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

Research report

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

a , a a b a*Jin-Ho Song , Yoon Young Jang , Yong Kyoo Shin , Moo Yeol Lee , Chung-Soo LeeaDepartment of Pharmacology, Chung-Ang University, College of Medicine, 221 Heuk-Suk Dong, Dong-Jak Ku, Seoul 156-756, South Korea

bDepartment 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) sodiumchannels in rat dorsal root ganglion neurons were studied using the whole-cell patch clamp technique. Thimerosal blocked the two typesof sodium channels in a dose-dependent manner. The inhibitory effect of thimerosal was much more pronounced in TTX-R sodiumchannels 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 ofsodium channels in the hyperpolarizing direction. The voltage dependence of activation of both types of sodium channels was shifted inthe depolarizing direction by thimerosal. The inactivation rate in both types of sodium channels increased after thimerosal treatment. Allthese 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 TTX-sensitive (TTX-S) as well as TTX-resistant (TTX-R)sodium channels [9,15,17]. Compared to TTX-S sodium

Voltage-gated sodium channel plays an important role in current TTX-R sodium current exhibits slower time coursegeneration and conduction of action potential in excitable of activation and inactivation, activates at higher voltage,cells. Sodium channels on the axon initial segment of and has a smaller single channel conductance. Pharmaco-neurons determine the threshold for the action potential logically TTX-R sodium channels are more sensitive to

21 21 21 21 21and affect the duration and frequency of repetitive firings. divalent cations (Co , Mn , Ni , Cd , Zn ) andAlso the release of neurotransmitters from presynaptic pyrethroid insecticide but less sensitive to lidocaine thannerve terminal is influenced by sodium channel activity. TTX-S sodium channels [20,21,25,28]. The TTX-R so-The function of sodium channels is subject to modulation dium channel in DRG neurons was cloned and its aminoby various toxins, therapeutic drugs and neuromodulators. acid sequence revealed some homology with a cardiac

Tetrodotoxin (TTX) is a potent neurotoxin that blocks sodium channel. According to in situ hybridization thisvoltage-gated sodium channels. Most sodium channels are channel was localized to DRG cells with smaller diametersblocked by TTX at the concentration range of 1–10 nM. [2,22,23].However, sodium channels that are not sensitive to TTX Protein cysteine residues are reactive to the cellularexist in various tissues and in different animal species [32]. redox state and participate in the regulation of cellularRat dorsal root ganglion (DRG) neurons are endowed with functions. The redox modification of cysteine sulfhydryl

groups has been shown to alter the function of various ionchannels. The activity of voltage-dependent potassium*Corresponding author. Tel.: 182-2-820-5686; fax: 182-2-817-7115.

E-mail address: jinhos@dragonar.nm.cau.ac.kr (J.-H. Song) channels was increased by oxidation but decreased by

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

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reduction [19]. Opposite results were observed for cal- and the osmolarity was 304 mosM/l on average. Ancium-activated potassium channels [8,14,31]. In addition, Ag–AgCl pellet /3 M KCl-agar bridge was used for theoxidants decreased the activity of calcium release chan- reference electrode. Membrane currents were recordednels, NMDA receptor channels and ATP-sensitive potas- using an Axopatch-1D amplifier (Axon instruments, Fostersium channels, but augmented that of voltage-gated cal- City, CA). Signals were digitized by a 12-bit analog-to-cium channels [1,5,6,27]. The present study was under- digital interface (Digidata 1200A, Axon Instruments),taken to elucidate the action of thimerosal, an oxidant filtered with a 8-pole lowpass Bessel filter at 5 kHz andknown to cause a formation of disulfide link between two sampled at 50 kHz using pCLAMP6 software (Axonthiol groups, on TTX-S and TTX-R sodium channels in rat Instruments) on an IBM-compatible PC. Series resistanceDRG neurons using the whole-cell patch clamp technique. was compensated 60–70%. Capacitative and leakage cur-

rents were subtracted by using a P1P/4 procedure [4]. Theliquid junction potential between internal and external

2. Materials and methods solution was an averaged 21.7 mV. The data shown in thispaper were corrected for the liquid junction potential. All

2.1. Cell preparation experiments were performed at 22–248C. Stock solutionsof thimerosal and dithiothreitol were made in distilled

