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. Author manuscript; available in PMC: 2007 Oct 1.
Published in final edited form as: Pain. 2006 Sep 7;127(1-2):73–83. doi: 10.1016/j.pain.2006.08.002

Preferential block of inactivation-deficient Na+ currents by capsaicin reveals a non-TRPV1 receptor within the Na+ channel

Sho-Ya Wang a, Jane Mitchell b, Ging Kuo Wang b,*
PMCID: PMC1995448  NIHMSID: NIHMS23375  PMID: 16962240

Abstract

Capsaicin elicits burning pain via the activation of the vanilloid receptor (TRPV1). Intriguingly, several reports showed that capsaicin also inhibits Na+ currents but the mechanisms remain unclear. To explore this non-TRPV1 action we applied capsaicin to HEK293 cells stably expressing inactivation-deficient rat skeletal muscle Na+ mutant channels (rNav1.4-WCW). Capsaicin elicited a conspicuous time-dependent block of inactivation-deficient Na+ currents. The 50% inhibitory concentration (IC50) of capsaicin for open Na+ channels at +30 mV was measured 6.8 ± 0.6 μM (n = 5), a value that is 10–30 times lower than those for resting (218 μM) and inactivated (74 μM) wild-type Na+ channels. On-rate and off-rate constants for capsaicin open-channel block at +30 mV were estimated to be 6.37 μM−1 s−1 and 34.4 s−1, respectively, with a calculated dissociation constant (KD) of 5.4 μM. Capsaicin at 30 μM produced ~70% additional use-dependent block of remaining rNav1.4-WCW Na+ currents during repetitive pulses at 1 Hz. Site-directed mutagenesis showed that the local anesthetic receptor was not responsible for the capsaicin block of the inactivation-deficient Na+ channel. Interestingly, capsaicin elicited little time-dependent block of batrachotoxin-modified rNav1.4-WCW Na+ currents, indicating that batrachotoxin prevents capsaicin binding. Finally, neuronal open Na+ channels endogenously expressed in GH3 cells were as sensitive to capsaicin block as rNav1.4 counterparts. We conclude that capsaicin preferentially blocks persistent late Na+ currents, probably via a receptor that overlaps the batrachotoxin receptor but not the local anesthetic receptor. Drugs that target such a non-TRPV1 receptor could be beneficial for patients with neuropathic pain.

Keywords: Capsaicin, Voltage-gated sodium channel, Open-channel block, Analgesia, Use-dependent block

1. Introduction

Capsaicin, a pungent ingredient of the hot pepper, has become an important tool in pain research. This drug at sub-micromolar concentrations specifically activates the vanilloid receptor (transient receptor potential V1 channel or TRPV1; Caterina et al., 1997). However, capsaicin at micro- to millimolar concentrations apparently modulates a wide variety of ion channels (for the list see Lundbaek et al., 2005). Interestingly, repeated topical applications of capsaicin cream (0.025–0.075%; or 82–246 μM) achieve modest pain relief in most patients with postherpetic neuralgia, postmastectomy pain, and diabetic neuropathy (Szallasi and Blumberg, 1999). The underlying mechanism for the efficacy of capsaicin in pain relief appears primarily due to TRPV1-mediated calcium influx, desensitization of nociceptive neurons, and “die-back” of nociceptive endings (Cherny and Portenoy, 1999; Sawynok, 2003).

Earlier reports showed that capsaicin at 1% applied onto the peripheral nerve reduced C-fiber afferent volleys (Petsche et al., 1983; Chung et al., 1985). Although axonal membranes of unmyelinated fibers may contain TRPV1 receptors (Bernardini et al., 2004), voltage-clamp experiments showed that capsaicin directly inhibits Na+ currents in specific types of ganglion neurons (Su et al., 1999; Liu et al., 2001). These findings implied that capsaicin may block action potentials through the Na+ channel route. Recently, Lundbaek et al. (2005) showed that capsaicin at 30 μM blocked 12% and 37%, respectively, of resting and inactivated rat skeletal muscle α-subunit Na+ channels (rNav1.4) expressed in HEK293 cells. Because of capsaicin’s general effects on many ion channels and on lipid bilayer stiffness, these authors concluded that the weak inhibition of rNav1.4 Na+ currents by capsaicin is due to changes in membrane elasticity.

