Thimerosal decreases TRPV1 activity by oxidation of extracellular sulfhydryl residues

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1 Neuroscience Letters 369 (2004) Thimerosal decreases TRPV1 activity by oxidation of extracellular sulfhydryl residues Yunju Jin a, Dong Kwan Kim a, Lee-Yong Khil b, Uhtaek Oh c, Jun Kim a, Jiyeon Kwak d, a Department of Physiology and Biophysics, Seoul National University College of Medicine, Seoul , South Korea b Department of Hygienic Chemistry, College of Pharmacy, Seoul National University, Seoul , South Korea c Sensory Research Center, National Creative Research Initiatives, College of Pharmacy, Seoul National University, Seoul , South Korea d Department of Physiology and Biophysics, Inha University College of Medicine, 253 Yonghyun-dong, Nam-gu, Incheon , South Korea Received 24 May 2004; received in revised form 10 July 2004; accepted 27 July 2004 Abstract TRPV1, a receptor for capsaicin, plays a key role in mediating thermal and inflammatory pain. Because the modulation of ion channels by the cellular redox state is a significant determinant of channel function, we investigated the effects of sulfhydryl modification on the activity of TRPV1. Thimerosal, which oxidizes sulfhydryls, blocked the capsaicin-activated inward current (I cap ) in cultured sensory neurons, in a reversible and dose-dependent manner, which was prevented by the co-application of the reducing agent, dithiothreitol. Among the three cysteine residues of TRPV1 that are exposed to the extracellular space, the oxidation-induced effect of thimerosal on I cap was blocked only by a point mutation at Cys621. These results suggest that the modification of an extracellular thiol group can alter the activity of TRPV1. Consequently, we propose that such a modulation of the redox state might regulate the physiological activity of TRPV Elsevier Ireland Ltd. All rights reserved. Keywords: Thimerosal; Sulfhydryl oxidation; Capsaicin; TRPV1; Dorsal root ganglion Capsaicin, the main pungent ingredient of hot peppers, produces burning pain and neurogenic inflammation through the excitation of small sensory neurons [3,25]. In cultured dorsal root ganglion (DRG) neurons, capsaicin activates a ligandgated, non-selective cation channel [19]. A cdna that encodes a channel that is activated by capsaicin was cloned recently, and was classified as TRPV1 [4,18]. The TRPV1 channel has properties that resemble those of the capsaicinactivated channel that is present in sensory neurons. In addition, TRPV1 is activated by heat and extracellular acid [4,27], and the lipid metabolic products of lipoxygenases and anandamide activate TRPV1 [13,23,31]. TRPV1-deficient mice exhibit reduced inflammatory thermal hyperalgesia, so TRPV1 appears to be essential for mediating thermal hyperalgesia that is induced by inflammation [5,6]. During inflammation or reperfusion injury, reactive oxygen species are generated, which affects the redox state of tissues [11]. Among the many amino acids that are present Corresponding author. Tel.: ; fax: address: kwak1014@inha.ac.kr (J. Kwak). within biologically active proteins, cysteine residues are reactive to the cellular redox state. Thus, redox modification of cysteinyl sulfhydryl (SH) groups due to a change in the redox state constitute an important mechanism for regulating cellular function. Oxidation of cysteine residues modulates the activity of the channels in N-methyl-d-aspartate receptors [2], GABA A receptors [1], ATP-sensitive K + channels [20], large conductance Ca 2+ -activated K + channels [7], and voltage-dependent Ca 2+ [17,26] and Na + channels [24]. Recently, intradermal injection of a reducing agent, dithiothreitol (DTT), was reported to induce thermal hyperalgesia, an effect that could be blocked by co-injection of an oxidizing agent, 5,5 -dithio-bis-(2-nitrobenzoic acid) (DTNB) [26]. These results suggested that changes in the redox state in peripheral tissues may influence the activity of ion channels that are expressed in sensory neurons. Because TRPV1 has been implicated in mediating inflammatory pain [5,6], it is possible that changes in the redox state in peripheral tissues modulate the activity of TRPV1. In the present study, we examined the effects of a sulfhydryl-oxidizing agent, thimerosal, on TRPV1-dependent currents in cultured DRG neurons. In ad /$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved. doi: /j.neulet