DRG neurons were isolated as described previously water at a concentration of 100 and 500 mM, respectively,[20,28]. Rats (2–6 days postnatal) were anesthetized in an and aliquots were stored at 2208C until used. They wereisoflurane-saturated chamber. The vertebral column was diluted in the external solution to the desired concen-then removed and cut longitudinally, generating two trations just before experiment. All chemicals were pur-

21hemisections, which were placed into sterile Ca - and chased from Sigma Chemical Co.21Mg -free phosphate-buffered saline (PBS, Sigma Chemi- TTX (100 nM) was used to separate TTX-R sodium

cal Co., St. Louis, MO). The ganglia were plucked from currents from TTX-S sodium currents. For the study ofbetween the vertebrae, and incubated in PBS containing TTX-S sodium channels, cells that expressed only TTX-Strypsin (2.5 mg/ml, Type IX, Sigma) at 378C for 30 min. sodium currents were used. TTX-S sodium currents wereAfter enzyme treatment, ganglia were rinsed twice with completely inactivated within 2 ms when currents wereculture media (Dulbecco’s modified Eagle medium (Gib- evoked by depolarizing steps to 0 mV, while TTX-RcoBRL, Grand Island, NY) supplemented with horse sodium currents persisted for more than 20 ms. Thus, theserum (10% v/v; Sigma)). Single cells were mechanically difference in kinetics was used to identify the type ofdissociated by trituration with a fire-polished Pasteur sodium current. A period of 5–10 min was allowed afterpipette in 2 ml culture media. The dissociated cells were the establishment of the whole-cell recording configurationplated onto poly-L-lysine (Sigma)-coated glass coverslips to ensure adequate equilibration between the internal(12 mm; Warner Instruments Co., Hamden, CT). Cells pipette solution and the cell interior and to obtain a stablewere incubated in the culture media in a 95% air /5% CO membrane current.2

atmosphere controlled at 368C for 2–7 h before patchclamp experiments. 2.3. Data analysis

2.2. Electrophysiological recording Data were analyzed by a combination of pCLAMP6programs and SigmaPlot (Jandel Scientific, San Rafael,

Cells attached to coverslips were transferred into a CA). Results are expressed as mean6S.E.M. and n repre-recording chamber on the stage of an inverted microscope. sents the number of the cells examined. Statistical signifi-Ionic currents were recorded under voltage-clamp con- cance was determined using (paired) Student’s t-test withditions by the whole-cell patch clamp technique [12]. P,0.05 considered significant.Suction pipettes (1–1.2 MV) were made of borosilicateglass capillary tubes (TW150F-4, World Precision Instru-ment, Sarasota, FL) using a two-step vertical puller (PP- 3. Results83, Narishige, Tokyo, Japan) and heat-polished with amicroforge (MF-83, Narishige). The pipette solution con- 3.1. Effects of thimerosal on the sodium currenttained (in mM): CsCl 125, NaF 20, HEPES 5, EGTA 5. amplitudeThe pH was adjusted to 7.2 with CsOH and the osmolaritywas 279 mosM/l on average. The external solution Tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resis-contained (in mM): NaCl 50, choline chloride 90, tetra- tant (TTX-R) sodium currents in rat dorsal root ganglionethylammonium chloride 20, D-glucose 5, HEPES 5, neurons (DRG) were separated using the criteria asMgCl 1, CaCl 1. Lanthanum (LaCl , 10 mM) was used described in Materials and methods. Typical TTX-S and2 2 3

to block calcium channel currents. The solution was TTX-R sodium currents are shown in Fig. 1. Activationadjusted to pH 7.4 with tetraethylammonium hydroxide and inactivation kinetics for TTX-S sodium currents were

J.-H. Song et al. / Brain Research 864 (2000) 105 –113 107

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 msfrom 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 externalsolution as indicated in the box. (B) Time course of thimerosal effect on TTX-R sodium current amplitude (n57). Currents were evoked by stepdepolarizations 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 washedout with thimerosal-free external solution as indicated in the box. Representative current traces recorded before and after thimerosal treatment are shown inthe right panel.

