABA-mediated Cl current. Benzodiazepines and barbiturates are known to allosterically potentiate GABA-mediated current (Hevers and Luddens, 1998). Conversely, convulsant drugs like picrotoxin (PTX), TBPS, and several insecticides are known to depress GABA-mediated current (Bloomquist, 1996).

It has been known for a number of years that PTZ inhibits GABA-activated channels (Macdonald and Barker, 1978). Initial radioligand binding studies suggested that the site of action of PTZ was the benzodiazepine site of the GABA_A receptor (Rehavi et al., 1982). Subsequent binding studies, however, indicated the site of action of PTZ was likely the picrotoxin site of the receptor (Ramamjaneyulu and Ticku, 1984; Squires et al., 1984). Based on these binding studies, it is now generally accepted that PTZ acts at the picrotoxin site of the channel. PTX and other presumed PTX-site ligands, including TBPS and the cyclodiene insecticides, are proposed to bind within the channel pore formed by the TM2 (ffrench-Constant et al., 1993; Zhang et al., 1994; Gurley et al., 1995; Xu et al., 1995). Consequently, PTZ presumably also mediates its inhibitory effect through interaction at the picrotoxin site within TM2.

The mechanism of block by PTX and other related compounds is still equivocal. Based on the use-dependent characteristics of PTX, it may act within the channel lumen to block the channel (Akaike et al., 1985; Inoue and Akaike, 1988). However, single-channel studies have demonstrated that PTX does not affect channel burst duration (Newland and Cull-Candy, 1992). Moreover, PTX-induced inhibition of the GABA_A receptor is voltage independent (Newland and Cull-Candy, 1992). These results are inconsistent with the conclusion that picrotoxin inhibits the receptor via a traditional open channel blocking mechanism. In addition, although it has been demonstrated that mutations of amino acids in the second transmembrane domain of the receptor inhibit the actions of PTX, it is not known if these amino acids are involved in binding, a transduction event subsequent to picrotoxin binding, or even accessibility of picrotoxin to its site of action. In addition to blocking GABA_A receptors, picrotoxin blocks a number of other ion channels, including GABA_C receptors (Wang et al., 1995; Zhang et al., 1995), glycine receptors (Pribilla et al., 1992), and glutamate-gated Cl channels (Etter et al., 1999). Consequently, an understanding of its actions should advance ion channel physiology in general. Thus, the precise site and mechanism of block by picrotoxin continues to be an important topic of investigation.

Although the mechanism of picrotoxin inhibition is still being debated, even less is known about PTZ-induced block of the GABA_A receptor. For instance, studies in recent years have demonstrated that the inhibitory actions of picrotoxin and other presumed picrotoxin-site ligands are affected by subunit configuration (Pribilla et al., 1992; Zhang et al., 1995; Bell-Horner et al., 2000); this work has helped to define the presumed sites of action of these ligands. However, no such studies have evaluated potential subunit-dependent effects of PTZ. In addition, assessments of PTZ-induced block in general are lacking. In this study, we have assessed the functional interaction of the convulsant PTZ with numerous configurations of GABA_A receptors, using whole-cell and single-channel patch clamp. Whereas our results indicate some aspects of PTZ-induced inhibition are similar to those observed with picrotoxin, we noted important disparities in the actions of PTZ and suggest the functional domains for the two drugs are comparable but not equivalent.

	Materials and Methods

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Cloned GABA_A Receptors. Human embryonic kidney cell lines (HEK293) stably expressing varying configurations of recombinant GABA_A receptors were studied in the present investigation. Cells expressing rat 122, 322, 622, 12, and 22 receptors (short isoform of the 2 subunit in all cases) were generously supplied by Pharmacia-Upjohn (Kalamazoo, MI). A detailed description of the preparation of HEK293 cells stably expressing GABA_A receptors has been published previously (Hamilton et al., 1993). Cells stably expressing human 122 receptors were also studied. A complete description of preparation of this cell line has been published previously (Hawkinson et al., 1996).

