Mercury Chloride Modulation of the GABAAReceptor–Channel Complex in Rat Dorsal Root Ganglion Neurons

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

140, 508–520 (1996)

0247

Mercury Chloride Modulation of the GABAA Receptor–Channel Complex in Rat Dorsal Root Ganglion Neurons CHAO-SHENG HUANG

AND

TOSHIO NARAHASHI

Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, Illinois 60611 Received November 3, 1995; accepted June 13, 1996

Mercury Chloride Modulation of the GABAA Receptor–Channel Complex in Rat Dorsal Root Ganglion Neurons. HUANG, C.-S., AND NARAHASHI, T. (1996). Toxicol. Appl. Pharmacol. 140, 508–521. Mercury compounds affect synaptic transmission through multiple actions on various receptors and ion channels. One of their target sites is the GABAA receptor–channel complex, which is stimulated by mercury chloride (1–10 mM) in a potent and efficacious manner. We studied the mechanisms by which mercury chloride modulates the activity of the GABA system of rat dorsal root ganglion neurons in primary culture using the whole-cell patch clamp technique. The active form of mercury chloride was determined by measuring mercury potentiation of GABA-induced currents as a function of extracellular pH and chloride concentration. Experiments with various chloride concentrations indicated that Hg2/ was far less potent than HgCl2 and HgCl20 4 . Experiments with pH changes indicated that the mercury chloride–hydroxyl complex, predominantly HgCl4(OH)30, was equally potent to noncomplexed mercury chloride, predominantly HgCl20 4 . Mercury chloride potentiation of GABA-induced currents was use dependent, increasing with the frequency of channel openings. However, the potentiation was independent of membrane potential suggesting that mercury chloride binds to an external site of the receptor. Also supporting an external binding site is the observation that preapplication of mercury chloride alone for up to 60-sec potentiated GABA-induced currents. This site appears to be distinct from the zinc binding site. Desensitization of GABA-induced currents was accelerated by mercury chloride. Mercury chloride potentiation of the currents was blocked by cysteine and iodoacetamide suggesting involvement of sulfhydryl groups in this action. q 1996 Academic Press, Inc.

Mercury compounds produce diverse behavioral disorders in humans and animals (Satoh, 1991). The neurological disorders caused by mercury are exemplified by Minamata disease in Japan (Takeuchi et al., 1962), the symptoms of which include impaired vision, speech and hearing, weakness of the extremities, and ataxia (Hunter et al., 1940; Chang, 1977). This indicates that the central nervous system is the target site of mercury. Mercury exerts multiple actions on the nervous system 0041-008X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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(Narahashi et al., 1991, 1994). Both methylmercury and mercury chloride increase the frequency of spontaneous miniature endplate potentials indicating stimulation of acetylcholine release (Atchison and Narahashi, 1982; Miyamoto, 1983; Cooper et al., 1984; Atchison, 1986; Traxinger and Atchison, 1987). This effect of methylmercury is due to intracellular release of Ca2/ from internal stores (Levesque and Atchison, 1987, 1988; Hare et al., 1993). Release of dopamine, norepinephrine, and 5-hydroxytryptamine is also stimulated by methylmercury (Mckay et al., 1986a,b; Nakazato et al., 1979). Methylmercury also suppresses nerve-evoked endplate potentials (Atchison and Narahashi, 1982; Atchison, 1986), and this effect is ascribed to block calcium entry to the nerve terminals (Atchison, 1986; Shafer et al., 1990). Calcium channels have indeed been shown to be blocked by both methylmercury and mercury chloride (Shafer and Atchison, 1991; Bu¨sselberg et al., 1991; Pekel et al., 1993; Weinsberg et al., 1995). Ligand binding to the muscarinic and nicotinic acetylcholine receptors (Eldefrawi et al., 1977), to the dopamine receptor (Bondy and Agrawal, 1980), and to the opioid receptors (Tejwani and Hanissian, 1990) are also inhibited by methylmercury and mercury chloride. Kainate-activated currents are suppressed by inorganic mercury with a Ki of 70 nM (Kiskin et al., 1986; Umbach and Gundersen, 1989), and GABA-induced chloride currents are augmented by mercury chloride with a high potency and efficacy (Arakawa et al., 1991). Whereas any of the aforementioned mercury actions on parameters related to synaptic transmission could be important to produce symptoms of poisoning, mercury chloride modulation of the GABAA receptor–chloride channel complex is deemed significant as the effect is highly potent. Mercury chloride potentiates GABA-induced currents even at 0.1 mM (Arakawa et al., 1991). Thus, the present study was designed to address three major questions pertaining to mercury chloride potentiation of GABA-induced currents. First, the binding site of mercury chloride on the GABA receptor and the use dependence of mercury modulation were analyzed. Second, the role of sulfhydryl groups in mer-

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cury action on the GABA system was studied. Mercury has a high affinity for sulfhydryl groups (Webb, 1966) and acts on various receptors and channels as described above. Therefore its interaction with sulfhydryl groups may play a pivotal role in its toxic actions. Third, mercury chloride forms various complexes with Cl0 and OH0 in a manner dependent upon Cl0 concentration and pH of the medium. Thus, the active forms of mercury chloride in modulating the GABA receptor were determined.