much faster than those for TTX-R sodium currents. Both current amplitude by 32.162.1% (n57) (Fig. 1B). Thetypes of sodium currents were blocked after bath applica- effect was not reversed upon washout.tion of thimerosal. The current amplitude decreased rapidlywithin 2–3 min after the drug application and then the rate 3.2. Dose- and holding potential-dependent inhibition ofof decrement slowed. The decrease of current amplitude sodium currents by thimerosalwas persistent and a steady-state level could not beobtained as long as the neurons were maintained under the Thimerosal blocked the two types of sodium channels inwhole-cell configuration (data not shown). In order to a dose-dependent manner (Fig. 2). When the membranefacilitate the quantification of data the thimerosal effects was held at 280 mV, thimerosal at 100, 300 and 1000 mMwere measured 5 min after the thimerosal application in reduced TTX-S sodium current amplitude by 21.863.3%the following experiments. (n59), 30.564.1% (n511) and 38.262.7% (n530), re-

Thimerosal at 1 mM blocked TTX-S sodium current spectively. At the same holding potential, thimerosal at 30,amplitude by 38.166.0% (n57) (Fig. 1A). The blocking 100, 300 and 1000 mM blocked TTX-R sodium currenteffect of thimerosal was not reversed after washout with amplitude by 24.262.4% (n57), 36.761.6% (n535),thimerosal-free external solution, rather the current am- 44.864.4% (n59) and 57.364.4% (n57), respectively.plitude decreased continuously. TTX-R sodium currents Thus TTX-R sodium currents were approximately tenwere more sensitive to thimerosal than TTX-S sodium times more sensitive to thimerosal than TTX-S sodiumcurrents. Thimerosal at 100 mM reduced TTX-R sodium currents. At 280 mV almost all TTX-R sodium channels

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on the two types of sodium channels resulted fromoxidative processes, dithiothreitol (DTT), a reducing agent,was applied after the thimerosal effect had developed. DTTpartially reversed the inhibitory effect of thimerosal on thetwo types of sodium channels (Fig. 3). The reversal byDTT was more efficient for TTX-S than TTX-R sodiumcurrents in spite of the fact that a higher concentration ofthimerosal was used in TTX-S sodium currents. DTT (1mM) alone slightly increased the current amplitudes ofboth types of sodium channels. After DTT treatmentthimerosal still reduced the current amplitude (TTX-S,thimerosal 1 mM, 2764%, n57; TTX-R, thimerosal 100mM, 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 isalso confirmed that TTX-R sodium channels are moresensitive to oxidation than TTX-S sodium channels.

3.4. Effects of thimerosal on sodium channelinactivation

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 holdingpotentials of 280 or 2100 mV as indicated in the figure. Symbolsindicate the remaining current amplitudes 5 min after thimerosal treat-ments, and numbers in parentheses represent the number of the cellsexamined.

are relieved from inactivation while a large portion ofTTX-S sodium channels are inactivated [26]. It is possiblethat the thimerosal effect is dependent on the level ofinactivated sodium channels, which is evident for somedrugs such as lidocaine and phenytoin. To measure theeffect of thimerosal on TTX-S sodium channels when theyare free of inactivation, the membrane was held at 2100mV. Under this holding potential thimerosal at 100, 300and 1000 mM reduced TTX-S sodium current amplitude by5.362.8% (n59), 11.062.4% (n59) and 17.961.9% (n5

20), respectively. Thus the effect of thimerosal wasattenuated when the membrane was held at a morenegative potential indicating that part of the inhibitoryaction of thimerosal was due to modulation of the inactiva-tion state of sodium channels. It was also evident that thesensitivity difference between the two types of sodiumchannels to thimerosal became greater when it was com-pared under the same conditions where the inactivation ofsodium channels was removed.

3.3. Reversal of thimerosal effect by dithiothreitolFig. 3. Reversal of thimerosal effect on TTX-S (A, n57) and TTX-R (B,n57) sodium current amplitude by dithiothreitol (DTT). Currents were

Thimerosal is a sulfhydryl oxidant that causes formation generated by step depolarizations to 0 mV from a holding potential ofof a disulfide bridge between two sulfhydryl groups. To 280 mV at an interval of 30 s. Thimerosal and DTT were appliedaddress the question that the inhibitory effect of thimerosal sequentially as indicated in the boxes.

