Inhibitory action of thimerosal, a sulfhydryl oxidant, on sodium channels in rat sensory neurons

Brain Research 864 (2000) 105–113 www.elsevier.com / locate / bres

Research report

Inhibitory action of thimerosal, a sulfhydryl oxidant, on sodium channels in rat sensory neurons a, a a b a Jin-Ho Song *, Yoon Young Jang , Yong Kyoo Shin , Moo Yeol Lee , Chung-Soo Lee a

Department of Pharmacology, Chung-Ang University, College of Medicine, 221 Heuk-Suk Dong, Dong-Jak Ku, Seoul 156 -756, South Korea b Department of Physiology, Chung-Ang University, College of Medicine, 221 Heuk-Suk Dong, Dong-Jak Ku, Seoul 156 -756, South Korea Accepted 23 February 2000

Abstract The effects of thimerosal, a sulfhydryl oxidizing agent, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion neurons were studied using the whole-cell patch clamp technique. Thimerosal blocked the two types of sodium channels in a dose-dependent manner. The inhibitory effect of thimerosal was much more pronounced in TTX-R sodium channels than TTX-S sodium channels. The effect of thimerosal was irreversible upon wash-out with thimerosal-free external solution. However, dithiothreitol, a reducing agent, partially reversed it. Thimerosal shifted the steady-state inactivation curves for both types of sodium channels in the hyperpolarizing direction. The voltage dependence of activation of both types of sodium channels was shifted in the depolarizing direction by thimerosal. The inactivation rate in both types of sodium channels increased after thimerosal treatment. All these effects of thimerosal would add up to cause a depression of sodium channel function leading to a diminished neuronal excitability.  2000 Elsevier Science B.V. All rights reserved. Themes: Excitable membranes and synaptic transmission Topics: Sodium channels Keywords: Sulfhydryl oxidation; Thimerosal; Tetrodotoxin-sensitive; Tetrodotoxin-resistant; Sodium channel; Dorsal root ganglion

1. Introduction Voltage-gated sodium channel plays an important role in generation and conduction of action potential in excitable cells. Sodium channels on the axon initial segment of neurons determine the threshold for the action potential and affect the duration and frequency of repetitive firings. Also the release of neurotransmitters from presynaptic nerve terminal is influenced by sodium channel activity. The function of sodium channels is subject to modulation by various toxins, therapeutic drugs and neuromodulators. Tetrodotoxin (TTX) is a potent neurotoxin that blocks voltage-gated sodium channels. Most sodium channels are blocked by TTX at the concentration range of 1–10 nM. However, sodium channels that are not sensitive to TTX exist in various tissues and in different animal species [32]. Rat dorsal root ganglion (DRG) neurons are endowed with *Corresponding author. Tel.: 182-2-820-5686; fax: 182-2-817-7115. E-mail address: [email protected] (J.-H. Song)

TTX-sensitive (TTX-S) as well as TTX-resistant (TTX-R) sodium channels [9,15,17]. Compared to TTX-S sodium current TTX-R sodium current exhibits slower time course of activation and inactivation, activates at higher voltage, and has a smaller single channel conductance. Pharmacologically TTX-R sodium channels are more sensitive to divalent cations (Co 21 , Mn 21 , Ni 21 , Cd 21 , Zn 21 ) and pyrethroid insecticide but less sensitive to lidocaine than TTX-S sodium channels [20,21,25,28]. The TTX-R sodium channel in DRG neurons was cloned and its amino acid sequence revealed some homology with a cardiac sodium channel. According to in situ hybridization this channel was localized to DRG cells with smaller diameters [2,22,23]. Protein cysteine residues are reactive to the cellular redox state and participate in the regulation of cellular functions. The redox modification of cysteine sulfhydryl groups has been shown to alter the function of various ion channels. The activity of voltage-dependent potassium channels was increased by oxidation but decreased by

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02198-3

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reduction [19]. Opposite results were observed for calcium-activated potassium channels [8,14,31]. In addition, oxidants decreased the activity of calcium release channels, NMDA receptor channels and ATP-sensitive potassium channels, but augmented that of voltage-gated calcium channels [1,5,6,27]. The present study was undertaken to elucidate the action of thimerosal, an oxidant known to cause a formation of disulfide link between two thiol groups, on TTX-S and TTX-R sodium channels in rat DRG neurons using the whole-cell patch clamp technique.