TABLE 1 Mercury Chloride Complexes at Different pH Values and Chloride Concentrations Cl0 concentration pH

1 nM

1 mM

142 mM

6 7 8

Hg2/ Hg2//Hg(OH)/ Hg(OH)/

HgCl2 HgCl2/HgCl2(OH)0 HgCl2(OH)0

HgCl20 4 30 HgCl20 4 /HgCl4(OH) 30 HgCl4(OH)

MATERIALS AND METHODS Materials. Dorsal root ganglia (DRG) were dissected from the lumbosacral area of newborn Sprague–Dawley rats (1–6 days old) under methoxyflurane anesthesia, and were transferred into Ca2/-/Mg2/-free phosphate buffered saline solution supplemented with 6 g/liter D-glucose. The ganglia were digested in this solution added with 2.5 mg/ml trypsin (type XI, Sigma, St. Louis, MO) for 25 min at 377C and then washed with Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% neonatal calf serum and 0.08 mg/ml gentamicin. The ganglia were dissociated acutely by repeated triturations using a fire-polished Pasteur pipette in 2.0 ml DMEM. The dissociated neurons were plated onto coverslips coated with poly-Llysin (0.1 mg/ml, Sigma). Cells were maintained in DMEM containing neonatal calf serum and gentamicin in a 90% air/10% CO2 atmosphere controlled at 367C. Neurons cultured for less than 2 days were used for experiments. Since no growth factors were added to the culture media, DRG neurons became less healthy beyond 2 days. Electrical recording and test solutions. Ionic currents were recorded under voltage clamp conditions using the whole-cell patch clamp technique (Hamill et al., 1981). Pipette electrodes were made from 1.5-mm (o.d.) borosilicate glass capillary tubes and had a resistance of 2 MV when filled with standard internal solution. The transmembrane voltage was clamped at 060 mV. Unless otherwise indicated, a period of 10 min was allowed following rupture of the membrane to ensure adequate equilibration between the internal pipette solution and the cell interior. Membrane currents passing through the electrode were recorded with an Axopatch amplifier (Axopatch1B, Axon Instruments, Foster City, CA), and currents were stored in an SX 386 computer (DELL Computer Company, Austin, TX) using pCLAMP 6 software (Axon Instruments). The external and internal solutions for whole-cell current recording were designed to eliminate sodium and potassium channel currents. The standard external solution contained (in mM) choline chloride 136, CaCl2 2, MgCl2 1, and N-2-hydroxyethylpiperazine-N*-2-ethanesulphonic acid (HEPES) 10. The standard internal solution contained (in mM) CsCl 140, CaCl2 1, ethyleneglycol bis-(b-aminoethylether)-N,N,N*N,*-tetraacetic acid (EGTA) 5, and HEPES 10. The pH of all solutions was adjusted to 7.3, unless otherwise indicated, with 1 M Tris (hydroxy-methyl)aminomethane (Tris base), and the osmolarity was raised to 290 mOsm with sucrose. Chloride concentration in the external solution was lowered by replacing choline chloride, calcium chloride, and magnesium chloride by sucrose, calcium gluconate, and magnesium gluconate, respectively. Test solutions were prepared on the day of experiment by diluting the following aqueous stock solutions with the standard external solution: 100 mM GABA, 1 mM mercury chloride, and 10 mM zinc chloride. Test solutions of N-ethylmaleimide, iodoacetamide, dithiothreitol, glutathione (reduced form), and cysteine were made on the day of experiment with external solution. All drugs were obtained from Sigma. Drug application system. A U-tube system was used for drug application (Fenwick et al., 1982; Krishtal and Pidoplichko, 1980). The recording chamber was continuously perfused with the normal external solution by gravity at a rate of 1–2 ml per min. The outlet hole of the U-tube was placed around 100 mm away from neurons, and was continuously perfused

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with a test solution by a perfusion pump. When an electromagnetic valve located between the U-tube outlet hole and the perfusion pump was closed by using a computer protocol, the test solution was allowed to flow through the outlet hole to neurons. Upon opening the electromagnetic valve again, the test solution was sucked back into the U-tube. The exchange time for the test solution was usually less than 100 msec. All experiments were carried out at a room temperature of 20–237C. Data analysis. The dose–response relationship of the peak GABAinduced currents was fitted to a sigmoid curve as calculated by the fourparametric logistic function equation (Tandel Scientific, Corte Madera, CA): Ip Å Imax[C n/(C n / K nd)]

(1)

where Ip represents the peak current recorded at GABA concentration C, and Imax is the maximal response; Kd and n denote the half-maximal effective concentration of GABA and the Hill coefficient, respectively. Statistical analysis was done by using the Student’s t test at a significant level of p Å 0.05. Data are given as mean { S.E.M. with the number of experiments (n).