Electrophysiology. Whole-cell and outside-out patch recordings were made at room temperature (22-25°C). Except during acquisition of current-voltage relationships, cells were voltage-clamped at 60 mV. Patch pipettes of borosilicate glass (1B150F; World Precision Instruments, Inc., Sarasota, FL) were pulled (Flaming/Brown, P-87/PC; Sutter Instrument Co., Novato, CA) to a tip resistance of 1 to 2.5 M for whole-cell recordings and 8 to 12 M for outside-out single-channel recordings. The pipette solution contained 140 mM CsCl, 10 mM EGTA, 10 mM HEPES, 4 mM Mg-ATP, pH 7.2. Coverslips containing cultured cells were placed in a small chamber (~1.5 ml) on the stage of an inverted light microscope (Olympus IMT-2; Olympus, Tokyo, Japan) and superfused continuously (5-8 ml/min) with the following external solution containing 125 mM NaCl, 5.5 mM KCl, 0.8 mM MgCl₂, 3.0 mM CaCl₂, 20 mM HEPES, 10 mM D-glucose, pH 7.3. GABA-induced Cl currents from the whole-cell or outside-out configuration of the patch clamp technique were obtained using an Axoclamp 200A amplifier equipped with a CV-4 headstage (Axon Instruments, Foster City, CA). For whole-cell recording, GABA-induced Cl currents were low-pass filtered at 5 kHz, monitored on an oscilloscope and a chart recorder (Gould TA240; Gould, Cleveland, OH), and stored on a computer (pClamp 6.0, Axon Instruments) for subsequent analysis. Series resistance compensation (60-80%) was applied at the amplifier. To monitor the possibility that access resistance changed over time or during different experimental conditions, we measured and stored on our digital oscilloscope the current response to a 5 mV voltage pulse at the initiation of each recording. This stored trace was continually referenced throughout the recording. If a change in access resistance was observed during the recording period, the patch was aborted, and the data were not included in the analysis. For single-channel recordings, the currents were filtered with a low-pass Bessel filter (80 dB/decade) at a cut-off frequency of 1 to 2 kHz and simultaneously recorded on a video cassette recording system (Sony SLV-420; Sony, Tokyo, Japan) via a digital data recorder (VR-10B CRC; Instrutech Corp., Great Neck, NY).

Experimental Protocol. For all single-channel recordings and whole-cell recordings in HEK293 cells, GABA with or without PTZ was prepared in the extracellular solution and then applied from independent reservoirs by gravity flow for 10 to 20 s to cells or membrane patches using a Y-shaped tube positioned within 100 µm of the cells or the membrane patch. With this system, the 10 to 90% rise time of the junction potential at the open tip is 12 to 51 ms. We did not attempt to study receptors lacking the  subunit, because this subunit may be required for plasma membrane targeting and functional expression (Connolly et al., 1996). Receptors were typically activated with roughly the EC₃₀ GABA concentration; this concentration was chosen because minimal desensitization, which may confound interpretation of results, was elicited. Once a control GABA response was determined, the effect of PTZ on the response was examined. Recovery from PTZ-induced inhibition was readily obtained, thus full inhibition curves for PTZ could generally be obtained from studying one cell. GABA applications were separated by at least 2-min intervals to ensure both adequate washout of GABA from the bath and recovery of receptors from desensitization, if present. Typically, to monitor GABA response, a GABA pulse was applied before, during, and after incubation.

Chemicals. GABA and PTZ were obtained from Sigma (St. Louis, MO). IMGBL was synthesized as described previously (Canney et al., 1991). GABA and PTZ stocks were made in double-distilled H₂O. IMGBL was made in dimethyl sulfoxide and diluted in saline so that the final dimethyl sulfoxide concentration (v/v) was <0.2%.