2. Materials and methods

2.1. Cell preparation DRG neurons were isolated as described previously [20,28]. Rats (2–6 days postnatal) were anesthetized in an isoflurane-saturated chamber. The vertebral column was then removed and cut longitudinally, generating two hemisections, which were placed into sterile Ca 21 - and Mg 21 -free phosphate-buffered saline (PBS, Sigma Chemical Co., St. Louis, MO). The ganglia were plucked from between the vertebrae, and incubated in PBS containing trypsin (2.5 mg / ml, Type IX, Sigma) at 378C for 30 min. After enzyme treatment, ganglia were rinsed twice with culture media (Dulbecco’s modified Eagle medium (GibcoBRL, Grand Island, NY) supplemented with horse serum (10% v / v; Sigma)). Single cells were mechanically dissociated by trituration with a fire-polished Pasteur pipette in 2 ml culture media. The dissociated cells were plated onto poly-L-lysine (Sigma)-coated glass coverslips (12 mm; Warner Instruments Co., Hamden, CT). Cells were incubated in the culture media in a 95% air / 5% CO 2 atmosphere controlled at 368C for 2–7 h before patch clamp experiments.

2.2. Electrophysiological recording Cells attached to coverslips were transferred into a recording chamber on the stage of an inverted microscope. Ionic currents were recorded under voltage-clamp conditions by the whole-cell patch clamp technique [12]. Suction pipettes (1–1.2 MV) were made of borosilicate glass capillary tubes (TW150F-4, World Precision Instrument, Sarasota, FL) using a two-step vertical puller (PP83, Narishige, Tokyo, Japan) and heat-polished with a microforge (MF-83, Narishige). The pipette solution contained (in mM): CsCl 125, NaF 20, HEPES 5, EGTA 5. The pH was adjusted to 7.2 with CsOH and the osmolarity was 279 mosM / l on average. The external solution contained (in mM): NaCl 50, choline chloride 90, tetraethylammonium chloride 20, D-glucose 5, HEPES 5, MgCl 2 1, CaCl 2 1. Lanthanum (LaCl 3 , 10 mM) was used to block calcium channel currents. The solution was adjusted to pH 7.4 with tetraethylammonium hydroxide

and the osmolarity was 304 mosM / l on average. An Ag–AgCl pellet / 3 M KCl-agar bridge was used for the reference electrode. Membrane currents were recorded using an Axopatch-1D amplifier (Axon instruments, Foster City, CA). Signals were digitized by a 12-bit analog-todigital interface (Digidata 1200A, Axon Instruments), filtered with a 8-pole lowpass Bessel filter at 5 kHz and sampled at 50 kHz using pCLAMP6 software (Axon Instruments) on an IBM-compatible PC. Series resistance was compensated 60–70%. Capacitative and leakage currents were subtracted by using a P1P/ 4 procedure [4]. The liquid junction potential between internal and external solution was an averaged 21.7 mV. The data shown in this paper were corrected for the liquid junction potential. All experiments were performed at 22–248C. Stock solutions of thimerosal and dithiothreitol were made in distilled water at a concentration of 100 and 500 mM, respectively, and aliquots were stored at 2208C until used. They were diluted in the external solution to the desired concentrations just before experiment. All chemicals were purchased from Sigma Chemical Co. TTX (100 nM) was used to separate TTX-R sodium currents from TTX-S sodium currents. For the study of TTX-S sodium channels, cells that expressed only TTX-S sodium currents were used. TTX-S sodium currents were completely inactivated within 2 ms when currents were evoked by depolarizing steps to 0 mV, while TTX-R sodium currents persisted for more than 20 ms. Thus, the difference in kinetics was used to identify the type of sodium current. A period of 5–10 min was allowed after the establishment of the whole-cell recording configuration to ensure adequate equilibration between the internal pipette solution and the cell interior and to obtain a stable membrane current.