The purpose of this report is to determine the state-dependent binding of capsaicin and its antagonist capsazepine in voltage-gated Na+ channels. Following the experiments of Lundbaek et al. (2005) and the modeling work of Lipkind and Fozzard (2005) we chose the rNav1.4 skeletal muscle Na+ channels to study the blocking action of capsaicin and capsazepine. The chemical structures of these drugs are shown in Fig. 1 along with the local anesthetic lidocaine. After initial characterizations, we focused our study on the open-channel block of capsaicin using inactivation-deficient Na+ channels since these channels generated large persistent late Na+ currents (Wang et al., 2003b; Wang et al., 2004). In addition, we used site-directed mutagenesis to determine whether the binding site for capsaicin overlaps with the local anesthetic receptor within the Na+ channel. Finally, we also examined the block of capsaicin in neuronal Na+ channels in rat pituitary GH3 cells. These cultured cells were reported to express Nav1.1, 1.2. 1.3, and 1.6 Na+ channel isoforms endogenously (Vega et al., 2003). Instead of using site-directed mutagenesis, we impaired neuronal Na+ channel fast inactivation by two conventional techniques (chloramine-T and sea anemone toxin). Our results suggest that a non-TRPV1 receptor is present within the rNav1.4 and neuronal Na+ channel isoforms. This capsaicin receptor in the Na+ channel may become a therapeutic target for the treatment of neuropathic pain.

Fig. 1.

Fig. 1

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Chemical structures of capsaicin, capsazepine, and lidocaine. Capsaicin and capsazepine are neutral drugs. Lidocaine has a tertiary amine, which is protonated in physiological solution.

2. Materials and methods

2.1. Cultures of GH3 cells and HEK293 cells stably expressing rNav1.4 wild-type and inactivation-deficient Na+ channels

Rat pituitary GH3 cells were purchased from American Type Culture Collection (Manassas, VA). Human embryonic kidney (HEK293) cell lines stably expressing rNav1.4 wild-type Na+ channels and inactivation-deficient Na+ channels (rNav1.4-L435W/L437C/A438W; or the WCW mutant) were reestablished from frozen vials as described (Wang et al., 2004). Cultured HEK293 cells and GH3 cells were maintained at 37 °C in a 5% CO2 incubator in DMEM (Life Technologies, Rockville, MD) containing 10% fetal bovine serum (HyClone, Logan, UT) and 1% penicillin and streptomycin solution (Sigma, St. Louis, MO).

2.2. Transient transfection of HEK293t cells with S6 mutants in the WCW cDNA construct

We created additional S6 mutant channels in the WCW cDNA background by site-directed mutagenesis as described (Wang et al., 2004). Among these mutant channels are WCW-F1579A, WCW-F1579K, WCW-L1280K, and WCW-N434K. These specific residues in rNav1.4 channels (F1579, L1280, and N434) are located in the middle of S6 segments and appear critical for local anesthetic binding (Nau and Wang, 2004). For transient transfection HEK293t cells were grown to ~50% confluence in DMEM (Life Technologies, Inc., Rockville, MD) containing 10% fetal bovine serum (HyClone, Logan, UT), 1% penicillin and streptomycin solution (Sigma, St. Louis, MO), 3 mM taurine, and 25 mM HEPES (Life Technologies, Inc.) and transfected by calcium phosphate precipitation. Transfection of mutant channels (5–10 μg) along with rat β1-pcDNA1 (10–20 μg) and reporter CD8-pih3m (1 μg) was adequate for current recording. Rat β1 subunit was included to increase the level of the channel expression. Control experiments indicated that coexpression of the β1 subunit does not affect the capsaicin binding affinities. Cells were re-plated 15 h after transfection in 35-mm dishes, maintained at 37 °C in a 5% CO2 incubator, and used after 1–4 days. Transfection-positive cells were identified with immunobeads (CD8-Dynabeads, Lake Success, NY).

