Abstract
The γ-aminobutyric acid (GABA) receptor is an important site of action of a variety of chemicals, including barbiturates, benzodiazepines, picrotoxin, bicuculline, general anesthetics, alcohols, and certain insecticides. Fipronil is the first phenylpyrazole insecticide introduced for pest control. It is effective against some insects that have become resistant to the existing insecticides. To elucidate the mechanism of fipronil interaction with the mammalian GABA system, whole-cell patch-clamp experiments were performed using rat dorsal root ganglion neurons in primary culture. Fipronil suppressed the GABA-induced whole-cell currents reversibly in both closed and activated states. The IC50 values and Hill coefficients for fipronil block of the GABAA receptor were estimated to be 1.66 ± 0.18 μM and 1.23 ± 0.14 for the closed receptor, respectively, and 1.61 ± 0.14 μM and 0.96 ± 0.06 for the activated receptor, respectively. The association rate and dissociation rate constants of fipronil effect were estimated to be 673 ± 220 M−1 s−1 and 0.018 ± 0.0035 s−1 for the closed GABAA receptor, respectively, and 6600 ± 380 M−1 s−1and 0.11 ± 0.0054 s−1 for the activated GABAA receptor, respectively. Thus, both the association and dissociation rate constants of fipronil for the activated GABAA receptor are approximately 10 times as large as those for the closed receptor. Experiments with coapplication of fipronil and picrotoxinin indicated that they did not compete for the same binding site to block the receptor. It is concluded that although fipronil binds to the GABAA receptor without activation, channel opening facilitates fipronil binding to and unbinding from the receptor.
γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. The GABA receptor is an important site of action of a variety of chemicals, including barbiturates, benzodiazepines, picrotoxin, bicuculline, general anesthetics, alcohols, and insecticides (Eldefrawi and Eldefrawi, 1987; Gant et al., 1987; Arakawa et al., 1991; Burt and Kamatchi, 1991; Yeh et al., 1991; Olsen et al., 1992; Ticku et al., 1992; Kurata et al., 1993; Marszalec et al., 1994).
Fipronil is the first phenylpyrazole insecticide introduced for pest control (Moffat, 1993) and the second generation of insecticides acting on the GABA receptor to block the chloride channel. The first generation includes lindane and cyclodiene insecticides such as dieldrin, which suppress GABA-induced currents (Nagata and Narahashi, 1994, 1995a,b; Nagata et al., 1994). Fipronil is effective against insects such as Colorado potato beetle and some cotton pests that have become resistant to most of the existing insecticides, and is much more toxic to insects than to mammals.
Blocking actions of fipronil on Rdl GABA receptors have been demonstrated by recording GABA-activated currents in aDrosophila cell line (Millar et al., 1994), and inXenopus oocytes expressing the Drosophila melanogaster Rdl GABA receptors (Buckingham et al., 1994; Hosie et al., 1995). Fipronil inhibited Cl− uptake activated by GABA (Cole et al., 1993). The specific binding of [3H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2] octane ([3H]EBOB), a probe of picrotoxin binding site, was strongly inhibited by fipronil in Musca domestica (Cole et al., 1993) and D. melanogaster (Cole et al., 1995). Based on Scatchard analysis, fipronil inhibition of [3H]EBOB binding was shown to be noncompetitive in nature (Cole et al., 1993). A dieldrin-resistant housefly strain with a low-affinity EBOB binding site was tolerant to fipronil (Cole et al., 1993), and mutant Rdl GABA receptors were markedly less sensitive to fipronil (Hosie et al., 1995). This indicates that fipronil acts on GABA receptors but the exact site and mechanism of action remain to be seen.
The physiological and pharmacological characteristics of recombinant ion channels expressed in oocyte and other expression systems are not necessarily the same as those of native neurons (Stühmer and Parekh, 1995; Cooper and Millar, 1997; Lewis et al., 1997; Sivilotti et al., 1997; Sweileh et al., 2000).36Cl− flux and [3H]EBOB binding experiments do not allow one to elucidate the mechanism of fast interaction of fipronil with GABA receptors whose conformation could change from closed to open states on the order of milliseconds. We performed whole-cell patch-clamp experiments with rat dorsal root ganglion (DRG) neurons in primary culture to elucidate the detailed mechanism of action of fipronil on the GABA receptor chloride channel complex. Several important aspects of fipronil effects have been unveiled. Fipronil reversibly suppressed GABA-induced currents in a concentration-dependent manner. Fipronil suppressed both closed and activated GABAAreceptors with the equal affinity, but its rates of binding to and unbinding from the receptor were accelerated by receptor activation.