2.3. Data analysis Data were analyzed by a combination of pCLAMP6 programs and SigmaPlot (Jandel Scientific, San Rafael, CA). Results are expressed as mean6S.E.M. and n represents the number of the cells examined. Statistical significance was determined using (paired) Student’s t-test with P,0.05 considered significant.

3. Results

3.1. Effects of thimerosal on the sodium current amplitude Tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons (DRG) were separated using the criteria as described in Materials and methods. Typical TTX-S and TTX-R sodium currents are shown in Fig. 1. Activation and inactivation kinetics for TTX-S sodium currents were

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Fig. 1. (A) Time course of thimerosal effect on TTX-S sodium current amplitude (n57). Currents were evoked by step depolarizations to 0 mV for 10 ms from a holding potential of 280 mV at an interval of 30 s. Thimerosal 1 mM was applied for 5 min and then washed out with thimerosal-free external solution as indicated in the box. (B) Time course of thimerosal effect on TTX-R sodium current amplitude (n57). Currents were evoked by step depolarizations to 0 mV for 40 ms from a holding potential of 280 mV at an interval of 30 s. Thimerosal 100 mM was applied for 5 min and then washed out with thimerosal-free external solution as indicated in the box. Representative current traces recorded before and after thimerosal treatment are shown in the right panel.

much faster than those for TTX-R sodium currents. Both types of sodium currents were blocked after bath application of thimerosal. The current amplitude decreased rapidly within 2–3 min after the drug application and then the rate of decrement slowed. The decrease of current amplitude was persistent and a steady-state level could not be obtained as long as the neurons were maintained under the whole-cell configuration (data not shown). In order to facilitate the quantification of data the thimerosal effects were measured 5 min after the thimerosal application in the following experiments. Thimerosal at 1 mM blocked TTX-S sodium current amplitude by 38.166.0% (n57) (Fig. 1A). The blocking effect of thimerosal was not reversed after washout with thimerosal-free external solution, rather the current amplitude decreased continuously. TTX-R sodium currents were more sensitive to thimerosal than TTX-S sodium currents. Thimerosal at 100 mM reduced TTX-R sodium

current amplitude by 32.162.1% (n57) (Fig. 1B). The effect was not reversed upon washout.

3.2. Dose- and holding potential-dependent inhibition of sodium currents by thimerosal Thimerosal blocked the two types of sodium channels in a dose-dependent manner (Fig. 2). When the membrane was held at 280 mV, thimerosal at 100, 300 and 1000 mM reduced TTX-S sodium current amplitude by 21.863.3% (n59), 30.564.1% (n511) and 38.262.7% (n530), respectively. At the same holding potential, thimerosal at 30, 100, 300 and 1000 mM blocked TTX-R sodium current amplitude by 24.262.4% (n57), 36.761.6% (n535), 44.864.4% (n59) and 57.364.4% (n57), respectively. Thus TTX-R sodium currents were approximately ten times more sensitive to thimerosal than TTX-S sodium currents. At 280 mV almost all TTX-R sodium channels

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on the two types of sodium channels resulted from oxidative processes, dithiothreitol (DTT), a reducing agent, was applied after the thimerosal effect had developed. DTT partially reversed the inhibitory effect of thimerosal on the two types of sodium channels (Fig. 3). The reversal by DTT was more efficient for TTX-S than TTX-R sodium currents in spite of the fact that a higher concentration of thimerosal was used in TTX-S sodium currents. DTT (1 mM) alone slightly increased the current amplitudes of both types of sodium channels. After DTT treatment thimerosal still reduced the current amplitude (TTX-S, thimerosal 1 mM, 2764%, n57; TTX-R, thimerosal 100 mM, 1863%, n56) but less so than thimerosal alone did. Thus the inhibitory effect of thimerosal in part, if not all, arises from oxidative reaction of sodium channels. It is also confirmed that TTX-R sodium channels are more sensitive to oxidation than TTX-S sodium channels.