2.3. Solutions and chemicals

Cells were perfused with an extracellular solution containing (in mM) 65 NaCl, 85 choline-Cl, 2 CaCl2, and 10 HEPES (titrated with tetramethylammonium-OH to pH 7.4). The pipette (intracellular) solution consisted of (in mM) 100 NaF, 30 NaCl, 10 EGTA, and 10 HEPES (titrated with cesium-OH to pH 7.2). Capsaicin and capsazepine were purchased from Sigma (St. Louis, MO). Drugs were dissolved in dimethylsulfoxide (DMSO) at 10 mM as stock solutions and stored at 4 °C. Final drug concentrations up to 100 μM were made by serial dilution. The highest DMSO concentration in the bath solution (1%) had little effect on Na+ currents. Chloramine-T was obtained from Aldrich Chemical Co. (Milwaukee, WI), dissolved in distilled water at 40 mM stock concentration, and used within a few hours after final dilution. Sea anemone toxin ATXII was purchased from Calbiochem (La Jolla, CA), dissolved in the extracellular solution at 100 μM stock concentration, and stored at −20 °C until needed. Both chloramine-T and ATXII were used to impair fast inactivation of Na+ channels (Hille, 2001).

2.4. Electrophysiology and data acquisition

The whole-cell configuration of a patch-clamp technique (Hamill et al., 1981) was used to record Na+ currents in HEK293 cells at room temperature (22 ± 2 °C). There were no endogenous TRPV1 channels that could be activated by capsaicin in this HEK expression system (Caterina et al., 1997). Electrode resistance ranged from 0.5 to 1.0 MΩ. Command voltages were elicited with pCLAMP9 software and delivered by Axo-patch 200B (Axon Instrument) or by EPC-7 (List Electronics). Cells were held at −140 mV and dialyzed for 10–15 min before current recording. The capacitance and leak currents were cancelled with the patch-clamp device and by P/−4 subtraction. Liquid junction potential was not corrected. Peak currents at +30 mV were 2–20 nA for the majority of cells. Access resistance was 1–2 MΩ under the whole-cell configuration; series resistance compensation of >85% typically resulted in voltage errors of ≤3 mV at +30 mV. Dose–response studies were performed at +30 mV for the outward Na+ currents. Such recordings allowed us to avoid the complication of series resistance artifacts and to minimize inward Na+ ion loading (Cota and Armstrong, 1989). Curve fitting was performed by Microcal Origin (Northampton, MA). An unpaired Student’s t-test was used to evaluate estimated parameters (Mean ± SEM or fitted value ± SE of the fit); P values of <0.05 were considered statistically significant.

3. Results

3.1. Block of resting and inactivated rNav1.4 Na+ channels by capsaicin

We first investigated the potency of capsaicin block in wild-type rat skeletal muscle rNav1.4 Na+ channels. The block of resting and inactivated Na+ channels was studied in the presence of capsaicin ranging from 1 to 100 μM. Traces of Na+ currents at various capsaicin concentrations were recorded with a 10-s conditioning pulse at −140 mV (Fig. 2A, for resting block) and at −70 mV (Fig. 2B, for inactivated block), respectively. Above 100 μM capsaicin the solutions turned turbid and were not included in this study. The pulse protocol is described in the legend to Fig. 2. Using these sets of data we constructed dose–response curves for capsaicin as shown in Fig. 2C. Block of resting rNav1.4 Na+ channels was weak. The estimated 50% inhibitory concentration (IC50) was 218 ± 13 μM and for 74 ± 1 μM for the resting and inactivated rNav1.4 Na+ channels, respectively (n = 5; Table 1). The potency difference in resting and inactivated block was ~3-fold. Despite the difference in the pulse protocol, our results are consistent with those reported by Lundbaek et al. (2005). As in their report, the accuracy of these values may be limited because of the inadequate concentration range. The inactivated-channel block by etidocaine is considerably more effective than the resting-channel block, by a factor of >10 (Ragsdale et al., 1994; Ragsdale et al., 1996). Such strong inactivated-channel block is found in most local anesthetics.

Fig. 2.