RESULTS

Active Forms of Mercury Chloride Complexes Mercury chloride exists in different complex forms in aqueous solutions as determined by the chloride concentration and the pH value (Webb, 1966; Hietanen and Sillen, 1952). At a very low chloride concentration of 1 nM, the major species is Hg2/, whereas HgCl2 and HgCl20 4 predominate at chloride concentrations of 1 and 142 mM, respectively. In addition, mercury chloride forms complexes with hydroxyl groups in solutions with different pH values. Using the Ka values of these mercury chloride hydroxyl complexes, the ratios of mercury chloride without hydroxyl groups to those with hydroxyl groups can be calculated. At a higher pH of 8.0, the main species of mercury chloride is those with hydroxyl groups, whereas at a low pH of 6.0, the predominant species is those without hydroxyl groups (Table 1). To determine the active forms of mercury chloride, its potency and efficacy were measured as a function of pH and chloride concentration. Effects of Extracellular pH Values on the GABA Dose–Response Relationship To determine if the pH value in the extracellular solution affects the affinity of GABAA receptor for GABA, the dose–

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0.9 { 0.25 (Fig. 2C, n Å 5–9). The maximal GABA response was increased by mercury chloride to 105.9 { 1.75% (n Å 4), 106.0 { 2.2% (n Å 6), and 108.2 { 5.72% (n Å 5) of control at pH 6.0, 7.0, and 8.0, respectively. The ratios of the Kd in the presence of 10 mM mercury chloride to that in the absence of mercury chloride were 0.724 { 0.013, 0.639 { 0.035, and 0.737 { 0.034 at pH 6.0, 7.0, and 8.0, respectively (Fig. 2D). These differences are not statistically significant (p ú 0.05). Thus, 10 mM mercury chloride increased the affinity of GABAA receptor for GABA regardless of pH. The mercury chloride complex with hydroxyl groups, predominantly HgCl4(OH)30, was equally potent to that without hydroxyl groups, predominantly HgCl20 4 , in potentiating GABA-induced currents. Mercury Chloride Complexes Are More Potent Than Mercury Divalent Cation FIG. 1. The dose–response relationships for GABA to induce currents at different pH values. Lowering or raising the pH from the neutral level decreases the affinity of GABA for the GABA receptor and decreases the stoichiometry of GABA binding.

response relationships for GABA-induced currents were fitted to a sigmoid curve using (1) (Fig. 1). The Kd for GABA was 180.2 { 17.1 mM with the Hill coefficient of 1.6 { 0.19 at pH 6.0 (n Å 4), 30.6 { 2.67 mM with the Hill coefficient of 2.18 { 0.437 at pH 7.0 (n Å 20), and 43.4 { 5.46 mM with the Hill coefficient of 1.2 { 0.15 at pH 8.0 (n Å 5). Thus, changing the pH of solution to either a lower or a higher value shifted the dose–response curve for GABA in the direction of higher GABA concentrations with a decrease in the stoichiometry of GABA binding. Effects of Mercury Chloride Complexes on the GABA Dose–Response Relationship at Different pH Levels To study the effects of mercury chloride complexes with or without hydroxyl groups on GABA-induced currents, the dose–response relationships for GABA were fitted to sigmoid curves at different pH values in the solutions containing 142 mM chloride with and without 10 mM mercury chloride. At pH 7.0, mercury chloride shifted the dose– response curve for GABA in the direction of lower GABA concentrations with the Kd shifted from 30.6 { 2.67 mM to 17.3 { 2.14 mM and with the Hill coefficient changed from 2.18 { 0.437 to 1.52 { 0.242 (Fig. 2B, n Å 6–20). At pH 6.0, mercury chloride, primarily in the forms without hydroxyl groups, decreased the Kd for GABA from 180.2 { 17.10 mM to 113.6 { 3.22 mM and the Hill coefficient from 1.6 { 0.19 to 0.8 { 0.13 (Fig. 2A, n Å 4). At pH 8.0, mercury chloride, mainly in the forms without hydroxyl groups, reduced the Kd for GABA from 43.4 { 5.46 mM to 33.0 { 1.20 mM and the Hill coefficient from 1.2 { 0.15 to