3.4. Effects of thimerosal on sodium channel inactivation It was shown that the thimerosal effect was dependent

Fig. 2. Dose-dependent effect of thimerosal on sodium current amplitude. Currents were evoked by step depolarizations to 0 mV from holding potentials of 280 or 2100 mV as indicated in the figure. Symbols indicate the remaining current amplitudes 5 min after thimerosal treatments, and numbers in parentheses represent the number of the cells examined.

are relieved from inactivation while a large portion of TTX-S sodium channels are inactivated [26]. It is possible that the thimerosal effect is dependent on the level of inactivated sodium channels, which is evident for some drugs such as lidocaine and phenytoin. To measure the effect of thimerosal on TTX-S sodium channels when they are free of inactivation, the membrane was held at 2100 mV. Under this holding potential thimerosal at 100, 300 and 1000 mM reduced TTX-S sodium current amplitude by 5.362.8% (n59), 11.062.4% (n59) and 17.961.9% (n5 20), respectively. Thus the effect of thimerosal was attenuated when the membrane was held at a more negative potential indicating that part of the inhibitory action of thimerosal was due to modulation of the inactivation state of sodium channels. It was also evident that the sensitivity difference between the two types of sodium channels to thimerosal became greater when it was compared under the same conditions where the inactivation of sodium channels was removed.

3.3. Reversal of thimerosal effect by dithiothreitol Thimerosal is a sulfhydryl oxidant that causes formation of a disulfide bridge between two sulfhydryl groups. To address the question that the inhibitory effect of thimerosal

Fig. 3. Reversal of thimerosal effect on TTX-S (A, n57) and TTX-R (B, n57) sodium current amplitude by dithiothreitol (DTT). Currents were generated by step depolarizations to 0 mV from a holding potential of 280 mV at an interval of 30 s. Thimerosal and DTT were applied sequentially as indicated in the boxes.

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on holding potential in Fig. 2, indicating that thimerosal affects the inactivation state of sodium channels. Further studies were performed to examine this. The effects of thimerosal on the steady-state inactivation curves for sodium channels are shown in Fig. 4. TTX-S sodium channels were inactivated completely at holding potential above 240 mV and were relieved from inactivation at holding potential below 2100 mV (Fig. 4A). Thimerosal at 1 mM for 5 min shifted the steady-state inactivation curve in the hyperpolarizing direction. The maximum current amplitude at a holding potential of 2110 mV was also reduced by about 15%. DTT at 1 mM partly reversed the shift of the steady-state inactivation curve caused by thimerosal, but it was not able to reverse the blocking effect of thimerosal on the maximum current amplitude at holding potential of 2110 mV. The data were fitted to the Boltzmann equation, I /Imax 5 1 / h1 1 exp[(Vh 2Vh 0.5 ) /k]j, where I is current amplitude, Imax is maximum current amplitude, Vh is holding potential, Vh 0.5 is the potential at which I is 0.5 Imax , and k is the slope factor. In control experiments, the half-maximum inactiva-