2 Y. Jin et al. / Neuroscience Letters 369 (2004) dition, using mutagenesis, we determined which amino acid is responsible for the redox modulation. DRG neurons that had been isolated from 2-day-old rats were used to record whole cell currents, as described previously [19]. Briefly, DRG from all levels of the spinal cord of neonatal rats was collected into cold culture medium that consisted of a 50:50 mixture of Dulbecco s modified Eagle s medium and Ham s F-12 solution (DMEM/F-12; Life Technologies, Grand Island, NY, USA), which contained 10% fetal bovine serum (FBS, Life Technologies), 1 mm sodium pyruvate (Sigma, St. Louis, MO, USA), and 100 U/ml streptomycin/penicillin (Sigma). Ganglia were incubated for 30 min at 37 C with 1 mg/ml collagenase (type II, Worthington, Freehold, NJ, USA), followed by a 30 min incubation with 2.5 mg/ml trypsin (Life Technologies). Dissociated cells were plated on coverslips that had been pretreated with 0.04 mg/ml polyethylenimine (Sigma) and nerve growth factor (Life Technologies) (25 ng/ml) was added. DRG neurons were incubated at 37 C in 95% air and 5% CO 2, and were used 1 3 days after plating. Mutations of each cysteine residue of rat TRPV1 were introduced into pcdna3.1-trpv1 with overlapping mutagenic primers using a site-directed mutagenesis kit (Quick Change TM ; Stratagene, La Jolla, CA, USA). Mutations were verified by sequencing the mutation sites. Wild-type and mutant TRPV1 were then transfected into HEK 293T cells (Lipofectamine TM 2000, Invitrogen/Life Technologies). HEK293 cells were maintained in modified Eagle s medium (MEM, Life Technologies), supplemented with 10% FBS and 100 U/ml penicillin/streptomycin, and were used 1 3 days after plating. Recordings were made from small DRG neurons in the whole-cell configuration at a holding potential of 60 mv. The pipette resistance was 5M. To record whole-cell currents, gigaseals were formed first, after which the membrane was ruptured by gentle suction. After a whole-cell configuration had been achieved, capacitative transients were canceled. Whole-cell currents were recorded with an EPC 7 amplifier (HEKA, Lambrecht, Germany) that was connected to an IBM-compatible computer via a TL-1 interface. In most cases, data were filtered at 3 khz using a low-pass Bessel filter. Recordings were acquired at a sample frequency of 1 khz. Data were analyzed using pclamp 6 (Axon Instruments, Union City, CA, USA). Electrophysiological experiments were performed at room temperature. All experimental solutions were adjusted to ph 7.4. The Ca 2+ -free extracellular solution (ECS) contained (in mm): 140 NaCl, 5 KCl, 1 MgCl 2, 10 HEPES, 20 glucose, and 10 EGTA. The pipette solution contained (in mm): 110 KCl, 2 MgCl 2, 1 CaCl 2, 10 HEPES, 11 BAPTA-K 3, 2 Mg-ATP, and 0.1 Na-GTP. Capsaicin was dissolved in absolute ethanol, and was stored as a 10 mm stock solution. Thimerosal was dissolved in water, and was stored as 100 mm and 1 M stock solutions, which were diluted freshly to appropriate concentrations at the time of an experiment. DTT, the reduced form glutathione (GSH), hydrogen peroxide (H 2 O 2 ), DTNB, and the oxidized form glutathione (GSSG) were prepared in the solution at an appropriate concentration immediately before use. CuCl 2 was dissolved in H 2 O (100 mm stock solution) and mixed with 1, 10-o-phenanthroline (300 mm stock solution in methanol) in a molar ration of 1:3. All chemicals were obtained from Sigma. All values are expressed as mean ± S.E.M. Paired Student s t-tests were performed for statistical comparisons. P < 0.05 was considered significant. Fig. 1A shows that extracellular application of capsaicin (0.3 M) for 20 s evoked inward currents in whole cells at a holding potential of 60 mv. Co-application of thimerosal (100 M) and capsaicin (0.3 