Data Analysis. For whole-cell recording, all data were recorded on a chart recorder and stored on a computer for subsequent off-line analysis (pClamp 6.0). GABA concentration-response profiles were constructed from whole-cell recordings and fitted to the following equation: I/I_max= [GABA]ⁿ/([GABA]ⁿ + EC₅₀ⁿ), where I and I_max represent the normalized GABA-induced current at a given concentration and the maximum current induced by a saturating [GABA], EC₅₀ is the half-maximal effective GABA concentration, and n is the Hill coefficient. PTZ inhibition-response relationship was fitted with the equation I/I_max = [PTZ]ⁿ/([PTZ]ⁿ + IC₅₀ⁿ), where I is Cl current amplitude normalized to control, IC₅₀ is the half-blocking concentration, and n is the Hill coefficient. A minimum of three (typically five to eight) individual experiments were conducted for each paradigm.

	Results

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Evaluation of GABA_A Receptor Subunit-Dependent Inhibition by PTZ. Other ligands that are presumed to bind at the picrotoxin site of the GABA_A receptor display some subunit-dependent effects (Bell-Horner et al., 2000). We thus evaluated whether changes in GABA_A receptor subunit isoforms influence the ability of PTZ to block the GABA_A receptor. Figure 1 illustrates PTZ inhibition in several configurations of the receptors. In all receptors tested, PTZ inhibited GABA-mediated current in a concentration-dependent manner. PTZ had similar effects on peak and steady-state currents and did not enhance current decay rate (Fig. 1A). This contrasts with the block induced by picrotoxin and the picrotoxin-site ligands TBPS and U-93631, which have minimal effects on peak amplitude but significantly enhance rate of current decay (Yakushiji et al., 1987; Dillon et al., 1993, 1995a). The PTZ-induced block reached steady state during the first drug application, as repeated applications of PTZ plus GABA did not result in further current inhibition. Table 1 shows IC₅₀ and Hill coefficients for PTZ inhibition in the receptors evaluated. PTZ displayed comparable efficacy and affinity at the different receptor configurations tested (Fig. 1B; Table 1). PTZ IC₅₀ values were near 1 mM (range = 0.6-2.2 mM) for all subunit configurations tested, including those with varying  subunit isoforms and those lacking an  or  subunit. Moreover, the IC₅₀ values were comparable in rat and human receptors.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of receptor subunit composition on PTZ-induced inhibition of GABAA receptors. A, examples of typical responses to PTZ in 122, 22, and 12 receptors. Initial trace in each example is response to GABA alone (EC30 concentration). PTZ inhibited GABA-gated current in a concentration-dependent manner. Drug application was 10 s in all cases. Current calibration is 500 pA for 122, 655 pA for 22, and 332 pA for 12, respectively. Time calibration is 20 s for all three receptors. B, mean concentration-response profiles for the effects of PTZ on varying configurations of recombinant GABAA receptors. IC50 values for PTZ ranged from 0.6 to 2.2 mM (Table 1). The h in h122 denotes human receptors; all others are rat receptors.