Materials and Methods
Culture of DRG Neurons.
The dorsal root ganglia were dissected from the lumbodorsal region of a newborn rat (2–5 days postnatal) and were immediately placed into Ca2+and Mg2+-free phosphate-buffered saline solution supplemented with 6 g/l glucose. The ganglia were digested in phosphate-buffered saline solution containing 2.5 mg/ml trypsin (Sigma, St. Louis, MO) for 20 min at 37°C. The ganglia were then dissociated by repeated triturations using a fire-polished Pasteur pipette in Dulbecco's modified Eagle's medium containing 0.1 mg/ml fetal bovine serum and 0.08 mg/ml gentamicin. The dissociated cells were placed on coverslips coated with poly(l-lysine). Neurons were maintained in Dulbecco's modified Eagle's medium containing serum and gentamicin in a 90% air, 10% CO2 atmosphere controlled at 37°C. Neurons cultured for 2 to 4 days were used for experiments.
Solutions and Test Chemicals.
Internal and external solutions were designed to eliminate sodium and potassium currents. The standard internal solution contained 140 mM CsCl, 1 mM MgCl2, 5 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The pH was adjusted to 7.3 with tris(hydroxymethyl)aminomethane (Tris base). The osmolarity of both internal and external solutions was adjusted to 290 mOsm with sucrose. The standard external solution contained 136 mM choline chloride, 2 mM CaCl2, 1 mM MgCl2, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and the pH was adjusted to 7.3 with Tris base.
GABA was first dissolved in distilled water to make stock solutions. Fipronil and picrotoxinin (Sigma) were dissolved in dimethyl sulfoxide. Fipronil was obtained from Rhône-Poulenc Yuka Agro K.K. (Akeno, Japan). These stock solutions were then diluted with the standard external solution. The final concentration of dimethyl sulfoxide in test solutions was 0.1% (v/v) or less, which had no adverse effect on GABA-induced currents.
Current Recording.
Membrane currents were recorded using the whole-cell patch-clamp technique (Hamill et al., 1981) at room temperature (22–24°C). Pipette electrodes were made from 0.8-mm (i.d.) borosilicate glass capillary tubes and fire-polished. The electrode had a resistance of 2 to 3 MΩ when filled with the standard internal solution. The membrane was clamped at −60 mV, and a 5-min period was allowed following rupture of the membrane to equilibrate the cell interior with pipette solution. Currents through the electrode were recorded by an Axopatch 200A amplifier (Axon Instruments, Foster City, CA), filtered at 5 kHz and digitized at 10 kHz through an analog-to-digital converter, and stored on the microcomputer hard disk for later analysis.
Drug Applications.
GABA was applied to the cell using two methods, short application and U-tube method. In the short application method, 300 μM GABA solution was pressure ejected for 10 ms onto the cell from a pipette connected to a Picospritzer (General Valve Corporation, Fairfield, NJ). Fipronil was perfused through the bath during the protocol.
In the U-tube method, GABA and fipronil were applied to the cell using a locally developed application system (Nagata and Narahashi, 1994). A computer-operated magnetic valve was controlled by the application program of pClamp 6 software. In the case of the U-tube method, the drug concentration around the receptor should be the same as that provided by the bath application because the whole cell was superfused with the solution coming out of the U-tube.
Data Analysis.
Whole-cell current records were analyzed by the pClamp version 6.0 software (Axon Instruments). The methods for kinetic analysis of GABA-induced currents, including those for calculating the association and dissociation rate constants, are given in respective sections under Results.
IC50 values and their slope factors (Hill coefficients) were calculated from the following equation:
The interaction between fipronil and picrotoxinin was analyzed in terms of a one-site model and a two-site model, as described in the respective sections under Results.
Results
Fipronil Suppression of the Closed GABAA Receptor.
When the membrane potential was held at −60 mV in the normal external solution, the application of GABA produced an inward current mediated by GABAA receptors. GABA responses were maintained at a stable level over a period of up to 60 min after rupture of the membrane.
The suppression of the closed GABAA receptor by bath application of 1 μM fipronil was assessed by a brief (10-ms) application of 300 μM GABA (Fig. 1A). Figure 1B is an example of an experiment showing the time course of changes in the amplitude of currents induced by test GABA pulses during 1 μM fipronil perfusion. The peak current amplitude gradually decreased to 66.1 ± 8.1% of the control (n = 5), and a complete recovery of currents was observed after washout with fipronil-free solution. Both the onset of current suppression during application of fipronil and the recovery from suppression after washing with drug-free solution progressed slowly, requiring several minutes (Fig. 1B).