M) decreased I cap to 24.3 ± 5.3% (n = 17) of the control response at a holding potential of 60 mv (Fig. 1A). The inhibitory effect of thimerosal on I cap was spontaneously reversible, as indicated by the observation that I cap recovered to 56.5 ± 8.5% of the control response after thimerosal had been washed out. The effect of thimerosal was concentration-dependent: as shown in Fig. 1B, when the concentration of thimerosal was increased from 0.3 to 300 M, the magnitude of the inhibition increased progressively, with a half-maximal inhibition concentration of 75.3 M. Because capsaicin-activated currents (I cap ) are desensitized in a Ca 2+ - dependent manner by repetitive applications of capsaicin [15], we used Ca 2+ -free ECS, and a pipette solution that contained 11 mm BAPTA, throughout the experiments to avoid tachyphylaxis. However, thimerosal seemed to show some inhibitory effect at the minimal concentration because weak tachyphylaxis still remained in our experimental condition. Voltage ramps from 80 to +80 mv were applied to cells (n = 8) for 1 s to determine whether thimerosal (100 M) affected the current voltage relationship. As shown in Fig. 1C, thimerosal blocked I cap at both negative and positive membrane potentials and the reversal potential was significantly shifted from 0.35 ± 1.8 to 8.61 ± 4.2 mv (P < 0.05). Thimerosal is endowed with an oxidative character by Hg 2+ [9]. Heavy metals such as Hg 2+ oxidize thiol groups and chelate with dibasic amino acids. Therefore, we suspected that the inhibitory effect of thimerosal on I cap (see above) might be the result of oxidation of the thiol groups that are present in TRPV1. To determine whether the inhibitory effect of thimerosal on I cap was caused by the oxidation of free thiol groups in TRPV1, we tested the effect of Hg 2+ and other sulfhydryl-specific oxidizing agents, including DTNB, GSSG, and H 2 O 2. As shown in Fig. 1D, application of Hg 2+ (1 mm), DTNB (0.5 mm), GSSG (0.5 mm), or H 2 O 2 (3 mm) significantly reduced I cap to 49.1 ± 4.9% (n =7,P < 0.01), 45.6 ± 4.2% (n = 12, P < 0.01), 49.1 ± 10.1% (n =4,P < 0.05), and 37.0 ± 4.2% (n =4,P < 0.01) of the control response, respectively. In addition, catalyst of the formation of disulfide linkage, copper-o-phenanthroline (Cu:Phe, 1:3) also decreased I cap to 41.2 ± 8.6% of the control (n =5, P < 0.01). These results suggest that thimerosal and other oxidizing agents probably inhibit I cap by oxidizing the free sulfhydryl groups of TRPV1. To determine whether the inhibitory effect of thimerosal on I cap was due to the oxidation of TRPV1, we firstly con-

3 252 Y. Jin et al. / Neuroscience Letters 369 (2004) Fig. 1. Effects of thimerosal, sulfhydryl-oxidizing agent, on I cap in cultured DRG neurons. (A) Co-application of thimerosal (TM, 100 M) and capsaicin (CAP, 0.3 M) to the bath solution caused a significant decrease in I cap (n = 17, P < 0.01). Bars over the representative trace indicate the application of capsaicin. (B) Thimerosal inhibited I cap in a concentration-dependent manner. Data are the mean ± S.E.M. reduction (%) in I cap by thimerosal at various concentrations ( M) of thimerosal in 3 17 experiments. (C) Current voltage relationship of I cap in the absence and presence of 100 M thimerosal. The basal currents in ECS were subtracted. The inset shows the I V relationship of I cap in HEK 293 T cells transfected wild-type TRPV1 (a: capsaicin, b: capsaicin + thimerosal). (D) Summary of the effects on I cap of various sulfhydryl-oxidizing agents, including Hg 2+, DTNB, GSSG, H 2 O 2, and Cu:Phe. Values at the top of each bar represent the number of experiments. Bars represent the mean ± S.E.M. Asterisks indicate a significant difference from the control response to capsaicin (P < 0.01). firmed the effect of DTT (a reducing agent) on I cap. Consistent with a previous report [29], co-application of DTT (50 mm) and capsaicin (0.3 M) in the ECS enhanced I cap reversibly to 231 ± 26.3% of the control response (Fig. 2A, n = 4) and extracellular mannitol up to 50 mm did not augment I cap in the DRG neurons (Fig. 2C). The enhancement of