Kinetic Parameters for PTZ Interaction with GABA_A Receptors. The above results demonstrated that the IC₅₀ for PTZ inhibition of GABA-gated current (millimolar range) is approximately 1000-fold higher than that reported for PTX (Krishek et al., 1996; Bell-Horner et al., 2000), which has in general been defined in the low-micromolar range. The rapid onset and recovery from PTZ inhibition, compared with PTX (Dillon et al., 1995a), suggested that this difference in functional affinity is at least in part due to changes in dissociation rate constants (k₁). To quantify the kinetic interactions of PTZ with the GABA-bound receptor, we used the following one-site model:
where R* is the GABA-bound receptor, R*PTZ is the PTZ-bound nonconducting receptor, and k₊₁ and k₁ are the drug association and dissociation rates, respectively. Interaction of the receptor with PTZ will proceed to equilibrium with an exponential time constant, , equal to 1/(k₊₁[PTZ] + k₁). Thus, based on this one-site model, a plot of the 1/ versus PTZ concentration should yield data that can be fitted to a linear function. The slope of the line will equal the k₊₁, and the y-intercept will equal the k₁ for PTZ interacting with GABA-bound receptors. This model has been used previously to define the kinetic parameters for other picrotoxin-site ligands (Dillon et al., 1993, 1995a). To define these parameters for PTZ, human 122 receptors were incubated with GABA at 5 µM; this concentration activated a stable current that displayed negligible desensitization. PTZ at varying concentrations (0.1-3 mM) was then coapplied with 5 µM GABA for 10 s. As shown in Fig. 2A, the PTZ-induced current decay was concentration-dependent. The reduction of initial current induced by PTZ was nicely fitted with a monoexponential function, yielding a  for inhibition at each concentration tested. By plotting the data as described above, we obtained an association rate (k₊₁) of 1.14 × 10³ M¹ s¹, a dissociation rate (k₁) of 0.476 s¹, and a dissociation constant (K_d = k₁/k₊₁) of 0.418 mM for PTZ in the presence of GABA (Fig. 2B). The k₁ for PTZ can also be estimated from the recovery of the GABA current upon removal of PTZ. Thus, the  for relaxation back to the control GABA current following PTZ inhibition was determined. This  was calculated to be 3.47 ± 0.16 s, the inverse of which yielded a k₁ of 0.3 ± 0.01 s¹. This estimate of k₁ is comparable with that determined using the one-site model, thus validating the use of the model to define these kinetic parameters.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Determination of the kinetic constants for PTZ interaction with GABA-bound receptors. A, receptors were continuously incubated with 5 µM GABA to elicit a stable nondesensitizing current. Varying concentrations of PTZ (0.1-3 mM), along with 5 µM GABA, were then applied to the cells. Upon PTZ application, GABA-induced currents decayed following a monoexponential function dependent on PTZ concentration. B, the reciprocal of the time constants from the monoexponential fitting was plotted as a function of PTZ concentrations. The solid line represents the best linear fit to the function 1/ = k+1[PTZ] + k1, producing the following kinetic parameters for PTZ: k1 = 0.476 s1; k+1 = 1.14 × 103 M1 s1; Kd = 0.418 mM.

Effect of PTZ on GABA Current Voltage Relationship. Because inhibition of ionic currents by many channel blockers is influenced by transmembrane voltage, we assessed the potential voltage dependence of PTZ-induced block of GABA_A receptors. In human 122 receptors, current activated by 10 µM GABA was recorded over a range of holding potentials in the presence or absence of 1 mM PTZ. GABA induced Cl current was outward-rectifying. PTZ did not significantly change the reversal potential (4.1 ± 1.0 mV in control and 4.4 ± 1.0 mV in the presence of 1 mM PTZ, p > 0.05, paired t test, n = 4) or the rectification profile. In addition, the magnitude of PTZ-induced inhibition was similar when tested at 60, 30, +30, and +60 mV (one-way ANOVA, p > 0.05, n = 4). Although not definitive, a lack of voltage dependence argues against a classical open channel blocking mechanism for PTZ.

Effect of PTZ on the GABA Concentration-Response Curve. Picrotoxin-induced block of GABA receptors has generally been defined as noncompetitive (Akaike et al., 1985; Yakushiji et al., 1987; Yoon et al., 1993) or mixed, having both noncompetitive and competitive components (Smart and Constanti, 1986; Krishek et al., 1996). The mechanism by which PTZ inhibits GABA-induced currents was studied by determining the concentration-response relationship of GABA in the absence and presence of PTZ (1, 5, and 20 mM) in human 122 receptors. Figure 3B shows that PTZ significantly increased the GABA EC₅₀ in a concentration-dependent manner (a 7-fold increase in GABA EC₅₀ in the presence of 20 mM PTZ, p < 0.05, one-way ANOVA), whereas the Hill coefficient was not significantly altered (p > 0.05, one-way ANOVA). Although there was a modest tendency for maximal GABA current to be reduced by PTZ, the difference was not significant (p > 0.05, paired t test, n = 4-5 for each PTZ concentration group). Similar results were obtained when experiments were conducted using rat 122 receptors (not shown). Thus, whereas picrotoxin is a mixed antagonist, the antagonism of GABA-gated current by PTZ is predominantly competitive in nature.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of PTZ (1 mM, 5 mM, and 20 mM) on the GABA concentration-response curve recorded from human 122 GABAA receptors. A, records illustrating effect of 5 mM PTZ on currents activated by 10, 100, and 1000 µM GABA. B, concentration-response curves for GABA with or without the presence of 1, 5, or 20 mM PTZ. Recordings were carried out at a holding potential of 60 mV. Amplitude is normalized to the maximum current activated by GABA in the absence of PTZ. Each data point is the average current from three to five cells. Note that PTZ increased the EC50 for GABA in a concentration-dependent manner but did not significantly affect the maximal GABA-gated current.