To examine whether the activation of the receptor by brief GABA test pulses influences fipronil inhibition of the closed receptor, experiments were carried out by comparing the suppression with repeated 10-ms applications of GABA (Fig. 2, records Aa and Ab, and open circles in B) with that obtained 15 min from the beginning of fipronil perfusion without applying GABA test pulses (Fig. 2, records Ac and Ad, and closed circles in B). Without GABA test pulses during a 15-min exposure to fipronil, the first test current amplitude was reduced to 68.4 ± 9.3% (n= 3) of control by bath perfusion of fipronil (Fig. 2, closed circle at d). This percentage of inhibition is almost the same as that caused by 1 μM fipronil with repeated channel activation by brief GABA test pulses (Fig. 2, open circle at b). Thus, the time course of suppression of the closed GABAA receptor by fipronil can be assessed by brief GABA test pulses.
Dose-Response Relationship for Fipronil Suppression of the Closed GABAA Receptor.
The dose-response relationship for fipronil suppression of the closed GABAA receptor was examined by 10-ms test pulses of 300 μM GABA as shown in Fig.3. Each current record of Fig. 3A was obtained when fipronil suppression reached a steady state at each concentration indicated. Fipronil suppressed the GABA-induced currents in a dose-dependent manner (Fig. 3B) with an IC50value estimated to be 1.66 ± 0.18 μM and a Hill coefficient of 1.23 ± 0.14 (n = 5).
Kinetic Parameters of Fipronil Interaction with the Closed GABAA Receptor.
Whereas fipronil was shown to bind to the closed GABAA receptor thereby blocking GABA-induced currents, it might bind to the activated GABAA receptor as well. A simple model (model 1) is proposed for fipronil interactions with the GABAA receptor in both states as shown in Fig.4, where R is the closed GABA receptor, G is the GABA molecule, R*G is the receptor bound and activated by GABA, F is the fipronil molecule, RF is the fipronil-bound closed receptor, R*GF is the fipronil-bound activated receptor,k+1 andk−1 are drug association and dissociation rates, respectively, without GABA, andk′+1 andk′−1 are drug association and dissociation rates, respectively, with GABA.
According to this model, when the receptor is not activated by GABA, fipronil is expected to interact mainly with the closed receptor. If the interaction of fipronil with the closed receptor follows a pseudo first order kinetics, the time constant (τ) for the onset of block is equal to 1/(k+1[F] +k−1). Similarly, if the interaction of fipronil with the activated receptor follows a pseudo first order kinetics the τ is equal to 1/(k′+1[F] +k′−1).
To obtain the fipronil effect on the closed GABAAreceptor, various concentrations of fipronil were applied to the bath. Fipronil suppression of current amplitude was monitored by 10-ms test pulses of 300 μM GABA at 10-s intervals. Bath application of fipronil caused concentration-dependent and time-dependent decreases in the availability of the closed receptor since there were gradual decreases in the amplitude of GABA test currents. To obtain the association and dissociation rates, the change in peak current amplitude was plotted as a function of fipronil incubation period, and the plots were fitted with a single exponential function (Fig.5A). The τ values were 74.9 ± 13.9, 45.0 ± 4.5, 34.9 ± 1.4, and 27.4 ± 0.68 s in the presence of 1, 3, 10, and 30 μM fipronil, respectively. This indicates the pseudo first order kinetics for interaction of fipronil with the closed receptor. The rate constant (1/τ) increased linearly with an increase in fipronil concentration during the incubation period (Fig. 5B). These data were analyzed using the model 1 described above (Fig. 4). Thus, the plot of 1/τ versus fipronil concentration yielded a k+1 of 673 ± 220 M−1 s−1, ak−1 of 0.018 ± 0.0035 s−1, and the calculated equilibrium dissociation constant (Kd =k−1/k+1) of 26 μM for fipronil interaction with the closed GABAA receptor.
Fipronil Suppression of the Activated GABAAReceptor.
To examine the fipronil block of the activated GABAA receptor, various concentrations of fipronil were coapplied with 30 μM GABA for 30 s. Coapplication of fipronil caused concentration-dependent decay in the current amplitude (Fig. 6A). Both kinetics and steady-state block were analyzed according to the right-hand side of model 1 (Fig. 4) in which fipronil interacts mainly with the activated receptor, R*G. The time constant of current decay (τ) is equal to 1/(k′+1[F] +k′−1).
Kinetic Parameters of Fipronil Binding to the Activated GABAA Receptor.
