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Modulation of CaV2.3 Calcium Channel Currents by Eugenol
1 Department of Physiology, School of Dentistry and Dental Research Institute, Seoul National University, 28-2 Yeongeon-Dong Chongno-Ku, Seoul, 110-749, Korea; and Correspondence: * corresponding author, odolbae{at}snu.ac.kr
Eugenol, a natural congener of capsaicin, is a routine analgesic agent in dentistry. We have recently demonstrated the inhibition of CaV2.2 calcium channel and sodium channel currents to be molecular mechanisms underlying the analgesic effect of eugenol. We hypothesized that CaV2.3 channels are also modulated by eugenol and investigated its mode of action using the whole-cell patch-clamp technique in a heterologous expression system. Eugenol inhibited calcium currents in the E52 cell line, stably expressing the human CaV2.3 calcium channels, where TRPV1 is not endogenously expressed. The extent of current inhibition was not significantly different between naïve E52 cells and TRPV1-expressing E52 cells, suggesting no involvement of TRPV1. In contrast, TRPV1 activation is prerequisite for the inhibition of CaV2.3 calcium channels by capsaicin. The results indicate that eugenol has mechanisms distinct from those of capsaicin for modulating CaV2.3 channels. We suggest that inhibition of CaV2.3 channels by eugenol might contribute to its analgesic effect.
Key Words: eugenol • voltage-gated calcium channels • CaV2.3 • R-type calcium channels
Calcium influx through voltage-gated calcium channels, which are activated by depolarization of the neuronal membrane, plays a pivotal role in neurotransmitter release. There are three types of voltage-gated calcium channels that play a large part in the process: CaV2.1 (P/Q-type) channels, CaV2.2 (N-type) channels, and CaV2.3 (R-type) channels (Catterall et al., 2003). In particular, CaV2.3 channels are distributed in subsets of dorsal root ganglion neurons (Wilson et al., 2000), and have been suggested to contribute to pain transmission in primary sensory neurons (Saegusa et al., 2000). We have suggested that CaV2.3 might have some role in nociception in the trigeminal system as well, since it is preferentially expressed in small-diameter trigeminal ganglion neurons (Fang et al., 2007). Eugenol is a pungent liquid, derived from clove oil, that has been widely used in dental clinics as an analgesic and anti-inflammatory agent (Jorkjend and Skoglund, 1990; Markowitz et al., 1992). Eugenol shares a common chemical structure with capsaicin, a vanillyl-like moiety that is believed to be essential for capsaicin-like activity (Sterner and Szallasi, 1999; Szallasi and Blumberg, 1999). The use of eugenol as an analgesic agent is not very intuitive, since it is known as a ligand for transient receptor potential vanilloid 1 (TRPV1) (Yang et al., 2003), a channel believed to be involved in nociception (Koltzenburg, 2004). Recently, we have explained its analgesic effect in terms of its inhibition of N-type (CaV2.2) calcium channels (Lee et al., 2005) and sodium channels (Park et al., 2006). We hypothesized that eugenol would also inhibit CaV2.3 calcium channels. We tested the modulatory mechanism of eugenol on CaV2.3 calcium channels in a heterologous expression system and compared it with that of capsaicin.
Cell Culture and Transfection E52 cell lines, the HEK293 cell line stably expressing CaV2.3 calcium channels with subunit  1E–32B /β1B, were use throughout the experiment (Williams et al., 1994). Cells were grown in DMEM (Invitrogen, Carlsbad, CA, USA) with 5% fetal bovine serum (Invitrogen) plus penicillin G (100 U/mL, Invitrogen), streptomycin sulfate (100 µg/mL, Invitrogen), and geneticin (500 µg/ml, Invitrogen). One day before the recording, cells were transfected with plasmids containing the cDNAs for the TRPV1 channel with the use of a commercial transfection kit (Welfect EX-plus, Welgene, Daegu, Korea), according to the manufacturer’s instructions. Cells were re-plated onto poly-L-lysine-coated glass coverslips on the day of recording and were used within 8 hrs after being re-plated.