Effect of Intracellular PTZ. It has been reported in the Xenopus oocyte expression system that PTZ is able to cross the plasma membrane of cells and possibly acts at an intracellular site(s) (Bloms-Funke et al., 1996). To test this, we examined effects of extracellularly applied PTZ in recombinant human 122 receptors when a near saturating PTZ concentration (20 mM) had already been added to the pipette solution and thus pre-equilibrated intracellularly. Because HEK cells are known to be coupled, these experiments were conducted on isolated single cells to ensure that PTZ was fully equilibrated in the cell under study. In the presence of intracellular PTZ, extracellular coapplication of 1 and 20 mM PTZ resulted in 45.5 ± 2.5% and 89.7 ± 5.2% inhibition of the current amplitude induced by 10 µM GABA (Fig. 4A; n = 6). This degree of inhibition was not different from that recorded in cells that were not exposed to intracellular PTZ (50 ± 2.7% inhibition by 1 mM PTZ and 91.4 ±1.5% by 20 mM PTZ, n = 5, p > 0.05, unpaired t test, Fig. 4B). In addition, the effects of extracellular application of PTZ were highly reversible and repeatable in the cells preloaded with PTZ. These data only conclusively demonstrate that the presence of PTZ inside the cell does not prohibit block by extracellular application; they do not rule out the possibility of an additional intracellular site of action of PTZ. To further assess the possibility that PTZ may have an intracellular site of action, we conducted the following experiments. GABA-activated currents were recorded from isolated single cells with or without intracellular 20 mM PTZ introduced into the cell via the patch pipette. The presence of 20 mM intracellular PTZ did not significantly influence the current amplitude induced by 10 µM GABA, as the current amplitude was 1539 ± 317 pA in PTZ-preloaded cells (n = 18) and 1476 ± 346 pA in control cells (n = 16, p > 0.05, unpaired t test). These results do not support the hypothesis that PTZ has an additional intracellular site of action. Moreover, the data also suggest that PTZ can only access its binding site to GABA_A receptors via an extracellular pathway.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of PTZ on GABA-activated currents in human 122 receptors in the presence or absence of intracellular PTZ. A, representative traces show the effect of extracellularly applied PTZ (1 or 20 mM) on the current induced by 10 µM GABA when 20 mM PTZ had already been introduced intracellularly through the patch pipette. Note that the inhibition induced by extracellular coapplication of PTZ was reversible and repeatable. B, histogram summarizing the mean effect of PTZ on the GABA response in human 122 GABAA receptors. The inhibition induced by PTZ was not different between the cells with (n = 5) and without (n = 4) intracellular PTZ preloading (p > 0.05, unpaired t test).

Effect of PTZ on GABA-Activated Single-Channel Currents. To explore at the single-channel level the effect of PTZ on GABA_A receptor gating kinetics, we recorded single-channel currents in the absence or presence of PTZ (1 mM, close to its IC₅₀ in the whole-cell recordings) using excised outside-out patches. Because the PTZ action among various subunit compositions or species was indistinguishable at the whole-cell level, we studied kinetics of single-channel activity in human 122 receptors only.