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To further investigate the potency of different mercury chloride complexes, mercury chloride potentiation of GABA-induced currents was studied as a function of chloride concentration and pH. At pH 7.0, 10 mM mercury chloride coapplied with 10 mM GABA potentiated GABA-induced currents 1.8 { 0.15 times, 3.6 { 0.07 times, and 3.8 { 0.27 times of controls at chloride concentrations of 1 nM [Hg2//Hg(OH)/], 1 mM [HgCl2/HgCl2(OH)0], and 142 mM 30 [HgCl20 4 /HgCl4(OH) ], respectively (Fig. 3A, n Å 4). At pH 6.0, potentiation was 1.13 { 0.07 times, 7.76 { 0.64 times, and 8.14 { 0.69 times of controls at chloride concentrations of 1 nM (Hg2/), 1 mM (HgCl2 ), and 142 mM (HgCl20 4 ), respectively (Fig. 3B, n Å 4). At pH 8.0, potentiation was 1.3 { 0.14 times, 1.6 { 0.16 times, and 1.9 { 0.16 times of controls at chloride concentrations of 1 nM [Hg(OH)/], 1 mM [HgCl2(OH)0], and 142 mM [HgCl4(OH)30], respectively (Fig. 3C, n Å 5). Thus, the mercury chloride complexes HgCl2 and HgCl20 4 are far more potent than mercury divalent cation Hg2/ (p õ 0.05). HgCl4(OH)30 and HgCl2(OH)0 are more potent than Hg(OH)/ (p õ 0.05). Mercury Chloride Potentiation of GABA-Induced Currents Is Use Dependent To determine whether mercury chloride potentiation of GABA-induced currents is use dependent, 1 mM mercury chloride was applied continuously to the bath while 10 mM GABA was applied through the U-tube at intervals of 20 sec (open circles), 1 min (filled triangles), and 2.5 min (filled circles). At the end of 7-min bath application of mercury chloride, GABA-induced currents, evoked at intervals of 20 sec, 1 min, and 2.5 min, were potentiated 11.7 { 0.85 times, 8.2 { 0.75 times, and 6.3 { 0.71 times, respectively (Fig. 4, n Å 6). Mercury chloride potentiation was clearly use dependent, increasing with increasing the frequency of channel openings.

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FIG. 2. Effects of mercury chloride on the dose–response curves for GABA. At pH 7.0, mercury chloride shifts the dose–response curve in the direction of lower GABA concentrations with the Kd decreased from 30.6 { 2.67 mM to 17.3 { 2.14 mM and with the Hill coefficient changed from 2.18 { 0.437 to 1.52 { 0.242 [(B) n Å 6–20]. At pH 6.0, mercury chloride decreases the Kd for GABA from 180.2 { 17.10 mM to 113.6 { 3.22 mM with the Hill coefficient decreased from 1.6 { 0.19 to 0.8 { 0.13 [(A) n Å 4]. At pH 8.0, the Kd for GABA decreases from 43.4 { 5.46 mM to 33.0 { 1.2 mM with the Hill coefficient decreased from 1.2 { 0.15 to 0.9 { 0.25 [(C) n Å 5–9]. Note that the maximal GABA response is increased slightly by mercury chloride. The ratios of the Kd for GABA in the presence of 10 mM mercury chloride to that in the absence of mercury chloride are 0.724 { 0.013, 0.639 { 0.035, and 0.737 { 0.034 in pH 6.0, 7.0, and 8.0, respectively (D). Mercury chloride with hydroxyl groups is equally potent to that without hydroxyl groups in potentiating GABA-induced currents.

Mercury Chloride Potentiation of GABA-Induced Currents Is Voltage Independent

Effects of Mercury Chloride Preapplication on GABAInduced Currents

Mercury chloride can exert its use-dependent potentiation of GABA-induced currents via binding to a site either in the channel pore or outside of the channel pore. Most mercury chloride complexes at a chloride concentration of 142 mM are in the charged forms. If mercury chloride binds to a site in the channel pore, its potentiating effect on GABA-induced currents should be affected by the driving force across the cell membrane. To address this question, the amplitudes of currents induced by 10 mM GABA (filled circles) and coapplication of 10 mM GABA and 10 mM mercury chloride (open triangles) are plotted as a function of the holding membrane potential in Fig. 5A. The degree of mercury chloride potentiation was independent of the membrane potential, indicating that mercury chloride binds to an external site of the receptor–channel (Fig. 5B, n Å 3).