Reverse Transcriptase/Polymerase Chain-reaction (RT-PCR)
Electrophysiological Recordings
Drugs
Statistical Analysis
Relevance of E52 Cell Lines to the CaV2.3 Calcium Channels We first confirmed the relevance of the E52 cell line to the CaV2.3 channels using electrophysiology and the RT-PCR technique. Whole-cell recordings of Ca2+ currents, with 5 mM Ba2+ as the charge carrier (IBa), were performed with solutions without Na+ and K+ ions, to eliminate other voltage-gated currents, such as Na+ and K+ currents. Ni2+, a selective blocker of CaV2.3 calcium channels, was applied to confirm the appropriate expression of CaV2.3 in E52 cells. Consistent with a previous report (Wu et al., 1998), application of 100 µM of NiCl2 inhibited more than 80% of IBa recorded from the E52 cell line (Figs. 1A, 1B  ). The pore-forming subunit of CaV2.3 channels is 1E (Catterall et al., 2003). The result from the RT-PCR with the primer targeted for the 1E subunit showed the presence of CaV2.3 channels in the E52 cell line (Fig. 1C).
Eugenol Inhibited CaV2.3 Calcium Channel Currents in a TRPV1-independent Manner We examined the effect of eugenol on CaV2.3 calcium channels stably expressed in E52 cells. Whole-cell recordings of Ca2+ currents were performed as described above. Application of 1 mM eugenol inhibited calcium currents from the E52 cell line without changing membrane holding currents (Fig. 2A ; 56.4 ± 4.68%, n = 5). Application of 1 mM of eugenol to the E52 cell line transfected with TRPV1 also inhibited calcium currents similarly (Fig. 2B; 50.8 ± 9.36%, n = 4). The difference in the current inhibition by eugenol between TRPV1-expressing E52 cells and naïve E52 cells was not statistically significant (P > 0.05).
Capsaicin Inhibited CaV2.3 Calcium Channel Currents through TRPV1 To compare the modes of action between eugenol and capsaicin, we also examined the effect of capsaicin on CaV2.3 calcium channels stably expressed in E52 cells. Application of 500 nM capsaicin to TRPV1-expressing E52 cells elicited inward currents (Fig. 3Ba ). The amplitudes of calcium currents were difficult to measure during the period of inward currents, because of the increased membrane-holding currents and outward currents elicited by test pulses to 0 mV (see point 2 in Fig. 3B). However, we observed the inhibition of calcium currents prolonged even after TRPV1 was desensitized and membrane-holding currents returned to the basal level (see point 3 in Fig. 3B). Comparing the amplitudes of calcium currents between point 1 and point 3 (Fig. 3B), we found that capsaicin inhibited calcium currents in the cells transfected with TRPV1 (80 ± 15%, n = 7). However, capsaicin neither elicited an inward current nor inhibited calcium currents in naïve E52 cells (Fig. 3A; –1.00 ± 3.39%, n = 5), which has no endogenously expressed TRPV1 (see APPENDIX).
CaV2.3 calcium channel inhibition by eugenol and capsaicin is summarized in Fig. 4 .
Eugenol has various uses in dental clinics as an analgesic during and after surgical procedures. We have shown, in a recent series of studies, that its analgesic effects might be due to its inhibition of CaV2.2 calcium channels (Lee et al., 2005) and voltage-gated sodium channels (Park et al., 2006). In the present study, we further investigated whether eugenol would also modulate CaV2.3 calcium channels, and its mode of action. In these experiments with the E52 cell line, which stably expresses CaV2.3 channels, we found that eugenol inhibited CaV2.3 calcium channel currents. Since activation of TRPV1 is known to inhibit voltage-gated calcium channels (Bleakman et al., 1990), and eugenol is known to activate TRPV1 (Yang et al., 2003), we then compared the results of naïve E52 cells and E52 cells transiently expressed with TRPV1. The extent of CaV2.3 calcium current inhibition was not significantly different between naïve E52 cells and TRPV1-expressing E52 cells. This finding suggests that the activation of TRPV1 by eugenol might provoke a different cascade that does not inhibit calcium currents, and that the inhibition of calcium currents by eugenol is independent of TRPV1 activation. Unlike eugenol, capsaicin inhibited CaV2.3 calcium currents only in TRPV1-expressing E52 cells, in agreement with our previously reported study on CaV2.2 channels (Lee et al., 2005). This implies that, although eugenol and capsaicin are alike in chemical structure, and both act as ligands for TRPV1 (Sterner and Szallasi, 1999; Szallasi and Blumberg, 1999), the mechanisms by which they interact with TRPV1 or CaV2.3 calcium channels might be different. In agreement with our results, capsaicin reduced