Introduction

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-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 a Drosophila cell line (Millar et al., 1994), and in Xenopus 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 GABA_A receptors with the equal affinity, but its rates of binding to and unbinding from the receptor were accelerated by receptor activation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

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(-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.

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 M 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.

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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Fipronil Suppression of the Closed GABA_A 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 GABA_A receptors. GABA responses were maintained at a stable level over a period of up to 60 min after rupture of the membrane.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Suppression of GABA-induced currents by fipronil. A, current records in response to 10-ms applications of 300 µM GABA before, during, and after application of 1 µM fipronil in the bath. a, control; currents evoked by every fifth test pulse are shown during perfusion of 1 µM fipronil, and currents evoked by every third test pulse are shown during washout of fipronil. B, time course of changes in peak current amplitude before, during, and after application of 1 µM fipronil. The peak amplitude of current gradually decreased during fipronil bath application. Recovery was observed after washing with fipronil-free solution. Letters a to c correspond to those in A.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Preapplication of fipronil suppresses GABA-induced currents without receptor activation. Fipronil 1 µM was superfused through bathing solution for 15 min with or without channel activation by GABA pulses. A, current records in response to 10-ms application of 300 µM GABA. a, control for b; b, current was recorded after 15-min application of fipronil with repeated GABA pulse applications; c, another control for d; d, current was recorded after 15-min preapplication of fipronil without GABA pulse application. B, time course of changes in current amplitude during the experiment shown in A. Letters a to d correspond to those in A. , repeated GABA pulse applications during fipronil superfusion. , without repeated GABA applications during fipronil superfusion. Both methods yielded the same degree of block.

Dose-Response Relationship for Fipronil Suppression of the Closed GABA_A Receptor. The dose-response relationship for fipronil suppression of the closed GABA_A 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 IC₅₀ value estimated to be 1.66 ± 0.18 µM and a Hill coefficient of 1.23 ± 0.14 (n = 5).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Dose-response relationship for the suppression of the closed GABAA receptor by bath application of fipronil. The currents induced by 10-ms applications of 300 µM GABA were suppressed by various concentrations of fipronil. The test current was measured after 15-min exposure to each concentration of fipronil, and the current reduction represented the steady-state fipronil block of the closed GABAA receptor. This inhibition was concentration-dependent (A) with an IC50 of 1.66 ± 0.18 µM and a Hill coefficient of 1.23 ± 0.14. Mean ± S.D. (n = 5) (B).

Kinetic Parameters of Fipronil Interaction with the Closed GABA_A Receptor. Whereas fipronil was shown to bind to the closed GABA_A receptor thereby blocking GABA-induced currents, it might bind to the activated GABA_A receptor as well. A simple model (model 1) is proposed for fipronil interactions with the GABA_A 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 and k₁ are drug association and dissociation rates, respectively, without GABA, and k'₊₁ and k'₁ are drug association and dissociation rates, respectively, with GABA.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Model 1. Fipronil interacts with both the closed and activated GABAA receptors. R, closed receptor; G, GABA; R*G, the receptor bound and activated by GABA; F, fipronil; RF, the fipronil-bound closed receptor; R*GF, the fipronil-bound activated receptor; k+1 and k1, drug association and dissociation rates, respectively, without GABA; and k'+1 and k'1, drug association and dissociation rates, respectively, with GABA.

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View larger version (23K): [in this window] [in a new window]   Fig. 5.   Kinetics of fipronil suppression of the closed receptor. Decreases in currents by various concentrations of fipronil during bath application are plotted as a function of incubation period. GABA pulses (300 µM, 10 ms) were applied every 10 s to monitor the change in current amplitude. A, time course of decrease in peak current amplitude by bath application of fipronil at 1, 3, 10, and 30 µM for 100 to 150 s. Changes in current amplitude relative to the first current amplitude are plotted as a function of incubation period for each fipronil concentration, and the plots are fitted with a single exponential function. B, reciprocal of the time constants obtained from a single exponential fitting is plotted as a function of fipronil concentration. The solid line represents the relation 1/ = k+1[F] + k1. The fit was less satisfactory than would be expected as the correlation coefficient was 0.89 with a degree of freedom of 2. The association and dissociation rates for fipronil interaction with the closed receptor are calculated to be k+1 = 673 ± 220 M1 s1 and k1 = 0.018 ± 0.0035 s1, respectively, and Kd is calculated to be 26 µM.

Fipronil Suppression of the Activated GABA_A Receptor. To examine the fipronil block of the activated GABA_A 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 () is equal to 1/(k'₊₁[F] + k'₁).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effects of fipronil at various concentrations on the decay phase of currents induced by long applications of GABA. A, GABA 30 µM was applied for 30 s to induce currents, and was coapplied with various concentrations of fipronil. The decay phase of currents was fitted with a single exponential function to obtain time constants. B, reciprocal of the time constants (1/) is plotted as a function of fipronil concentration to calculate the association and dissociation rate constants for fipronil interaction with the activated receptor. Data points are best fitted to the solid line according to the equation 1/ = k'+1[F] + k'1. The correlation coefficient of 0.995 gives a significant level of P < 0.05. k'+1 = 6600 ± 380 M1 s1; k'1 = 0.11 ± 0.0054 s1; the calculated Kd = 17 µM (mean ± S.D., n = 3-7).

Kinetic Parameters of Fipronil Binding to the Activated GABA_A 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  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/ versus fipronil concentration was fitted with the equation of a linear regression (Fig. 6B). We obtained a value of 6600 ± 380 M¹ s¹ for k'₊₁, 0.11 ± 0.0054 s¹ for k'₁, and the calculated equilibrium dissociation constant (K_d = 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 the K_d value.

Dose-Response Relationship for Fipronil Suppression of the Activated GABA_A Receptor. To obtain the dose-response relationship for fipronil suppression of the activated GABA_A 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).

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Dose-response relationship for the fipronil suppression of the activated GABAA receptors. Currents were induced by 30-s coapplication of 30 µM GABA and fipronil, and measured at 25 s from the beginning of coapplication. 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 (mean ± S.D., n = 3-5).

Interactions between Fipronil and Picrotoxinin. Two models for fipronil and picrotoxinin interactions at the GABA_A 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. K_F and K_P 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):

(1)

where R_T is the total receptor, and P and F represent picrotoxinin and fipronil concentrations, respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 8.   One-site model and two-site model for interactions of fipronil (F) and picrotoxinin (P) with the GABAA receptor (R). RF, fipronil-bound receptor; RP, picrotoxinin-bound receptor; RPF, picrotoxinin- and fipronil-bound receptor; KF and KP, the equilibrium dissociation constants for fipronil and picrotoxinin binding, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 9.   Effect of picrotoxinin on fipronil suppression of GABA-induced currents. Dose-response relationships for fipronil suppression of GABA-induced currents were determined in the absence () of picrotoxinin and the presence () of 1 µM (A) and 3 µM (B) picrotoxinin. Picrotoxinin at 1 µM caused 34.0 ± 9.7% block of the current (n = 4) (A) and at 3 µM caused 51.8 ± 12.1% block of the current (n = 4) (B). Dose-response relationships in the presence of picrotoxinin were fitted by two equations described in the text. Dotted line depicts the fit of data with logistic eq. 1 (one-site model). Solid line depicts the fit of data with logistic eq. 2 (two-site model). The KP and KF values were set to 1.60 and 1.66 µM, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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).

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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.