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tion potential (Vh 0.5 ) was estimated to be 271.562.0 mV and the slope factor (potential required for an e-fold change) was 6.3860.43 mV (n58). Thimerosal at 1 mM shifted Vh 0.5 by 25.760.7 mV (P,0.001) and subsequent application of 1 mM DTT reversed the shift by 14.860.7 mV (P,0.001). Slope factors were 6.6260.28 and 6.5960.30 mV in thimerosal-treated group and subsequent DTT-treated group, respectively, but the differences were not statistically significant. Similar results were observed in TTX-R sodium channels (Fig. 4B). Almost all TTX-R sodium channels were inactivated at holding potential above 220 mV and were free of inactivation at holding potential below 270 mV. Thimerosal shifted the steady-state inactivation curve in the hyperpolarizing direction and reduced the maximum current amplitude. Both effects were partly reversed by DTT. In control experiments the steady-state inactivation curve was best fitted by the Boltzmann equation when Vh 0.5 was 239.761.8 mV and the slope factor was 5.5460.34 mV (n58). Thimerosal at 100 mM shifted the curve by 24.760.4 mV (P,0.001) and DTT at 1 mM reversed the shift by 11.760.6 mV (P,0.05). The slope factor was increased to 6.9060.40 mV (P,0.001) by thimerosal but the effect was not significantly reversed by DTT (6.7260.65 mV).

3.5. Effects of thimerosal on sodium channel activation

Fig. 4. Effects of thimerosal on the steady-state inactivation curves for TTX-S (A, n58) and TTX-R (B, n58) sodium channels. The membrane potential was held at various levels for 20 s, and then current was evoked by a step depolarization to 0 mV. The peak current amplitude is plotted as a function of the holding potential. I, current amplitude; I max , maximum control current amplitude; (d) control; (j) thimerosal 1 mM (A) or 100 mM (B) for 5 min; (m) DTT 1 mM for 5 min after thimerosal treatment.

Effects of thimerosal on the current–voltage relationship and the conductance–voltage curve are illustrated in Fig. 5A for TTX-S sodium channels and in Fig. 5B for TTX-R sodium channels. TTX-S sodium currents were evoked by 10-ms depolarizing steps to various levels from a holding potential of 280 mV. Test potentials ranged from 255 to 150 mV in 5-mV increments and were delivered at a frequency of 0.2 Hz. Thimerosal at 1 mM for 5 min reduced TTX-S sodium current amplitude throughout the entire test potentials but the degree of block was more evident at lower test potentials (Fig. 5A). Also the current– voltage curve was shifted to the right direction. This is shown more clearly in the conductance–voltage curve. In control experiments, the half-maximum conductance (Vg0.5 ) was calculated to be 231.160.7 mV and the slope factor was 5.4760.30 mV (n58). Thimerosal changed these values by 14.461.4 mV (P,0.05) and 11.2060.38 mV (P,0.01), respectively. In TTX-R sodium currents similar results were observed as in TTX-S sodium currents (Fig. 5B). TTX-R currents were evoked by 40-ms depolarizing steps to various levels from a holding potential of 280 mV. Test potentials ranged from 235 to 150 mV in 5-mV increments and were delivered at a frequency of 0.2 Hz. Thimerosal at 100 mM blocked TTX-R sodium current at all test potentials, the effect being more pronounced at lower test potentials. In the absence of thimerosal Vg0.5 was 210.661.7 mV and the slope factor was 5.3460.25 mV (n57). Thimerosal

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Fig. 5. (A) Representative current–voltage relationship curves and the conductance–voltage relationship curves for TTX-S sodium channels before (s) and after (d) thimerosal 1 mM for 5 min treatment (n58). (B) Representative current–voltage relationship curves and the conductance–voltage relationship curves for TTX-R sodium channels before (s) and after (d) thimerosal 100 mM for 5 min treatment (n57). The conductance–voltage curves are drawn according to the Boltzmann equation, G /Gmax 5 1 / h1 1 exp[(Vg0.5 2Vg) /k]j, where G is conductance, Gmax is maximum conductance, Vg is test potential, Vg0.5 is the potential at which G is 0.5 Gmax , and k is the slope factor.

shifted the values by 14.060.8 mV (P,0.01) and 11.9860.45 mV (P,0.01), respectively.