J.-H. Song et al. / Brain Research 864 (2000) 105 –113 109

on holding potential in Fig. 2, indicating that thimerosal tion potential (Vh ) was estimated to be 271.562.0 mV0.5

affects the inactivation state of sodium channels. Further and the slope factor (potential required for an e-foldstudies were performed to examine this. The effects of change) was 6.3860.43 mV (n58). Thimerosal at 1 mMthimerosal on the steady-state inactivation curves for shifted Vh by 25.760.7 mV (P,0.001) and subsequent0.5

sodium channels are shown in Fig. 4. application of 1 mM DTT reversed the shift by 14.860.7TTX-S sodium channels were inactivated completely at mV (P,0.001). Slope factors were 6.6260.28 and

holding potential above 240 mV and were relieved from 6.5960.30 mV in thimerosal-treated group and subsequentinactivation at holding potential below 2100 mV (Fig. 4A). DTT-treated group, respectively, but the differences wereThimerosal at 1 mM for 5 min shifted the steady-state not statistically significant.inactivation curve in the hyperpolarizing direction. The Similar results were observed in TTX-R sodium chan-maximum current amplitude at a holding potential of 2110 nels (Fig. 4B). Almost all TTX-R sodium channels weremV was also reduced by about 15%. DTT at 1 mM partly inactivated at holding potential above 220 mV and werereversed the shift of the steady-state inactivation curve free of inactivation at holding potential below 270 mV.caused by thimerosal, but it was not able to reverse the Thimerosal shifted the steady-state inactivation curve inblocking effect of thimerosal on the maximum current the hyperpolarizing direction and reduced the maximumamplitude at holding potential of 2110 mV. The data were current amplitude. Both effects were partly reversed byfitted to the Boltzmann equation, I /I 5 1/ h1 1 DTT. In control experiments the steady-state inactivationmax

exp[(Vh 2Vh ) /k]j, where I is current amplitude, I is curve was best fitted by the Boltzmann equation when0.5 max

maximum current amplitude, Vh is holding potential, Vh Vh was 239.761.8 mV and the slope factor was0.5 0.5

is the potential at which I is 0.5 I , and k is the slope 5.5460.34 mV (n58). Thimerosal at 100 mM shifted themax

factor. In control experiments, the half-maximum inactiva- curve by 24.760.4 mV (P,0.001) and DTT at 1 mMreversed the shift by 11.760.6 mV (P,0.05). The slopefactor was increased to 6.9060.40 mV (P,0.001) bythimerosal but the effect was not significantly reversed byDTT (6.7260.65 mV).

3.5. Effects of thimerosal on sodium channel activation

Effects of thimerosal on the current–voltage relationshipand the conductance–voltage curve are illustrated in Fig.5A for TTX-S sodium channels and in Fig. 5B for TTX-Rsodium channels. TTX-S sodium currents were evoked by10-ms depolarizing steps to various levels from a holdingpotential of 280 mV. Test potentials ranged from 255 to150 mV in 5-mV increments and were delivered at afrequency of 0.2 Hz. Thimerosal at 1 mM for 5 minreduced TTX-S sodium current amplitude throughout theentire test potentials but the degree of block was moreevident at lower test potentials (Fig. 5A). Also the current–voltage curve was shifted to the right direction. This isshown more clearly in the conductance–voltage curve. Incontrol experiments, the half-maximum conductance(Vg ) was calculated to be 231.160.7 mV and the slope0.5

factor was 5.4760.30 mV (n58). Thimerosal changedthese values by 14.461.4 mV (P,0.05) and 11.2060.38mV (P,0.01), respectively.