Fig. 2

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Block of resting and inactivated rNav1.4 wild-type Na+ currents by capsaicin. (A) The resting-channel block was determined by holding the cells at −140 mV. Currents at various concentrations of capsaicin were measured by a 5-ms test pulse at +30 mV, delivered at 30-s intervals. The block reached its steady state within 2–3 min. (B) The inactivated-channel block was determined by delivering a 10-s conditioning pulse to −70 mV before the 5-ms test pulse at +30 mV as described in Wang et al. (2003a). (C) Dose–response curve was constructed by using data shown in A (resting block; closed squares) and in B (inactivated block; open squares), plotted against the concentration, and fitted with a Hill equation. The IC50 value for resting block was estimated 218.4 ± 12.7 μM (Hill coefficient 1.33 ± 0.08) (■, n = 5) using a Hill equation. The IC50 value for inactivated block was 74.1 ± 0.6 μM (1.65 ± 0.02) (□, n = 5).

Table 1.

IC50 values for resting, open, and inactivated block of wild-type and mutant rNav1.4 Na+ channels by capsaicin

Capsaicin block of Na+ channels Resting-channel block IC50 in μM [Hill coefficient] Open-channel block IC50 in μM [Hill coefficient] Inactivated-channel block IC50 in μM [Hill coefficient]
Wild-type 218 ± 13 [1.33 ± 0.08] N.A. 74 ± 1 [1.65 ± 0.02]
WCW channel 57 ± 2 [2.44 ± 0.12] 6.8 ± 0.6 [1.43 ± 0.14] N.A.
WCW/F1579K 75 ± 3 [2.63 ± 0.29] 9.3 ± 0.6 [0.94 ± 0.05] N.A.
WCW/F1579A 66 ± 3 [2.34 ± 0.18] 13.3 ± 1.1 [1.63 ± 0.19] N.A.
WCW/L1280K 31 ± 1 [2.23 ± 0.22] 7.3 ± 1.0 [0.99 ± 0.13] N.A.
WCW/N434K 87 ± 1 [2.34 ± 0.05] 4.5 ± 0.6 [1.18 ± 0.17] N.A.
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IC50 values were estimated by fitting the dose–response curve as described in Figs. 2C and 3B. Values averaged from five separate cells (n = 5) were best fitted with a Hill equation. N.A., not applicable. Values of Hill coefficient are in brackets.

3.2. Open-channel block by capsaicin in inactivation-deficient mutant Na+ channels

Since normal Na+ channels open briefly for ~0.5 ms and are then rapidly inactivated (Aldrich et al., 1983), we used rNav1.4-WCW inactivation-deficient Na+ channels to study the block of the open channel by capsaicin. These channels open persistently during a 50-ms depolarization as shown in Fig. 3A (control trace); a large fraction of Na+ currents were maintained even at the end of the 50-ms pulse. Unlike its weak resting-and inactivated-channel block in wild-type Na+ channels, capsaicin blocked the inactivation-deficient Na+ channel effectively and elicited a time-dependent block of rNav1.4-WCW Na+ currents at +30 mV in a concentration-dependent manner as shown in Fig. 3A. Analyses of capsaicin block at various voltages ranging from −30 to +50 mV showed that the steady-state block of the maintained currents is not voltage dependent. The dose–response curves for the resting- and open-channel block (Fig. 3B; peak vs. steady-state block) yield resting-and open-channel IC50 values [Hill coefficient] of 57 ± 2 μM [2.44 ± 0.12, n = 5] and 6.8 ± 0.6 μM [1.43 ± 0.14, n = 5], respectively. These values are listed in Table 1 for comparison. We also measured the on-and off-rate as shown in Fig. 4A. This analysis was based on a one-to-one binding scheme as previously described in detailed for flecainide (Wang et al., 2003a; Ramos and O’Leary, 2004). The on-rate was estimated at 6.37 μM−1 s−1, whereas the off-rate was 34.4 s−1; the ratio of off-rate/on-rate was calculated as the equilibrium dissociation constant (KD) with a value of 5.4 μM for the open-block of rNav1.4-WCW Na+ currents. These results from the dose–response curve and from on- and off-rate kinetics are consistent with the notion that one capsaicin molecule blocks one open Na+ channel.

Fig. 3.