To obtain the association and dissociation rates, currents shown in Fig. 6A were fitted with a single exponential function. The time constant of current decreased with an increase in fipronil concentration. The τ values were 11.09 ± 3.66, 10.10 ± 3.83, 7.16 ± 0.71, 5.48 ± 1.19, and 3.38 ± 0.69 s in the presence of fipronil at 0, 1, 3, 10, and 30 μM, respectively. Again, these data were analyzed using the same model as described above (Fig. 4) by simply replacing the term for the closed receptor with the activated receptor. The plot of 1/τ versus fipronil concentration was fitted with the equation of a linear regression (Fig. 6B). We obtained a value of 6600 ± 380 M−1 s−1 fork′+1, 0.11 ± 0.0054 s−1 for k′−1, and the calculated equilibrium dissociation constant (Kd =k′−1/k′+1) of 16 μM for fipronil block of the activated receptor. These results show that GABA activation enhanced the association rate of fipronil for the receptor 9.8-fold, and at the same time increased the dissociation rate 6.2-fold, leading to a 1.5-fold decrease in theKd value.
Dose-Response Relationship for Fipronil Suppression of the Activated GABAA Receptor.
To obtain the dose-response relationship for fipronil suppression of the activated GABAA receptor, sustained currents induced by long coapplication of GABA and fipronil (Fig. 6A) were measured at 25 s after the beginning of application. The reduction of this current was assumed to be mainly due to fipronil block of the activated receptor. Fipronil suppressed the GABA-induced currents in a dose-dependent manner with an IC50 estimated to be 1.61 ± 0.14 μM and a Hill coefficient of 0.96 ± 0.06 (n = 3–5) (Fig. 7).
Interactions between Fipronil and Picrotoxinin.
Two models for fipronil and picrotoxinin interactions at the GABAA receptor are proposed, one-site model and two-site model (Fig. 8), where R is receptor, P is picrotoxinin, and F is fipronil. RF is the fipronil-bound receptor, RP is the picrotoxinin-bound receptor, and RPF is the picrotoxinin- and fipronil-bound receptor.KF andKP are the equilibrium dissociation constants for fipronil and picrotoxinin binding, respectively. In one-site model, fipronil and picrotoxinin compete with each other for the same binding site. For two-site model, fipronil and picrotoxinin have their own binding sites, and they can independently bind to the respective binding site to inhibit GABA-induced current. The dose-dependent suppression of GABA-induced current by fipronil in the presence of picrotoxinin was analyzed by two equations, one for one-site model (eq. 1) and the other for two-site model (eq. 2):
To determine the site of action of fipronil on the GABAA receptor, competition experiments were performed using picrotoxinin (Fig. 9). To examine the interaction between fipronil and picrotoxinin at the receptor, picrotoxinin at 1 or 3 μM was applied to the bath solution together with various concentrations of fipronil. Picrotoxinin at 1 μM caused 34.0 ± 9.7% block of GABA-induced currents (n = 4), and picrotoxinin at 3 μM caused 51.8 ± 12.1% block of GABA-induced currents (n = 4). Dose-response relationships for the fipronil suppression of GABA-induced currents with and without picrotoxinin are shown in Fig.9, and are fitted by the above-mentioned two eqs. 1 and 2. Dotted line depicts the fit of data with logistic eq. 1 (one-site model), and solid line depicts the fit of data with logistic eq. 2 (two-site model). TheKP andKF values were set to 1.60 and 1.66 μM, respectively. RT was set to 100% as the total GABAA receptor. It is clear that the two-site model clearly gives better fitting to the data than the one-site model. It is concluded that fipronil and picrotoxinin do not share a common binding site.
Discussion
Fipronil Suppression of GABA-Induced Current.
Fipronil has been found to exert an inhibitory action on the GABA-activated chloride channel current in DRG neurons. Fipronil blocks GABA-induced currents slowly and reversibly, and the inhibitory effect of fipronil does not require channel opening, indicating that fipronil acts on the closed GABA receptor. However, receptor activation facilitates fipronil block. The fipronil suppression is in keeping with the previous results based on 36Cl− uptake (Cole et al., 1993) and GABA-induced currents in recombinant GABA (Rdl) receptors expressed in Xenopus oocytes (Buckingham et al., 1994; Hosie et al., 1995).
According to model 1 (Fig. 4), the time constant for current decay due to block of the closed receptor is equal to 1/(k+1[F] +k−1) and that due to block of the activated receptor is equal to 1/(k′+1[F] +k′−1). Analysis of the data using this model revealed that the association and dissociation rates for GABA-activated receptors were 9.8- and 6.2-fold greater than those for the closed receptor. Thus, receptor activation facilitates fipronil binding to and unbinding from the receptor. However, the affinity of fipronil for the GABAA receptor is not altered by the receptor activation. This conclusion is supported by the observation that the IC50 values for the activated and closed receptors are similar. TheKd values determined from the kinetic analysis were about 10-fold greater than the IC50values determined by the equilibrium analysis for both the resting and activated receptors. The exact reason for this difference remains to be determined.