In TTX-R sodium currents similar results were observedas in TTX-S sodium currents (Fig. 5B). TTX-R currentswere evoked by 40-ms depolarizing steps to various levelsfrom a holding potential of 280 mV. Test potentials

Fig. 4. Effects of thimerosal on the steady-state inactivation curves for ranged from 235 to 150 mV in 5-mV increments andTTX-S (A, n58) and TTX-R (B, n58) sodium channels. The membrane were delivered at a frequency of 0.2 Hz. Thimerosal at 100potential was held at various levels for 20 s, and then current was evoked mM blocked TTX-R sodium current at all test potentials,by a step depolarization to 0 mV. The peak current amplitude is plotted as

the effect being more pronounced at lower test potentials.a function of the holding potential. I, current amplitude; I , maximummaxIn the absence of thimerosal Vg was 210.661.7 mV andcontrol current amplitude; (d) control; (j) thimerosal 1 mM (A) or 100 0.5

mM (B) for 5 min; (m) DTT 1 mM for 5 min after thimerosal treatment. the slope factor was 5.3460.25 mV (n57). Thimerosal

110 J.-H. Song et al. / Brain Research 864 (2000) 105 –113

Fig. 5. (A) Representative current–voltage relationship curves and theconductance–voltage relationship curves for TTX-S sodium channelsbefore (s) and after (d) thimerosal 1 mM for 5 min treatment (n58).(B) Representative current–voltage relationship curves and the conduct- Fig. 6. Effect of thimerosal on the time course of sodium channelance–voltage relationship curves for TTX-R sodium channels before (s) inactivation. (A) TTX-S sodium channels. Pre-pulses to 240 mV from aand after (d) thimerosal 100 mM for 5 min treatment (n57). The holding potential of 2100 mV were applied for various duration and wereconductance–voltage curves are drawn according to the Boltzmann immediately followed by depolarizing steps to 0 mV (n58). (B) TTX-Requation, G /G 5 1/ h1 1 exp[(Vg 2Vg) /k]j, where G is conductance,max 0.5 sodium channels. Pre-pulses to 220 mV from a holding potential of 280G is maximum conductance, Vg is test potential, Vg is the potential atmax 0.5 mV were applied for various duration and were immediately followed bywhich G is 0.5 G , and k is the slope factor.max depolarizing steps to 0 mV (n57). The current amplitude is plotted as a

function of the pre-pulse duration.

shifted the values by 14.060.8 mV (P,0.01) and11.9860.45 mV (P,0.01), respectively. 10 s. At pre-pulse of 100 ms control currents were

inactivated 41.565.8% while 53.965.7% of currents were3.6. Effects of thimerosal on the inactivation rate of inactivated after thimerosal 100 mM treatment for 5 minsodium channels (P,0.001, n57) (Fig. 6B).

Effects of thimerosal on the time dependent inactivation 3.7. Effects of thimerosal on the recovery of sodiumof sodium channels are shown in Fig. 6. TTX-S sodium channels from inactivationcurrents started to inactivate 0.4 ms after the pre-pulsepotential change to 240 mV from a holding potential of TTX-S sodium channels were inactivated by 5 s de-2100 mV. The current amplitude then decreased exponen- polarizing step to 240 mV from a holding potential oftially and disappeared almost completely after 10-s pre- 2100 mV and then repolarized to 2100 mV for variouspulse. After treatment of thimerosal 1 mM for 5 min the duration followed by a step depolarization to 0 mV. Theinactivation rate increased compared to control. At pre- resultant current amplitude was plotted as a function of thepulse of 10 ms, 50.165.8% of sodium currents were repolarizing duration (Fig. 7A). TTX-S sodium currentsinactivated in control experiments while 72.262.9% of started to recover from inactivation after 1 ms repolariza-sodium currents were inactivated after thimerosal treatment tion and attained a maximum amplitude after 20 s repolari-(P,0.001, n58) (Fig. 6A). zation. Thimerosal at 1 mM for 5 min had a negligible

TTX-R sodium currents started to inactivate 2 ms after effect on the recovery time course.pre-pulse potential change to 220 mV from a holding For TTX-R sodium channels the inactivating potential ofpotential of 280 mV and were completely inactivated after 220 mV for 5 s from a holding potential of 280 mV and

J.-H. Song et al. / Brain Research 864 (2000) 105 –113 111

channel inactivation [18]. N-ethylmaleimide (NEM),which forms a covalent bond to cysteinyl sulfhydryl group,induced a slow sodium channel inactivation with theactivation process unaltered [24]. Nitrosylation of cysteinylsulfhydryl groups caused a reduction of TTX-S and TTX-R sodium current in rat nodose ganglia, and shifted thesteady-state sodium channel inactivation curve in thehyperpolarizing direction [16].