Fig. 3

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Block of rNav1.4-WCW inactivation-deficient Na+ currents by capsaicin. (A) Representative current traces were recorded at various capsaicin concentrations ranging from 1.0 to 100 μM. Cells were depolarized by a 50-ms test pulse at +30 mV. Pulses were delivered at 30-s intervals. Without drug, the control trace shows the presence of large persistent late Na+ currents. With drug, the time-dependent block becomes evident. Holding potential was set at −140 mV. (B) Peak and late currents (near the end of the test pulse) as shown in A were measured at various capsaicin concentrations. Data were normalized to the control amplitude in the absence of drug and best fitted with a Hill equation. The IC50 value for the peak current was 57.5 ± 1.8 μM (Hill coefficient, 2.44 ± 0.14) (■, n = 5). For the maintained late current the IC50 was 6.8 ± 0.6 μM (1.43 ± 0.14) (□, n = 5).

Fig. 4.

Fig. 4

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Kinetic analyses of the open-channel block by capsaicin. (A) The time-dependent block by capsaicin at each concentration as shown in Fig. 3A was normalized with respect to the control current trace without drug and fitted by an exponential function. The τ value (n = 5) was then inverted, and plotted against the capsaicin concentration. The on-rate (6.37 ± 0.34 μM−1 s−1) corresponds to the slope of the fitted line and the off-rate (34.4 ± 3.7 s−1) corresponded to the y-intercept. (B) The recovery time course of the open-channel block by capsaicin (closed circles) was measured at −140 mV with various intervals after a 50-ms depolarization to +30 mV. Most of the time-dependent block (71%) recovered rapidly with a τ value of 21.7 ± 1.5 ms (n = 5). The remaining current (22%) recovered slowly with a fitted τ value of 16.6 ± 3.6 s. Without capsaicin presence, most Na+ currents recovered rapidly (open circles) under an identical pulse protocol.

Recovery from the open-channel block induced by capsaicin was determined at −140 mV by a two-pulse protocol as shown in the inset of Fig. 4B. A majority of the time-dependent open-channel block by 30 μM capsaicin recovered rapidly, with a τ value of 21 ms (~74%). The rapid recovery of the open-channel block by capsaicin at −140 mV implies that (1) capsaicin binding is weak at the holding potential where the channel is in its resting state and (2) capsaicin is not “trapped” within the closed channel.

3.3. Capsazepine block of resting, inactivated, and open Na+ channel

Capsazepine antagonizes the action of capsaicin in TRPV1 channels. However, capsazepine was able to reverse mechanical hyperalgesia (Walker et al., 2003). In addition, Urban et al. (2000) reported that a non-pungent capsaicin analogue, SDZ 249–665, is a potent orally active, analgesic/anti-hyperalgesic agent. Thus, activation of the TRPV1 receptor by capsaicin might not be a prerequisite for analgesia. We measured the resting-, inactivated-, and open-channel block using the same protocols as shown in Figs. 2 and 3. The IC50 values are listed in Table 1 for comparison. We noticed that capsazepine is more effective in blocking the resting and inactivated Na+ channels. Nonetheless, the open-channel block by capsazepine is quite similar to that by capsaicin. Fig. 5A shows the block of the WCW currents at various capsazepine concentrations. The IC50 for the open-channel block is estimated to be 6.5 ± 0.3 μM (Fig. 5B; n = 5), nearly identical to that of capsaicin Table 2.

Fig. 5.

Fig. 5

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Block of rNav1.4-WCW inactivation-deficient Na+ currents by capsazepine. (A) Representative current traces are shown at various capsazepine concentrations (0–100 μM). Cells were depolarized by a 50-ms test pulse at +30 mV. Pulses were delivered at 30-s intervals with a holding potential of −140 mV. (B) Peak and maintained late currents near the end of the test pulse as shown in A were measured at various capsazepine concentrations. Data were normalized to the control saline response, plotted against the concentration, and fitted with a Hill equation. The IC50 value for the peak current was 26.3 ± 4.1 μM (Hill coefficient, 1.5 ± 0.3) (■, n = 5). For the maintained late current the IC50 was 6.5 ± 0.3 μM (1.6 ± 0.1) (□, n = 5).