I cap by DTT was apparent only at DTT concentrations that were greater than 10 mm (data not shown). Pretreatment with DTT, at a concentration that did not affect I cap (10 mm), blocked the inhibitory effect of thimerosal on I cap (Fig. 2B). These results indicate that the sulfhydryl reducing agent, DTT, protected free sulfhydryl groups in TRPV1 from the thimerosal-induced oxidation. Because thimerosal is thought to be relatively impermeable to the cell membrane, we predicted that thimerosal acted on TRPV1 at the extracellular surface of the cell. However, we could not exclude the possibility that small amounts of thimerosal might cross the cell membrane at physiological ph to act on TRPV1 from within the cell. Consequently, to determine whether thimerosal could act within the cell, we assessed whether including 4 mm GSH in the pipette solution would block the inhibitory effect of thimerosal on I cap. Despite the presence of GSH within the cell, the reduction of I cap by thimerosal (100 M) persisted (31.7 ± 3.4% of the control response, n = 6, Fig. 2D), indicating that the inhibitory effect of thimerosal on I cap was due to the oxidation of free sulfhydryl groups on the extracellular surface of TRPV1. As shown in Fig. 3A, TRPV1 has three extracellular cysteine residues (at positions 616, 621, and 634) in its primary sequence. Cys634 is located in the putative p-loop region of TRPV1, and the other two cysteines are located in the linker that connects the S5 segment to the p-loop. To determine which of the cysteine residues in TRPV1 was involved in the modulatory effect of thimerosal on I cap, we mutated each of the three cysteine residues to a serine or alanine residue by site-directed mutagenesis. Wild-type or mutant TRPV1 was heterologously expressed in HEK293 cells after transient transfection. As shown in Fig. 3B, 100 M thimerosal reduced I cap to 61.5 ± 4.6% of the control response (n =5) in HEK293 cells that expressed wild-type TRPV1. These results suggested that the site of oxidation by thimerosal was located within TRPV1 itself, rather than on an auxiliary subunit. In addition, I V relationship of wild type TRPV1 was not different from that of native channels in DRG neurons (Fig. 1C, inset). In HEK293 cells that expressed the C634S mutant, application of 0.3 M capsaicin produced an inward current. In these cells, co-application of 100 M thimerosal and capsaicin significantly decreased I cap to 53.0 ± 10.7% of the control response (n =5,P < 0.05). Similarly, in cells

4 Y. Jin et al. / Neuroscience Letters 369 (2004) Fig. 2. DTT enhanced I cap reversibly and prevented the thimerosal-induced decrease in I cap in cultured DRG neurons. (A) Co-application of DTT (50 mm) and capsaicin (0.3 M) to the bath solution enhanced I cap reversibly. The bar chart summarizes the effects of DTT on I cap (n =4)at a holding potential of 60 mv. Bars represent the mean ± S.E.M. Asterisks indicate a significant difference from the first bar (P < 0.01). (B) Upper traces are representative current traces illustrating how pretreatment with DTT prevented the reduction of I cap by thimerosal. Bar graph summarizes the relative responses to thimerosal (1, 100 M) in the presence (filled bar) and absence (open bar) of 10 mm DTT. Bars represent the mean ± S.E.M. Asterisks indicate a significant difference from the adjacent open bar (P < 0.01). (C) Mannitol (10 mm) added to ECS did not significantly change I cap. (D) The bar chart summarizes the effects of intracellular and extracellular GSH on I cap at a holding potential of 60 mv. Values at the top of each bar represent the number of experiments. Bars represent the mean ± S.E.M. that expressed the C616S mutant, thimerosal caused a significant reduction in I cap to 82.4 ± 3.2% of the control response (n =6,P < 0.01), although the magnitude of the reduction was smaller than the reduction that occurred in cells that expressed wild-type TRPV1 or the C634S mutant. By contrast, thimerosal failed to reduce I cap in cells that expressed the C621A mutant (I cap = 97.5 ± 2.1% of the control response in the presence of thimerosal; n = 