Thimerosal is a disinfectant and causes oxidation ofcysteinyl sulfhydryl groups leading to the formation ofdisulfide bonds between neighboring sulfhydryl groups.The present study showed that thimerosal depressed bothTTX-S and TTX-R sodium channels in rat DRG neuronsin a dose dependent manner. The inhibition of sodiumcurrent was rapid within 2–3 min after the application ofthimerosal, then slowed down but hardly reached a steadystate. Blockage of sodium current by thimerosal was notreversed by washout with drug-free external solution, butpartially reversed by DTT, a sulfhydryl reducing agent.This result strongly suggests that thimerosal depressed thesodium currents by oxidative mechanism. Since thimerosalis hydrophilic and impermeable to lipophilic cellularmembranes, thimerosal seems to act on the extracellularsurface of the sodium channel or pore region, where itoxidizes cysteines and renders structural or functionalchanges to the sodium channel protein.

TTX-S and TTX-R sodium channels showed a differen-tial sensitivity to thimerosal. At the holding potential of

Fig. 7. Effect of thimerosal on the recovery of sodium channels from 280 mV, which is near the resting membrane potential,inactivation. (A) TTX-S sodium channels. Pre-pulse was applied to 240 TTX-R sodium channels were approximately ten timesmV for 5 s from a holding potential of 2100 mV, and then membrane

more sensitive to the inhibitory action of thimerosal thanpotential was repolarized to 2100 mV for various duration which wasTTX-S sodium channels. At this holding potential almostfollowed by depolarizing steps to 0 mV (n55). (B) TTX-R sodiumall TTX-R sodium channels are relieved from inactivation,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 but a large portion of TTX-S sodium channels are in the280 mV for various duration which was followed by depolarizing steps inactivated state (Fig. 4). Thimerosal shifted the steady-to 0 mV (n55). The current amplitude is plotted as a function of the

state sodium channel inactivation curve in the hyper-repolarizing duration.polarizing direction. When the holding potential was 2100mV where almost all TTX-S sodium channels are relieved

repolarizing potential of 280 mV were used. TTX-R from inactivation, the degree of inhibition caused bysodium currents started to recover from inactivation after thimerosal became far less than that measured at the 28010 ms repolarization and attained a maximum amplitude mV holding potential. So the sensitivity difference betweenafter 30 s repolarization. Thimerosal at 100 mM for 5 min the two types of sodium channels becomes even greater ifslightly shifted the recovery time curve to right direction the comparison is made under the equal condition in termsbut the change was not significant (Fig. 7B). of the inactivation. The differential sensitivity was also

observed in the reversal of thimerosal effect by DTT. DTTat 1 mM reversed the effect of 1 mM thimerosal on TTX-S

4. Discussion sodium channels in terms of the current amplitude and thesteady-state inactivation curve more effectively than that of

Sodium channels are subject to modulation by oxidative 100 mM thimerosal on TTX-R sodium channels.or covalent modification of sulfhydryl containing amino The rate of the sodium channel inactivation was acceler-acids. Chloramine-T, an methionine oxidant, abolished the ated by thimerosal. Unlike the other effects of thimerosalsodium channel inactivation irreversibly and shifted the described above, this was more pronounced in TTX-S thansteady-state sodium current inactivation curve in the TTX-R sodium channels. However, thimerosal did notdepolarizing direction with no effect on the sodium affect the recovery rate of sodium channels from inactiva-channel activation [29,30]. Another methionine oxidant tion.cyanogen bromide, however, did not affect the sodium Thimerosal interfered with the activation process of the

112 J.-H. Song et al. / Brain Research 864 (2000) 105 –113

sodium current in SNS-null and wild-type small sensory neurons, J.sodium channels. Thus thimerosal shifted the activationNeurosci. 19 (RC43) (1999) 1–6.curves of both types of sodium channels in the depolariz- 21[8] T.J. DiChiara, P.H. Reinhart, Redox modulation of hslo Ca -

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[18] G.S. Oxford, C.H. Wu, T. Narahashi, Removal of sodium channelat a depolarized holding potential we used. Thus it isinactivation in squid giant axons by n-bromoacetamide, J. Gen.highly probable that the TTX-R sodium channel wePhysiol. 71 (1978) 227–247.

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