To further locate the target site of mercury chloride on the GABAA receptor–channel complex, mercury chloride was applied alone to the cell for various durations while the chloride channel was closed, and after washout of mercury chloride, GABA was applied to the cell. If GABA-induced currents are potentiated after mercury preapplication, it implies that mercury chloride targets a site outside of the channel. If mercury chloride binding site is in the channel pore, preapplication of mercury chloride will not potentiate GABA-induced currents because mercury chloride has no access to its binding site while the channel is closed. Preapplications of 10 mM mercury chloride alone through the Utube for 5 sec (Fig. 6A,a), 60 sec (Fig. 6A,b), and 120 sec (Fig. 6A,c) potentiated 10 mM GABA-induced currents 1.65 { 0.105 times (n Å 7), 2.45 { 0.562 times (n Å 4), and

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FIG. 3. Mercury chloride complexes are more potent than mercury divalent cation. At pH 7.0, 10 mM mercury chloride coapplied with 10 mM GABA 30 potentiates GABA-induced currents 1.8 { 0.15 times, 3.6 { 0.07 times, and 3.8 { 0.27 times in 142 mM chloride [HgCl20 4 /HgCl4(OH) ], 1 mM chloride 0 2/ / [HgCl2 /HgCl2(OH) ], and 1 nM chloride [Hg /Hg(OH) ], respectively [(A) n Å 4]. At pH 6.0, 10 mM mercury chloride potentiation of GABA-induced currents is 1.13 { 0.07 times, 7.76 { 0.64 times, and 8.14 { 0.69 times in 1 nM chloride (Hg2/), 1 mM chloride (HgCl2 ), and 142 mM chloride (HgCl20 4 ), respectively [(B) n Å 4]. At pH 8.0, 10 mM mercury chloride potentiates the currents 1.3 { 0.14 times, 1.6 { 0.16 times, and 1.9 { 0.16 times in 1 nM chloride [Hg(OH)/], 1 mM chloride [HgCl2(OH)0], and 142 mM chloride [HgCl4(OH)30], respectively [(C) n Å 5]. Thus, mercury chloride 2/ 30 and HgCl2(OH)0 are more efficacious than complexes (HgCl2 ; HgCl20 4 ) are more efficacious than mercury cation (Hg ) (p õ 0.05). HgCl4(OH) Hg(OH)/ (p õ 0.05).

1.65 { 0.458 times of controls (n Å 5), respectively (Fig. 6B). Therefore, mercury chloride’s binding site may be located outside of the channel pore. Mercury chloride potentiation of GABA-induced currents reached a maximum after 60-sec preapplication but decreased at 120 sec (Fig. 6B). This decrease may be due to desensitization of the GABAA receptor by prolonged mercury chloride preapplication. The transient increase and subsequent decrease of currents are further demonstrated in Fig. 6C in which currents induced by coapplications of 10 mM GABA and 10 mM mercury chloride at an interval of 1 min gradually increased reaching a peak and decreased thereafter due to desensitization. After washout for 15 min, GABA-induced currents remained

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small. Thus, mercury chloride effect is irreversible after washout. Mercury Chloride Accelerates GABA-Induced Desensitization To further demonstrate acceleration of desensitization of GABA-induced currents by mercury chloride, a long 60-sec application protocol was used in the absence and presence of 10 mM mercury chloride (Fig. 7A). The decay phase of the current induced by 10 mM GABA could be fitted by a double exponential function. Mercury chloride decreased the time constants of both fast and slow phases of current desen-

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FIG. 4. Mercury chloride potentiation of GABA-induced currents is use dependent. With 1 mM mercury chloride in the bath, 10 mM GABA was applied through the U-tube at intervals of 20 sec (open circles), 1 min (filled triangles), and 2.5 min (filled circles). At the end of 7-min mercury chloride bath application, GABA-induced currents are potentiated 11.7 { 0.85 times, 8.2 { 0.75 times, and 6.3 { 0.71 times of controls as evoked at intervals of 20 sec, 1 min, and 2.5 min, respectively (n Å 6).

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steady/peak current amplitude ratio of 35 mM GABA-induced currents were 23.1 { 1.44 sec (n Å 3) and 43.9 { 3.14% (n Å 3–4), respectively, and those of currents by coapplication of 10 mM GABA and 30 mM NEM were 13.7 { 0.34 sec (n Å 3) and 33.1 { 1.91% (n Å 3–4), respectively (Fig. 8D). Thus, IAA and NEM potentiate GABA-induced currents, and NEM accelerates desensitization of GABAinduced currents. To further demonstrate the hypothesis that sulfhydryl groups are involved in mercury chloride potentiation of GABA-induced currents, 100 mM cysteine, a sulfhydryl reducing agent, was used. The current induced by 10 mM GABA was potentiated by cysteine only slightly (Fig. 9). Coapplication of 10 mM mercury chloride and 10 mM GABA potentiated the current, but cysteine abolished mercury chloride potentiation of the current (Fig. 9). The effect of cysteine was reversible after washout with the solution devoid of