Site of Action of Fipronil.
The GABA receptor comprises several binding sites, including those for GABA, barbiturates, benzodiazepines, and picrotoxin (Olsen et al., 1992, Yoon et al., 1993). The activation of the GABA receptor increased the association rate of picrotoxinin for the GABA-bound receptor about 100 times (Dillon et al., 1995). Picrotoxin also displayed a greater affinity for GABA-bound open channels, and may act as an allosteric modulator (Smart and Constanti, 1986; Newland and Cull-Candy, 1992; Yoon et al., 1993). These studies suggest that picrotoxin preferentially interacts with the activated GABA receptors and stabilizes the receptors in a desensitized or closed state. The present study shows that receptor activation facilitates fipronil binding to and unbinding from the receptor, but does not affect the affinity of fipronil for the receptor. Therefore, fipronil and picrotoxinin may act as allosteric modulators at different sites to block the GABA receptor.
Scatchard analysis of radioligand binding data showed that fipronil noncompetitively inhibited the binding of the antagonist [3H]EBOB to membranes of the housefly, M. domestica (Cole et al., 1993, 1995). However, the potency to displace [3H]EBOB binding was much higher in insects than in mammals, with IC50 values of 3 nM and 1.1 μM, respectively (Hainzl et al., 1998). The latter value for mammals is in the same order of magnitude as IC50values of 1.66 and 1.61 μM obtained in the present electrophysiological experiments for the closed and activated GABAA receptors, respectively. Low sensitivity of the mammalian GABAA receptor to fipronil was also shown in vivo using quantitative autoradiography of [3H]EBOB binding (Kamijima and Casida, 2000).
Since GABAA receptors are known to comprise at least six α-subunits, three β-subunits, three γ-subunits, and one δ-subunit (McKernan and Whiting, 1996), those present in the brain may respond to a drug in a manner different from those in DRG neurons. Whereas the 13 GABAA receptor subunits allow for the possible existence of more than 10,000 pentameric subunit combinations, the actual number of major subunit combinations is likely to be less than 10, and the largest population of GABAA receptors in the brain contain the α1-, β2-, and γ2-subunits (McKernan and Whiting, 1996). In contrast, the neonatal rat DRG neurons appear to contain the α2-subunits (Persohn et al., 1991; Serafini et al., 1998). Thus, it remains to be seen how the brain GABAA receptors respond to fipronil.
Previous studies also showed that cyclodiene insecticides and picrotoxin may share a common binding site (Matsumura and Ghiasuddin, 1983; Eldefrawi and Eldefrawi, 1987; Bloomquist et al., 1992; Nagata and Narahashi, 1994). In the present study, picrotoxinin was applied to the receptor with various concentrations of fipronil to determine their interactions. Data were fitted using kinetic models, and the two-site model fit better than the one-site model (Fig. 9). This result leads to the conclusion that fipronil and picrotoxinin have their own binding sites, and do not share the common binding site.
The dieldrin-resistant housefly strain is tolerant to fipronil although the resistance ratios are much less for fipronil than for dieldrin (Cole et al., 1993, 1995). Mutant dieldrin-resistant GABA (Rdl) receptors expressed in Xenopus oocytes was markedly less sensitive to fipronil than the wild-type receptors (Hosie et al., 1995). It remains to be determined whether dieldrin and fipronil interact with each other at a distinct binding site to block the GABA receptor.
Acknowledgments
We thank Julia Irizarry and Yukiko Sato for secretarial assistance, and Nayla Hassan for technical assistance.
Footnotes
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Send reprint requests to: Dr. Toshio Narahashi, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL 60611. E-mail: tna597{at}northwestern.edu
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↵1 Current address: Brain Science Institute, The institute of Physical and Chemical Research, Waco 351-0198, Japan.
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This work was supported in part by a grant-in-aid for developmental scientific research (No. 10760027 and 09460023) from the Ministry of Education, Science, Culture, and Sports of Japan; by a research fellowships of the Japan Society for the Promotion of Science for Young Scientists; and a grant from the National Institutes of Health (NS 14143).
- Abbreviations:
- GABA
- γ-aminobutyric acid
- EBOB
- 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane
- DRG
- dorsal root ganglion
- Received June 26, 2000.
- Accepted November 2, 2000.
- The American Society for Pharmacology and Experimental Therapeutics