7). These results suggest that thimerosal acted primarily on the Cys621 residue that is located in the external aperture of the channel pore that is formed by TRPV1. The kinetics of I cap in cells that expressed the C621A mutant differed from those of cells that expressed wild-type TRPV1. As shown in the representative trace in Fig. 3B, C621A mutant TRPV1 was activated rapidly by capsaicin, and closed slowly, relative to cells that expressed wild-type TRPV1 or the native capsaicin channels in DRG neurons. The average opening (τ O ) and closing (τ C ) time constants for the wild-type TRPV1 were 19.3 ± 2.6 and 21.9 ± 2.8 s (n = 10), respectively, which were not significantly different to those of the native capsaicin channel in DRG neurons (15.9 ± 1.0 and 16.1 ± 1.0 s for τ O and τ C, respectively; n = 5). However, the activation of C621A mutant TRPV1 was rapid: τ O decreased significantly to 4.6 ± 0.7 s (n = 10, P < 0.01). The closure of the C621A mutant TRPV1 channel was too slow to determine τ C. These results implicated Cys621 in modulating the gating properties of TRPV1. In the present study, we investigated whether changes in redox state would modulate the activity of TRPV1 channels in response to capsaicin. The oxidation of TRPV1 greatly reduced the response of TRPV1 to capsaicin. By contrast, application of the reducing agent, DTT, augmented the response of TRPV1 to capsaicin. In addition, we determined that the Cys621 residue is the SH group on the extracellular surface of TRPV1 that is targeted for modulation in response to a change in redox state. Because the activity of TRPV1 in sensory neurons was down-regulated by oxidizing agents, it is likely that a portion of each TRPV1 channel remains in a reduced state (which would produce the highest probability that the channels would open) until the redox state of the local environment is altered by pathological changes, such as ischemia or inflammation. Our study was focused on the effects of oxidation of cysteines on the extracellular side of TRPV1, because thimerosal has limited membrane permeability and reacts specifically with thiol groups through addition of a mercury ethyl group ( C 2 H 5 Hg) to free sulfhydryl group of cysteine residues [9]. Although thimerosal seems to be not a good oxidizing agent to imitate the oxidative stress that naturally happens, the down regulation of I cap could reflect possible change in TRPV1 activity by oxidative stress during ischemia and inflammation because H 2 O 2 that is more physiological oxidant and other various oxidizing agents showed the same decreasing effects on the TRPV1 activity and their effects were prevented in the presence of DTT. The present study also revealed that the effects of thimerosal and DTT on I cap could be reversed spontaneously in cultured DRG neurons. It has been reported that the facilitatory effects of DTT on I heat and T-type Ca 2+ currents in DRG neurons recovered to a control level after the washout of DTT [26,29]. Such a spontaneous recovery from agents that modify sulfhydryl groups implies that there might be endogenous

5 254 Y. Jin et al. / Neuroscience Letters 369 (2004) Fig. 3. Mutation of Cys621 blocked the thimerosal effect on I cap in transfected HEK 293T cells. (A) Predicted membrane topology of rat VR1 (TRPV1). Cysteine residues that are likely exposed to extracellular environment are indicated. (B) Alignment of sequences in the vicinity of p-loop region of TRPV1 from rat (rtrpv1), chicken (ctrpv1), rabbit (otrpv1), guinea pig (gtrpv1) and human (htrpv1). Gray box indicated conserved or identical residues. (C) Wild-type TRPV1 and mutant channels were expressed in HEK293 cells. Co-application of thimerosal (100 M) and capsaicin (0.3 M) decreased I cap in HEK293 cells expressing wild-type channels and C634S mutant channels. Mutation of Cys621 abolished the inhibitory effects of thimerosal on I cap. (D) Summary of the effects of thimerosal on I cap in HEK293 cells expressing wild-type or mutant channels. Values at the top of each bar represent the number of experiments. Bars represent the mean ± S.E.M. Asterisks indicate