sitization from 4.3 { 0.74 sec to 2.6 { 0.33 sec (n Å 6) and from 20.0 { 2.70 sec to 10.6 { 1.46 sec (n Å 5), respectively (Fig. 7B). The ratio of steady/peak current amplitudes was decreased by 10 mM mercury chloride from 47.3 { 4.13% to 22.8 { 4.01% (Fig. 7C, n Å 6). Thus, mercury chloride accelerates GABA-induced current desensitization. Effects of Sulfhydryl Modifying Agents on GABA-Induced Currents N-ethylmaleimide (NEM), cysteine, and iodoacetamide (IAA) oxidize free sulfhydryl groups of ion channel proteins, whereas dithiothreitol (DTT) and glutathione reduce the intramolecular disulfide bonds and yield free sulfhydryl groups to channel proteins (Petronilli et al., 1994; Chiamvimonvat et al., 1995). IAA (Fig. 8A, n Å 4–5) or NEM (Fig. 8B, n Å 5–6) coapplied with 10 mM GABA potentiated GABAinduced currents in a dose-dependent manner. The concentration of NEM to produce a maximal potentiation was 30 mM. The sulfhydryl reducing agents, glutathione and DTT, and the sulfhydryl oxidizing agent, cysteine, potentiated GABA-induced currents only slightly at a concentration of 100 mM (data not shown). Since current desensitization is accelerated by an increase in peak amplitude, the rate of desensitization of the current potentiated by NEM was compared with the same amplitude of current induced by a higher GABA concentration. The current induced by coapplication of 30 mM NEM and 10 mM GABA reached the same peak as that induced by 35 mM GABA alone, yet it showed an accelerated desensitization (Fig. 8C). The decay phase of the current could be fitted by a single exponential function. The time constant and the

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FIG. 5. Mercury chloride potentiation of GABA-induced currents is voltage independent. Currents induced by 10 mM GABA (filled circles) and coapplication of 10 mM GABA and 10 mM mercury chloride (open triangles) at different holding membrane potentials are normalized to that induced by 10 mM GABA at 060 mV (A). Mercury chloride potentiation is voltage independent [(B) n Å 3].

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FIG. 6. Preapplication of mercury alone potentiates GABA-induced currents. Preapplication of 10 mM mercury chloride alone for 5 sec [(A) a], 60 sec [(A) b], and 120 sec [(A) c] potentiates 10 mM GABA-induced currents 1.65 { 0.105 times (n Å 7), 2.45 { 0.562 times (n Å 4), and 1.65 { 0.458 times (n Å 5) of controls, respectively (B). (C) Mercury chloride desensitizes the GABAA receptor irreversibly. Currents induced by coapplications of 10 mM GABA and 10 mM mercury chloride gradually increased reaching a maximum, and decreased thereafter. After washout for 15 min, the GABAinduced current still remains decreased.

cysteine. On the other hand, DTT or glutathione at 100 mM did not affect mercury chloride potentiation of GABA-induced currents (data not shown). Thus, free sulfhydryl groups, but not disulfide bonds, are involved in mercury chloride potentiation of GABA-induced currents. Increasing the concentration of IAA beyond 30 mM in-

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creases the osmolarity of the solution significantly. Since increased osmolarity alone decreased GABA-induced currents (data not shown), the potentiation effect of IAA on GABA-induced currents was corrected by calculating the ratio of the current induced by coapplication of IAA and GABA to the current induced by GABA alone with the

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(Fig. 10B) (p õ 0.05). IAA block of mercury chloride potentiation is exerted presumably through competition for sulfhydryl groups with mercury chloride. Zinc Chloride Does Not Affect Mercury Chloride Potentiation of GABA-Induced Currents Zinc chloride decreases GABA-induced currents through binding to a cationic binding site on the GABA receptor– channel complex (Draguhn et al., 1990; Narahashi et al., 1994). To determine if mercury chloride targets on this cation binding site, mercury chloride potentiation of GABAinduced currents was measured in the absence and presence of zinc chloride (Fig. 11A). Mercury chloride 10 mM potentiated GABA-induced currents 6.8 { 0.60 times of control, and zinc chloride at 1/10/30 mM did not affect the potentiation (Fig. 11B) (p õ 0.05). Thus, mercury chloride binds to a site other than the zinc binding site on the GABA receptor– channel complex. DISCUSSION

Active Forms of Mercury Chloride

FIG. 7. (A) Mercury chloride accelerates desensitization of GABAinduced currents. (B) Mercury chloride 10 mM decreases the time constants of fast and slow phases of desensitization of currents induced by 30 mM GABA from 4.3 { 0.74 sec of control to 2.6 { 0.33 sec (n Å 6), and from 20.0 { 2.70 sec to 10.6 { 1.46 sec (n Å 5), respectively. (C) The ratios of steady/peak current amplitudes are decreased by 10 mM mercury chloride from 47.3 { 4.13% to 22.8 { 4.01% (n Å 6).