high-voltage-activated calcium channel currents of rat dorsal root ganglion neurons through the mediation of capsaicin receptor activation (Bleakman et al., 1990), which indicates the involvement of TRPV1 in the inhibitory effects of capsaicin on calcium channels. We have also demonstrated the involvement of TRPV1 in the inhibition of calcium channels by capsaicin in trigeminal ganglion neurons (Kim et al., 2006). It is interesting to note that 2 vanilloid compounds—capsaicin and eugenol—which share similar chemical structures and contain the vanillyl-moiety, have different molecular mechanisms for their inhibitory effects on CaV2.3. Our results suggest that the vanillyl-moiety, which is known to be important for capsaicin-like action of vanilloid compound (Sterner and Szallasi, 1999), might not determine the modulation of calcium channels by vanilloid compounds, including capsaicin and eugenol. In our study, we used BAPTA as the Ca2+ chelating agent to eliminate the participation of calcium-mediated processes inside the cells. We found that eugenol and capsaicin still inhibited CaV2.3 channels in this condition. However, it has been demonstrated that BAPTA abolishes the inhibitory effects of TRPV1 on voltage-gated calcium channels in dorsal root ganglion neurons (Wu et al., 2005; 2006), which is not consistent with our observation. According to those reports, activation of TRPV1 by capsaicin inhibited calcium currents through Ca2+-dependent calcineurin. We have provided several instances arguing against the roles of intracellular calcium for calcium channel inhibition in trigeminal ganglion neurons and heterologously expressed systems (Kim et al., 2006). Although the reason for the discrepancies between results is not clear at present, direct gating of calcium channels by TRPV1 or a local increase in calcium near TRPV1 and calcium channels might be more important in our system (Kim et al., 2006). In line with the distinctive modes of action produced by eugenol and capsaicin, it is also interesting to note that activation of TRPV1 by eugenol did not elicit inward currents with the sodium-free extracellular solution, whereas it did with sodium-containing extracellular solution (Yang et al., 2003). In contrast, capsaicin elicited inward currents in TRPV1-expresssing cells, regardless of the presence of the sodium ion in extracellular solution. Given that TRPV1 is known as a non-selective cation channel, it is yet to be elucidated why eugenol and capsaicin produce different response patterns. It is possible that the interaction of the TRPV1 with a specific ligand might contribute to the differential ion permeability of the channel. However, further work is required to determine if this is the case. Altogether, eugenol inhibited CaV2.3 channels, and the inhibition was not dependent on the activation of TRPV1. Due to the lack of TRPV1 involvement in its inhibitory effects, an analogue of eugenol might be developed to substitute for capsaicin as a potential analgesic drug without an irritating burning sensation.
This research was supported by a grant (R01-2004-000-10384-0) from the Basic Research Program of the Korea Science & Engineering Foundation and a grant (M103KV010015-06K2201-01510) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea. We thank Dr. Miller (Northwestern University, Chicago, IL, USA) for the kind gift of E52 cell lines.
+ authors contributing equally to this work  A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/2/137/DC1. Received for publication July 20, 2007. Revision received October 14, 2007. Accepted for publication October 20, 2007.
Journal of Dental Research, Vol. 87, No. 2,
137-141 (2008)
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ABSTRACT
INTRODUCTION
. We compensated for series resistance and whole-cell capacitance. Data were low-pass-filtered at 2 kHz, sampled at 10 kHz, and collected by rClamp (custom-made patch clamp program using Delphi 5.0, Borland, Inc., Austin, TX, USA). Continuous chart data were recorded with iWorx 118 (Harvard Instrument, Dover, NH, USA) and LabScribe software (Harvard Instrument) installed in a different computer. The pipette solution for IBa contained (in mM): CsCl, 100; MgCl2, 1; HEPES, 10; BAPTA, 10; Mg-ATP, 3.6; phosphocreatine, 14; GTP, 0.1; and creatine phosphokinase, 50 units/mL; pH was adjusted to 7.4 by CsOH. The bath solution for IBa contained (in mM): tetraethylammonium chloride (TEACl), 151; HEPES, 10; BaCl2, 5; MgCl2, 1; and glucose, 10; pH was adjusted to 7.4 with TEAOH. Test pulses to 0 mV from a holding potential of –80 mV were delivered every 10 sec to evoke IBa. 