Table 2.

IC50 values for resting, open, and inactivated block of wild-type and inactivation-deficient Na+ channels by capsazepine

Capsazepine block of rNav1.4 Na+ channels Resting-channel block IC50 in μM [Hill coefficient] Open-channel block IC50 in μM [Hill coefficient] Inactivated-channel block IC50 in μM [Hill coefficient]
Wild-type 46.4 ± 3.8 [1.84 ± 0.23] N.A. 15.3 ± 1.0 [1.69 ± 0.18]
WCW Na+ channel 26.3 ± 4.1 [1.54 ± 0.35] 6.5 ± 0.3 [1.59 ± 0.10] N.A.
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IC50 values were estimated by the dose–response curve as described in Fig. 5B. Values averaged from five different cells (n = 5) were best fitted with a Hill equation. N.A., not applicable. Values of Hill coefficient are in brackets.

3.4. Use-dependent block by capsaicin in inactivation-deficient mutant Na+ channels

No use-dependent block of peak Na+ currents by capsaicin was observed in trigeminal ganglion neurons when stimulated at 0.5–5 Hz (Liu et al., 2001). However, at a higher frequency (10–66 Hz), use-dependent phenotype of rNav1.4 Na+ channels by capsaicin was observed (Lundbaek et al., 2005). Because of the rapid recovery time course for the open-channel block by capsaicin in inactivation-dependent Na+ currents (Fig. 4B), we should see minimal use-dependent block at 1 Hz during repetitive pulses. Applying repetitive +50-mV/20-ms pulses at 1 Hz, we observed a small use-dependent reduction of rNav1.4-WCW Na+ currents in the absence of capsaicin (Fig. 6A). Unexpectedly, we observed a large use-dependent block by 30 μM capsaicin in inactivation-deficient mutant Na+ currents under the same pulse protocol (Fig. 6B). The additional use-dependent block amounted to 70.7 ± 1.4% in peak amplitude (Fig. 6C; closed circles; n = 7). In comparison, without capsaicin present, the reduction in inactivation-deficient Na+ currents during repetitive pulses reached only 17.5 ± 0.3% of the peak amplitude (Fig. 6C; open circles; n = 5).

Fig. 6.

Fig. 6

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Use-dependent block of inactivation-deficient Na+ channels induced by capsaicin. Traces of rNav1.4-WCW Na+ currents were recorded during a repetitive pulse protocol in the absence (A) and in the presence (B) of 30 μM capsaicin. The pulse protocol is shown on the top panel in (A). Traces shown are from the pulse number 1, 5, 10, 15, 30, 45, and 60. Peak currents were measured, normalized against the peak amplitude from pulse 1, and plotted against the pulse number (C). Open circles represent the normalized peak currents from data without capsaicin as shown in (A); the closed circles are from data with 30 μM capsaicin as shown in (B). The use-dependent block was fitted by a single exponential function with a τ value of 7.6 ± 0.2 pulses (n = 7) with a component of 70.6 ± 1.4%. Recovery from the use-dependent block induced by 30 μM capsaicin was measured at various time intervals up to 540 s (D). The recovery time course was fitted by a two-exponential function and the fitted values were provided in Section 3.

Recovery from this use-dependent block by 30 μM capsaicin was extremely slow; it took about 10 min for the channels to return to their drug-free resting state at the holding potential of −140 mV (Fig. 6D). About 45% of use-dependent block recovered with a τ value of 24 s, whereas 55% recovered with a τ value of 161 s. In comparison, recovery from the open-channel block by 30 μM capsaicin during a single 50-ms pulse was much faster (τ = 21 ms) than that from the use-dependent block. The fast and slow recovery from the time-dependent block and the use-dependent block induced by capsaicin, respectively, resemble those found in the fast and slow block of rNav1.2 Na+ channels induced by the acetyl-KIFMK-amide (Eaholtz et al., 1999). The mechanism of this slow recovery phenotype will be addressed in Section 4.