equivalent osmolarity adjusted by addition of sucrose. Thus, IAA potentiation of GABA-induced currents was larger than the calculation by using the control current with unadjusted osmolarity. IAA at 100 mM abolished mercury chloride potentiation of GABA-induced currents (Fig. 10A). Mercury chloride 10 mM coapplied with 10 mM GABA potentiated the current 1.84 { 0.127 times of control, whereas this potentiating effect was decreased by IAA at 30 and 100 mM

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Most cases of mercury neurotoxicity in humans are caused by methylmercury, which has been shown to directly modulate ion channels (Shafer et al., 1990; Shafer and Atchison, 1991; Quandt et al., 1982; Shrivastav et al., 1976; Arakawa et al., 1991). Methylmercury can be converted to inorganic mercury such as mercury chloride via biotransformation in animals (Evans et al., 1977). The percentage of inorganic mercury out of the total mercury in the methylmercury-intoxicated human brain is as high as 72 to 88% (Friberg and Mottet, 1989). Since mercury chloride in its uncharged form (HgCl2 ) is highly membrane permeable (Gutknecht, 1981), it can penetrate the blood brain barrier causing neurotoxicity. Therefore, inorganic mercury converted from methylmercury outside the brain could reach the brain. Part of methylmercury neurotoxicity may be exerted by inorganic mercury. The dominant species of mercury chloride complexes are HgCl20 in physiological chloride concentrations and Hg2/ 4 in extreme low chloride concentrations (Table 1). Hg2/ is far less potent than HgCl20 4 in potentiating GABA-induced currents, indicating that HgCl20 4 plays a major role in mercury chloride neurotoxicity (Fig. 3). Furthermore, mercury chloride forms complexes with hydroxyl groups at high extracellular pH (Table 1). Mercury chloride–hydroxyl complexes decrease the Kd for GABA to an equal extent to mercury chloride without hydroxyl groups (Fig. 2), suggesting that both forms can contribute to mercury chloride neurotoxicity in either alkaline or acidic brain insult conditions. Similar to mercury chloride, other polyvalent cations such as zinc exist in several chloride and hydroxyl complexes

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FIG. 8. Effects of sulfhydryl modifying agents on GABA-induced currents. NEM or IAA, coapplied with 10 mM GABA potentiates GABA-induced currents in a dose-dependent manner (A and B). There is an optimal concentration of NEM, 30 mM, in potentiating GABA-induced currents (B). The current amplitude induced by coapplication of 30 mM NEM and 10 mM GABA reaches the same amplitude as that induced by 35 mM GABA alone but exhibits an accelerated desensitization (C). The time constants and steady/peak current amplitude ratios of 35 mM GABA-induced currents are reduced by coapplication of 10 mM GABA and 30 mM NEM from 23.1 { 1.44 sec to 13.7 { 0.34 sec (n Å 3) and from 43.9 { 3.14% to 33.1 { 1.91% (n Å 3–4), respectively (D).

depending on the extracellular chloride concentration and pH (Hahne and Kroontje, 1973). It is important to bear in mind that these chloride and hydroxyl complexes may have different potency and affinity to exert effects on ion channels. Extracellular pH is also known to modulate the activity of ligand-gated channels such as NMDA receptor–channels (Tang et al., 1990; Traynelis and Cull-Candy, 1990) and acetylcholine receptor–channels (Imoto et al., 1988). This may be caused by a cluster of pH-sensitive amino acids with charges opposite to those of the permeant ions at the channel mouth (Barnard et al., 1987; Imoto et al., 1988; Schofield et al., 1987), increasing the driving force for ion permeation due to local accumulation of permeant ions near the mouth of the channel. In brain slices, the activation of the GABAA receptors causes extracellular alkalinization and intracellular acidification (Chen and Chesler, 1991; Kaila et al., 1990, 1992, 1993). Changes

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in the extracellular pH can also affect outward rectification of GABA-induced currents (Robello et al., 1994). At the mouth of the GABA receptor–channel, basic lysine and arginine are accumulated, giving positive charges via protonation (Barnard et al., 1987; Schofield, 1987). Acidification decreases the affinity of GABA for the GABAA receptor drastically, whereas alkalinization not only decreases the affinity but also changes the stoichiometry of GABA binding (Fig. 1). Thus, changes in extracellular pH may also protonate or deprotonate the critical amino acids in the vicinity of GABA binding site, thereby affecting the affinity and stoichiometry of GABA for the GABAA receptor. Site of Action of Mercury Chloride Mercury chloride potentiation of GABA-induced currents is use dependent but voltage independent (Figs. 4 and 5).

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FIG. 9. Cysteine blocks mercury chloride potentiation of GABA-induced currents. With 100 mM cysteine applied to the bath, the control current amplitude induced by 10 mM GABA is increased only slightly. In the absence of cysteine, coapplication of 10 mM mercury chloride and 10 mM GABA potentiates the current, whereas potentiation subsides after introducing 100 mM cysteine to the bath. After washout of cysteine, potentiation reappears.