1

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Effect of PTZ on GABA-activated single-channel current. A, representative single-channel currents activated by 5 µM GABA or 5 µM GABA plus 1 mM PTZ in an outside-out patch recorded from human recombinant 122 GABAA receptors. Single-channel currents are shown at increasing time resolution for 0 µM (a1, a2), 5 µM GABA (b1-3), and 5 µM GABA plus 1 mM PTZ (c1-3). The patch contained two channels. Single-channel currents were recorded at a holding potential of 40 mV and low-pass filtered at 1 kHz. B, amplitude histogram in the absence and presence of PTZ. The data were constructed from the digitized data (53 kHz, 20 s) of same patch presented in A. A fitting with three Gaussian distributions revealed that the patch contained two channels (mean amplitude ~1.70 pA). In the presence of PTZ, note that channels exist in the open state much less frequently, but their amplitudes were not significantly altered.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effect of PTZ on the distributions of open (A) and closed (B) duration of human recombinant 122 GABAA receptors. Distributions were normalized and overlaid to display relative frequency distributions for comparison. Each histogram is derived from pooled data obtained from six patches. A, normalized linear-binned open duration frequency distribution histogram for GABA (5 µM) and GABA plus PTZ (1 mM). The open distributions were placed into 1-ms bins over a range of 1 to 27 ms. Single-channel open events were binned on a conventional scale using 20 bins per decade resolution. Histograms for open duration were best fitted with the sum of two exponential functions for experimental conditions with time constants (and relative areas) of 2.6 ms (0.855) and 10.7 ms (0.145) at control and 2.9 ms (0.883) and 10.7 ms (0.117) in the presence of PTZ. B, closed duration frequency histograms for 5 µM GABA were altered by 1 mM PTZ. Logarithmic-binned frequency histograms of closed duration were best fitted with sums of three exponential functions with time constants (and relative areas) of 3.4 ms (0.490), 31.8 ms (0.335), and 239.1 ms (0.169) at control and 3.3 ms (0.430), 43.7 ms (0.360), and 355 ms (0.209) in the presence of PTZ. All the curves were drawn according to the fits.

View this table: [in this window] [in a new window]   TABLE 2 Effect of PTZ (1 mM) on single-channel properties of human 122 recombinant GABAA receptors (outside-out patches were voltage-clamped at 40 mV)

Effect of IMGBL on PTZ Inhibition. The -butyrolactone derivative IMGBL is an antagonist of the action of picrotoxin (Holland et al., 1990; Yoon et al., 1993). We used IMGBL as a probe to determine whether functional domains for PTZ and picrotoxin overlap. IMGBL (5 mM) was chosen because it efficiently blocked picrotoxin inhibition of GABA-activated current in rat 122 receptors (Fig. 7, A and C). IMGBL also significantly reduced the inhibition of GABA-activated current produced by 1 mM and 20 mM PTZ (p < 0.05, paired t test, n = 5-7). Application of IMGBL alone (n = 2) had no effect on the whole-cell recording, and it did not affect GABA-activated current (Fig. 7B; n = 6). Protection of PTZ inhibition with the picrotoxin antagonist IMGBL supports the suggestion that PTZ and picrotoxin share a related functional domain.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Effect of IMGBL on PTZ-induced inhibition of GABA currents in rat 122 receptors. A, representative traces showing the effect of IMGBL (5 mM) on current inhibition of GABA-activated current produced by 10 µM PTX or 20 mM PTZ. Note that IMGBL markedly reduced the degree of PTX- or PTZ-induced inhibition of the response to 30 µM GABA. B, IMGBL (5 mM) did not elicit any effect on basal properties of the membrane or on GABA-activated current. C, graph summarizing the mean effect of IMGBL on PTX- or PTZ-induced inhibition of GABA current. Note that IMGBL significantly reduced the inhibition of GABA current produced by PTX or PTZ (p < 0.05, paired t test, n = 5-7).

	Discussion

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Based largely on radioligand binding studies, PTZ is believed to interact with the picrotoxin site of the GABA_A receptor (Ramamjaneyulu and Ticku, 1984; Squires et al., 1984). Remarkably few studies, however, have evaluated directly the actions of PTZ on GABA_A receptors and its mechanism and molecular site of action. Thus, in an attempt to more precisely define these parameters, we used an array of experimental manipulations to assess quantitatively the mechanism and site of action of PTZ.