3.5. Capsaicin blocks Na+ currents via a receptor that is distinct from the local anesthetic receptor

The local anesthetic receptor was initially delimited by Ragsdale et al. (1994). This binding site with a bound local anesthetic has been modeled within the S6 inner cavity of the rNav1.4 Na+ channel by Lipkind and Fozzard (2005). Such information allows us to test whether capsaicin block of Na+ currents is via the local anesthetic receptor. Four S6 mutants were created in WCW background: F1579K, F1579A, L1280K, and N434K. These mutants affect local anesthetic binding significantly, particularly on the open-channel block (Nau and Wang, 2004). Fig. 7A shows the block of WCW/F1579K mutant currents at various capsaicin concentrations. The IC50 values for the open-channel block of various LA-resistant mutant channels ranged from 4.5 to 13.3 μM as determined by the dose–response curve (e.g., Fig. 7B). A Hill coefficient close to unity (0.99–1.32; Table 1) was also found in WCW/N434K, WCW/L1280K, and WCW/F1579K mutant channels. The reason for the deviation of the rNav1.4-WCW/F1579A mutant channel (Hill coefficient, 1.63) is unclear. For the resting channel the capsaicin block was modest with the IC50 values ranging from 31 to 87 μM. These experimental results are comparable to that of the WCW Na+ channels (Table 1). Our results on LA-resistant mutants indicate that capsaicin does not exert its effect via the local anesthetic receptor.

Fig. 7.

Fig. 7

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Block of rNav1.4-WCW/F1579K mutant Na+ currents by capsaicin. (A) Representative current traces are shown at various capsaicin concentrations. Cells were depolarized by a 50-ms test pulse at +30 mV. Pulses were delivered at 30-s intervals. Holding potential was set at −140 mV. (B) Peak and maintained late currents at the end of the test pulse were measured at various capsaicin concentrations as shown in A. Data were normalized to the control saline response, plotted against the capsaicin concentration, and fitted with a Hill equation. The IC50 value for the peak current was 74.7 ± 3.3 μM (Hill coefficient, 2.63 ± 0.29) (■, n = 5). For the maintained late current the IC50 was 9.3 ± 0.6 μM (0.94 ± 0.05) (□, n = 5).

3.6. Minimal time-dependent block of BTX-modified rNav1.4-WCW Na+ currents by capsaicin

BTX is a potent Na+ channel activator and, when bound, BTX eliminates both fast and slow inactivation and can keep the normal wild-type Na+ channel open persistently (Hille, 2001). BTX also shifts the activation threshold toward the hyperpolarizing direction by 30–50 mV. We therefore tested whether capsaicin can block the open BTX-modified Na+ channel. We included 5 μM BTX in the pipette solution and applied ~500 repetitive pulses at 2 Hz to facilitate the binding of BTX to the open state of rNav1.4-WCW Na+ channels. The BTX-modified Na+ currents were recorded at +50 mV with a 50-ms duration as shown in Fig. 8A. A reduction (16.8 ± 1.8%, n = 4) of peak BTX-modified Na+ currents by capsaicin was the same as that of the resting block of WCW peak Na+ currents (16.5 ± 1.9%, n = 5; Fig. 3). However, application of 30 μM capsaicin failed to block the late BTX-modified rNav1.4-WCW Na+ currents. There was no apparent time-dependent block induced by capsaicin; the amplitude of the late Na+ currents after capsaicin treatment was comparable to the corresponding peak value. Even with a prolonged depolarization, the lack of time-dependent block in BTX-modified rNav1.4-WCW mutant Na+ channels was observed at all voltages tested (Fig. 8B). The time-dependent block by capsaicin was also absent in BTX-modified rNav1.4 wild-type Na+ channels (data not shown). This phenotype is very different from that of inactivation-deficient WCW mutant currents at 30 μM capsaicin, which are inhibited by more than 80% at +50 mV (Fig. 3A). We conclude that the BTX-modified open rNav1.4-WCW Na+ channels are resistant to capsaicin block.

Fig. 8.