a significant difference from the control value (P < 0.01). factors that regulate the redox state of the sulfhydryl groups of proteins in sensory neurons. The membrane-impermeant nature of thimerosal suggested that an oxidation site was probably located on the external surface of TRPV1, rather than on the intracellular surface. Rat TRPV1 has three extracellular cysteine residues that are located at positions 616, 621, and 634 between transmembrane domains 5 and 6. Among these cysteine residues, Cys634 is conserved in TRPV1 of several species, including rat [4], chicken [14], rabbit [8], guinea pig [21], and human [12] (Fig. 3B). Therefore, Cys634 is a candidate target for oxidative modulation by thimerosal. However, replacement of Cys634 by a serine residue in the present study failed to abolish the inhibitory effect of thimerosal on I cap. This result is probably due to the size of thimerosal: thimerosal is thought to be larger than methanethionsulfonate (MTS) reagents because it has an aromatic ring within its structure. As cysteinyl residues that were located 20% across the electrical field from the outside of the cell could not be modified by MTS reagents [30], it is likely that Cys634 may be located too deep within the channel pore to be accessible to thimerosal. The inhibitory effect on I cap of the single divalent cation, Hg 2+, was completely abolished in the C634S mutant (unpublished data). Therefore, we could not exclude the possibility that Cys634 is also a target for oxidation in vivo, but it is more likely that the remaining TRPV1 cysteine residues, Cys621 and Cys616, are the sites of oxidation by thimerosal. Unlike Cys634, Cys621 and Cys616 are not conserved in TRPV1 in other species. However, except in rat, other mammalian TRPV1 sequences have a cysteine residue at the 620 position (Fig. 3B). Therefore, this cysteine residue, which is located in the linker between the S5 region and the p-loop of mammalian TRPV1, would appear to be a likely target for the oxidative effects of thimerosal. Indeed, our results revealed that only the mutation of Cys621 could completely abolish the thimerosal-induced decrease in I cap. Based on these results, we surmise that Cys621 in rat TRPV1 may be crucial for the modulation of I cap by thimerosal. Recently, Tousova et al. [28] reported that Cu:Phe, catalyst of disulfide bond formation blocked the TRPV1 in transfected HEK 293T cells and in cultured DRG neurons. However, they suggested that Cu:Phe inhibited TRPV1 not by oxidation of extracellular cysteine residues but by plugging into the extracellular mouth of the channel because mutations of cysteines did not abolish the effects of Cu:Phe. We also found that Cu:Phe retained its blocking effect in the presence of DTT in DRG neurons (data not shown). Taken together these results, it is suggested that mechanism involved in Cu:Phe-induced blockade of I cap may be different from that of thimerosal action. In addition, we found that mutation of Cys621 resulted both in a significant increase in the rate of activation of I cap (Fig. 3B) and a

6 Y. Jin et al. / Neuroscience Letters 369 (2004) blockade of oxidative modification. It has been reported that the S5-P-S6 region is a basic pore module that governs key properties of ion permeation [22]. Amino acid residues such as Cys621 that are located at the extracellular end of the S5 and S6 segments (close to the membrane water interface) could modulate the attributes of the pore [10,16]. Thus, it is conceivable that Cys621, which is located in the linker between the S5 region and p-loop of TRPV1, may regulate the conductance or gating properties of the channel, as well as being the site of oxidative modulation. In summary, because TRPV1 has been implicated in the transmission of pain, and serves as a sensor for multimodal noxious stimuli [5,16,23], the modulation of I cap by extracellular oxidizing agents reported here will contribute to revealing the mechanisms that regulate the activity of TRPV1 during pathologic conditions such as inflammation and ischemia. 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