3.6. Effects of thimerosal on the inactivation rate of sodium channels Effects of thimerosal on the time dependent inactivation of sodium channels are shown in Fig. 6. TTX-S sodium currents started to inactivate 0.4 ms after the pre-pulse potential change to 240 mV from a holding potential of 2100 mV. The current amplitude then decreased exponentially and disappeared almost completely after 10-s prepulse. After treatment of thimerosal 1 mM for 5 min the inactivation rate increased compared to control. At prepulse of 10 ms, 50.165.8% of sodium currents were inactivated in control experiments while 72.262.9% of sodium currents were inactivated after thimerosal treatment (P,0.001, n58) (Fig. 6A). TTX-R sodium currents started to inactivate 2 ms after pre-pulse potential change to 220 mV from a holding potential of 280 mV and were completely inactivated after

Fig. 6. Effect of thimerosal on the time course of sodium channel inactivation. (A) TTX-S sodium channels. Pre-pulses to 240 mV from a holding potential of 2100 mV were applied for various duration and were immediately followed by depolarizing steps to 0 mV (n58). (B) TTX-R sodium channels. Pre-pulses to 220 mV from a holding potential of 280 mV were applied for various duration and were immediately followed by depolarizing steps to 0 mV (n57). The current amplitude is plotted as a function of the pre-pulse duration.

10 s. At pre-pulse of 100 ms control currents were inactivated 41.565.8% while 53.965.7% of currents were inactivated after thimerosal 100 mM treatment for 5 min (P,0.001, n57) (Fig. 6B).

3.7. Effects of thimerosal on the recovery of sodium channels from inactivation TTX-S sodium channels were inactivated by 5 s depolarizing step to 240 mV from a holding potential of 2100 mV and then repolarized to 2100 mV for various duration followed by a step depolarization to 0 mV. The resultant current amplitude was plotted as a function of the repolarizing duration (Fig. 7A). TTX-S sodium currents started to recover from inactivation after 1 ms repolarization and attained a maximum amplitude after 20 s repolarization. Thimerosal at 1 mM for 5 min had a negligible effect on the recovery time course. For TTX-R sodium channels the inactivating potential of 220 mV for 5 s from a holding potential of 280 mV and

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Fig. 7. Effect of thimerosal on the recovery of sodium channels from inactivation. (A) TTX-S sodium channels. Pre-pulse was applied to 240 mV for 5 s from a holding potential of 2100 mV, and then membrane potential was repolarized to 2100 mV for various duration which was followed by depolarizing steps to 0 mV (n55). (B) TTX-R sodium channels. Pre-pulse was applied to 220 mV for 5 s from a holding potential of 280 mV, and then membrane potential was repolarized to 280 mV for various duration which was followed by depolarizing steps to 0 mV (n55). The current amplitude is plotted as a function of the repolarizing duration.

repolarizing potential of 280 mV were used. TTX-R sodium currents started to recover from inactivation after 10 ms repolarization and attained a maximum amplitude after 30 s repolarization. Thimerosal at 100 mM for 5 min slightly shifted the recovery time curve to right direction but the change was not significant (Fig. 7B).

4. Discussion Sodium channels are subject to modulation by oxidative or covalent modification of sulfhydryl containing amino acids. Chloramine-T, an methionine oxidant, abolished the sodium channel inactivation irreversibly and shifted the steady-state sodium current inactivation curve in the depolarizing direction with no effect on the sodium channel activation [29,30]. Another methionine oxidant cyanogen bromide, however, did not affect the sodium