Several lines of evidence obtained in the present report support the contention that the site of action of PTZ is similar to the picrotoxin site of the receptor. First, we demonstrate that the -butyrolactone compound IMGBL, shown previously to antagonize the actions of picrotoxin (Holland et al., 1990), can also block the inhibitory actions of PTZ. Both findings are consistent with a common site of action of the two convulsant agents; second, single-channel recordings from human 122 GABA_A receptors demonstrated that PTZ inhibition of GABA_A receptors is due to a decrease in the frequency of channel opening. PTZ did not affect mean-channel open duration or single-channel conductance. These effects of PTZ at the single-channel level are comparable with those elicited by picrotoxin (Newland and Cull-Candy, 1992) and the picrotoxin-site ligands dieldrin (Ikeda et al., 1998) and U-93631 (Dillon et al., 1995b).

Additional characteristics of block are common to both PTZ and PTX. For instance, block by both PTX (Yakushiji et al., 1987; Newland and Cull-Candy, 1992; Yoon et al., 1993) and PTZ (present results) is voltage-independent. Moreover, we show that PTZ can only gain access to its binding site extracellularly. This is evident by the finding that intracellular PTZ, at a near saturating concentration, did not alter GABA-activated current. Our results conflict with those of Bloms-Funke et al. (1996), who found that intracellular PTZ could inhibit GABA_A receptors expressed in Xenopus oocytes. The discrepancy is likely due to the differences in experimental preparations. Intracellular picrotoxin was reported to block GABA currents in one study (Akaike et al., 1985) but had no effect in a separate study (Cull-Candy et al., 1987).

The fact that a number of characteristics of block by PTZ and PTX are similar does not indicate that the two drugs share a common site of action. Studies have shown that amino acids at the 2' and 6' positions in the cytoplasmic end of TM2 are involved in picrotoxin blockade of the GABA_A receptor (ffrench-Constant et al., 1993; Gurley et al., 1995; Xu et al., 1995). Gurley et al. (1995) demonstrated that picrotoxin sensitivity was abolished when the 6' threonine of the 2 subunit was mutated to phenylalanine (T6'F). We have recently shown that this mutation also abolishes the inhibitory actions of PTZ (Dibas and Dillon, 2000). The dependence of both PTZ and PTX on the nature of this 6' TM2 residue for GABA_A receptor block provides a physical basis for the similarity of action of the two drugs and supports the suggestion that they interact at the same or overlapping domains.

Although several lines of evidence support a common site of action of PTZ and picrotoxin, there are several important distinguishing features between the two compounds, some of which shed additional light on their respective functional domains. The affinity of PTZ is roughly 1000-fold lower than that of PTX and other presumed PTX-site ligands (Dillon et al., 1993, 1995a; Krishek et al., 1996). Our kinetic analysis illustrates that this is predominantly due to differences in the dissociation rate (k₁). In the present experiments, we defined the PTZ k₁ in GABA-bound receptors at 0.476 s¹ and k₊₁ at 1.14 × 10³ M¹ s¹. The same parameters for PTX were shown to be 0.0058 s¹ and 1.3 × 10⁴ M¹ s¹, respectively (Dillon et al., 1995a). The equilibrium dissociation constants (K_d) are thus 418 µM for PTZ and 443 nM for PTX. By definition, the formation of drug-receptor complexes at the equilibrium concentration of a drug is equivalent to the dissociation of drug-receptor complexes. Although the association rate (k₊₁) for PTZ is about 10-fold lower on a molar basis than that of PTX, at an equilibrium concentration the k₊₁ is equivalent to the k₁ (0.476 s¹ for PTZ versus 0.0058 s¹ for PTX). Thus, the much greater effect on initial peak current observed with PTZ inhibition, compared with PTX inhibition (Yakushiji et al., 1987; Yoon et al., 1993; Dillon et al., 1995), can be fully explained by the differences in kinetic constants described here.