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whereas forming dithiols from vicinal thiols by the oxidizing reagent, 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), decreases the NMDA receptor activity (Aizemann et al., 1989; Moriyoshi et al., 1991; Lei et al., 1992; Tang and Aizenman, 1993). Furthermore, sulfhydryl group modification affects the gating kinetics or ion permeability of rat brain IA K/ channels (Ruppersberg et al., 1991), sodium channels (Mitrovic et al., 1993; Backx et al., 1992; Satin et al., 1991), L-type calcium channels (Chiamvimonvat et al., 1995), and calcium-activated nonselective cation channels (Koivisto et al., 1993). Sulfhydryl modifying reagents, NEM and iodoacetamide, potentiate GABA-induced currents (Figs. 8A and B), indicating the involvement of free sulfhydryl groups in GABAA receptor activity. Sulfhydryl groups have a high affinity for mercury chloride (Webb, 1966). Mercury chloride inhibition of kainate receptors is abolished by cysteine, glutathione, and DTT (Umbach and Gundersen, 1989; Kiskin et al., 1986) and site-directed mutagenesis of cysteine 189 to serine blocks mercury chloride inhibition of CHIP28 water channel (Pres-

Since the charged mercury tetrachloride predominates in 142 mM chloride solution (Table 1), it is unlikely that mercury chloride will bind to a site inside the channel pore. In support of this notion, mercury chloride preapplication in the absence of GABA potentiates GABA-induced currents (Fig. 6), suggesting that mercury chloride binds to an extracellular site of the GABAA receptor–channel complex. Mercury chloride may increase the affinity of GABA for the receptor allosterically via binding to the closed state of the GABAA receptor channel, a reminiscent of picrotoxin use-dependent block of the GABAA receptor (Newland and Cull-Candy, 1992). However, there are at least two independent binding sites on the GABAA receptor–channel complex for the polyvalent cations (Ma and Narahashi, 1993). Zinc and copper suppress GABA-induced currents via a negative modulatory binding site, whereas lanthanum exerts its potentiating effect on GABA-induced currents through a positive modulatory site. Zinc chloride does not share with mercury chloride for the binding site (Fig. 11). Role of Sulfhydryl Groups in the Activity and Mercury Chloride Modulation of GABAA Receptor The sulfhydryl groups play an important role in the channel protein functions. Adjacent sulfhydryl groups of ion channels can hold different subunit polypeptides together via the disulfide bond (Borsotto et al., 1985; Curtis and Catterall, 1984; Schmidt et al., 1985, 1987). The redox modulatory site of the NMDA receptor is modulated by sulfhydryl reagents (Aizenman et al., 1989; Lazarewicz et al., 1989). Breaking disulfide bonds by the sulfhydryl reducing agent, DTT, increases the activity of the NMDA receptor,

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FIG. 10. Iodoacetamide abolishes mercury chloride potentiation of GABA-induced currents. Mercury chloride coapplied with GABA fails to potentiate the currents in the presence of 100 mM iodoacetamide (A). Mercury chloride 10 mM coapplied with 10 mM GABA potentiates the current 1.84 { 0.127 times of controls, whereas the potentiating effect is abolished in the presence of increasing concentrations of iodoacetamide (B) ( p õ 0.05).

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accelerate desensitization of GABA-induced currents via locking the GABAA receptor–channel complex from the closed state to the desensitized state. REFERENCES

FIG. 11. Zinc chloride does not compete with mercury chloride for the binding site on the GABAA receptor–channel complex. Mercury chloride potentiates GABA-induced currents in the absence and presence of zinc chloride (A). Mercury chloride 10 mM potentiates GABA-induced currents 6.8 { 0.60 times of controls despite the presence of zinc chloride at various concentrations (B) (p õ 0.05).

ton et al., 1993). Consistent with these observations, mercury chloride potentiation of GABA-induced currents is abolished by cysteine and attenuated in the presence of iodoacetamide (Figs. 9 and 10), but not by glutathione or DTT (data not shown), suggesting the involvement of free sulfhydryl groups, but not disulfide bonds, in mercury chloride potentiation of GABA-induced currents. Similar to its irreversible potentiation of GABA-induced currents (Arakawa et al., 1991), mercury chloride acceleration of GABA-induced current desensitization is irreversible (Fig. 6C). Long 120-sec preapplication of mercury chloride in the absence of GABA attenuates mercury chloride potentiation of GABA-induced currents (Fig. 6B), suggesting that mercury chloride can also bind to the closed state of the GABA receptor–channel complex and shifts it to a desensitized state. NEM accelerates GABA-induced current desensitization (Figs. 8B and C), indicating that sulfhydryl groups may be involved in the desensitization kinetics. The high affinity of mercury chloride to the sulfhydryl groups may account for the irreversibility of its action to potentiate and

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