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channel inactivation [18]. N-ethylmaleimide (NEM), which forms a covalent bond to cysteinyl sulfhydryl group, induced a slow sodium channel inactivation with the activation process unaltered [24]. Nitrosylation of cysteinyl sulfhydryl groups caused a reduction of TTX-S and TTXR sodium current in rat nodose ganglia, and shifted the steady-state sodium channel inactivation curve in the hyperpolarizing direction [16]. Thimerosal is a disinfectant and causes oxidation of cysteinyl sulfhydryl groups leading to the formation of disulfide bonds between neighboring sulfhydryl groups. The present study showed that thimerosal depressed both TTX-S and TTX-R sodium channels in rat DRG neurons in a dose dependent manner. The inhibition of sodium current was rapid within 2–3 min after the application of thimerosal, then slowed down but hardly reached a steady state. Blockage of sodium current by thimerosal was not reversed by washout with drug-free external solution, but partially reversed by DTT, a sulfhydryl reducing agent. This result strongly suggests that thimerosal depressed the sodium currents by oxidative mechanism. Since thimerosal is hydrophilic and impermeable to lipophilic cellular membranes, thimerosal seems to act on the extracellular surface of the sodium channel or pore region, where it oxidizes cysteines and renders structural or functional changes to the sodium channel protein. TTX-S and TTX-R sodium channels showed a differential sensitivity to thimerosal. At the holding potential of 280 mV, which is near the resting membrane potential, TTX-R sodium channels were approximately ten times more sensitive to the inhibitory action of thimerosal than TTX-S sodium channels. At this holding potential almost all TTX-R sodium channels are relieved from inactivation, but a large portion of TTX-S sodium channels are in the inactivated state (Fig. 4). Thimerosal shifted the steadystate sodium channel inactivation curve in the hyperpolarizing direction. When the holding potential was 2100 mV where almost all TTX-S sodium channels are relieved from inactivation, the degree of inhibition caused by thimerosal became far less than that measured at the 280 mV holding potential. So the sensitivity difference between the two types of sodium channels becomes even greater if the comparison is made under the equal condition in terms of the inactivation. The differential sensitivity was also observed in the reversal of thimerosal effect by DTT. DTT at 1 mM reversed the effect of 1 mM thimerosal on TTX-S sodium channels in terms of the current amplitude and the steady-state inactivation curve more effectively than that of 100 mM thimerosal on TTX-R sodium channels. The rate of the sodium channel inactivation was accelerated by thimerosal. Unlike the other effects of thimerosal described above, this was more pronounced in TTX-S than TTX-R sodium channels. However, thimerosal did not affect the recovery rate of sodium channels from inactivation. Thimerosal interfered with the activation process of the

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sodium channels. Thus thimerosal shifted the activation curves of both types of sodium channels in the depolarizing direction and increased the value of the slope factor. The reversal potential for sodium currents was near 125 mV and was not changed by thimerosal treatment. Therefore it is not likely that the sulfhydryl modification by thimerosal affects the ion selectivity of sodium channels. Most of the sodium channel functions examined in this study were modulated by thimerosal in favor of suppression. Dorsal root ganglia are primary sensory neurons. Among them C- and Ad-type neurons are smaller in size than other cell types and participate in nociception. They are sensitive to pain inducing agents such as bradykinin, serotonin, capsaicin, prostaglandins and adenosine, and express mostly TTX-R sodium channels [3,10,11,13]. Our data show that TTX-R sodium channels are more sensitive to thimerosal than TTX-S sodium channels, implying that transmission of nociception among others might be more susceptible to sulfhydryl oxidation. Two TTX-R sodium channels, SNS / PN3 and NaN, are expressed in DRG neurons [2,7,22]. NaN channel has a hyperpolarized voltage dependence of activation compared with SNS / PN3 channel. The activation kinetics of TTX-R sodium current we examined showed the characteristics of SNS / PN3 channel current. Moreover, NaN channels inactivates at potentials positive to 2120 mV and may not be detectable at a depolarized holding potential we used. Thus it is highly probable that the TTX-R sodium channel we studied is SNS / PN3 type.

Acknowledgements This work was supported by the Research Grant of Chung-Ang University in 1999 to Jin-Ho Song.

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