A somewhat unexpected but notable finding was that PTZ antagonized GABA-activated current exclusively via competitive inhibition. This contrasts with PTX-induced block, which displays noncompetitive (Akaike et al., 1985; Yakushiji et al., 1987; Yoon et al., 1993) and competitive components (Smart and Constanti, 1986; Krishek et al., 1996) of block. The PTZ-induced block is unlikely to be a true competition at the GABA binding site, as PTZ does not inhibit binding of [³H]muscimol to GABA_A receptors (Ticku and Maksay, 1983). The competitive component of picrotoxin block may be due to its ability to allosterically stabilize the receptor in an inactivated state, instead of true competition at the agonist binding site (Smart and Constanti, 1986, and below). A similar mechanism is likely to account for PTZ-induced block. Other examples of allosterically mediated "competitive" antagonism have been demonstrated (Bertrand et al., 1992; Lynch et al., 1995). Indeed, picrotoxin itself blocks human glycine 1 receptors through allosteric competitive inhibition (Lynch et al., 1995); an arginine residue at the extracellular region of TM2 was shown to be involved in this antagonism. With regard to PTX and PTZ block of the GABA_A receptor, the 6' threonine of TM2 may be involved in their ability to allosterically stabilize the receptor in a nonconducting state.

A difference between PTZ and PTX inhibition that provides considerable information about their relative functional domains is revealed by our assessment of subunit-dependent effects of PTZ. In general, picrotoxin has been considered to display relatively nonspecific interactions with various configurations of GABA_A receptors (Newland and Cull-Candy, 1992; Krishek et al., 1996). However, recent work has shown that GABA_A receptors lacking an  subunit are significantly (10- to 20-fold) more sensitive to PTX than those composed of subunits (Bell-Horner et al., 2000). This is also true for the PTX-site ligand U-93631 (Bell-Horner et al., 2000) and the insecticide dieldrin (C. L. Bell-Horner and G. H. Dillon, in preparation). Interestingly, our present results demonstrate that PTZ inhibition is not enhanced in receptors lacking an  subunit. The enhanced sensitivity to PTX, U-93631, and dieldrin is presumably due to the relative abundance of alanines at the 2' position in TM2 (Bell-Horner et al., 2000). This amino acid position is equivalent to that shown by ffrench-Constant et al. (1993) to confer resistance to dieldrin in mutant Drosophila GABA receptors that have serine instead of alanine in this position. A recent model (Zhorov and Bregestovski, 2000) hypothesizes on the interaction of PTX in the Cl channel. According to the model, PTX enters deep into the channel, and its hydrophobic domain interacts with residues at the TM2 2' position, whereas its electronegative domain hydrogen bonds with threonine residues at the 6' position. Considering that both the 2' and 6' positions have been implicated in block by PTX, U-93631, and dieldrin, this model is plausible for the actions of these antagonists. As noted, the 6' residue is also involved in PTZ inhibition (Dibas and Dillon, 2000). However, the current results would suggest the postulated hydrophobic interactions at the 2' position are of minor significance for the actions of PTZ. The model by Zhorov and Bregestovski was formulated to describe noncompetitive blockade. Considering that the 6' position is involved in blockade for both PTX and PTZ (which displays only competitive antagonism), it is possible that this site is responsible for the allosteric competitive blockade for both compounds. Two possibilities could mediate the noncompetitive PTX blockade. It could be mediated at a distinct, undefined site; two sites of action for PTX have been proposed (Yoon et al., 1993). Alternatively, PTX interaction at the 2' position may induce a structural change at the gate of the channel (Xu et al., 1995), which results in noncompetitive antagonism. The lack of effect of the 2' position on PTZ-induced block is consistent with this view. Additional studies are necessary to more fully delineate the domains of the two ligands.

In summary, our results support the contention that the central nervous system convulsant pentylenetetrazole competitively antagonizes the GABA_A receptor, likely through an allosteric interaction in the Cl channel. A number of characteristics of PTZ-induced blockade are similar to that induced by picrotoxin, and block by both ligands is affected by the 6' position of TM2. However, the different characteristics of PTZ inhibition that we have described, compared with PTX inhibition, indicate the domains of interaction cannot be identical. Our results further underscore the complexity of the convulsive site.