Fig. 8

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Absence of time-dependent block in BTX-modified rNav1.4-WCW Na+ channels by capsaicin. (A) About 500 repetitive pulses (+50-mV for 20 ms at 2 Hz) were applied first to facilitate BTX binding. Traces of BTX-modified rNav1.4-WCW Na+ currents at +50 mV were then recorded before and after 30 μM capsaicin (labeled in A). The pipette solution contained 5 μM BTX. Although capsaicin inhibited ~15% of Na+ currents, no time-dependent block during the 50-ms pulse was evident. (B) Traces of BTX-modified rNav1.4-WCW Na+ currents were recorded at various voltages ranging from −120 to +50 mV for 800 ms. Again, no time-dependent block was evident during the 800-ms pulse. Activation threshold was near −80 mV, showing that BTX shifted the activation gating of WCW channels by −30 mV (Wang et al., 2004). Experiments were repeated in four separate cells.

3.7. Block of neuronal inactivation-deficient Na+ currents by capsaicin

GH3 cells are known to express neuronal Nav1.1, 1.2, 1.3, and 1.6 isoforms (Vega et al., 2003). Kinetics of these Na+ currents have been extensively studied before (Cota and Armstrong, 1989). Preliminary screening experiments using GH3 cells indicated that capsaicin blocks resting and inactivated Na+ channels weakly, comparable to the data shown in Fig. 1 for rNav1.4 Na+ channels. To examine whether neuronal open Na+ channels are sensitive to capsaicin block, we impaired fast Na+ channel inactivation by two conventional methods. First, GH3 cells were briefly treated with 0.5 mM chloramine-T for ~3 min and washed with the reagent-free external solution for ~5 min as described before (Wang and Wang, 1992). Under these conditions, a sizeable fraction of Na+ channels remained open at the end of a 50-ms pulse as shown in Fig. 9A (trace 1 before vs. trace 2 after chloramine-T). We found that these persistent open Na+ channels were highly sensitive to capsaicin block (trace 3). The time-dependent block of capsaicin was also evident during the prolonged pulse. Most of maintained Na+ currents (86.6 ± 2.2%, n = 8) were blocked by 30 μM at the end of the pulse (Fig. 9A, trace 3). This magnitude of capsaicin block corresponds to an IC50 value of 4.0 μM as estimated by the Langmuir isotherm.

Fig. 9.

Fig. 9

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Capsaicin block of neuronal inactivation-impaired Na+ channels in GH3 cells. (A) Traces of Na+ currents were recorded at +30 mV before (trace 1) and after (trace 2) 0.5 mM chloramine-T treatment. Trace 2 was taken 5 min after chloramine-T treatment was terminated by washing with reagent-free external solution. Treatment of chloramine-T was limited to 3 min. Notice that a fraction of Na+ channels remained open during the 50-ms pulse. Capsaicin at 30 μM blocked a majority of the maintained current during the 50-ms pulse in a time-dependent manner (trace 3) (86.6 ± 2.2%, n = 8). In contrast, peak currents were only slightly inhibited (9.1 ± 1.7%, n = 8). (B) Traces of Na+ currents were recorded before (trace 1) and after (trace 2) external 1 μM ATXII treatment for ~3 min. Again, a significant fraction of Na+ channels remained open during the 50-ms pulse at +50 mV. With 1 μM ATXII presence, capsaicin at 30 μM also blocked a majority of the maintained current during the 50-ms pulse (trace 3) (89.8 ± 1.2 %, n = 7). However, the peak currents were reduced little (6.9 ± 1.1%, n = 7).

Second, GH3 cells were treated with 1 μM sea anemone toxin ATXII for ~3 min, which induced maintained currents at the end of a 50-ms pulse as shown in Fig. 9B (trace 1 before vs. trace 2 after 1 μM ATXII). These persistent open Na+ channels were again highly sensitive to capsaicin block, resulting in an apparent time-dependent inhibition of maintained Na+ currents (Fig. 9B, trace 3). At 30 μM capsaicin, 89.8 ± 1.2% (n = 7) of maintained Na+ currents induced by 1 μM ATXII were blocked (Fig. 9B, trace 3), with an estimated IC50 value of 3.4 μM. In contrast, peak Na+ currents in both Figs. 9A and B (trace 3) were relatively resistant to capsaicin block, indicating again that the resting-channel block by capsaicin was weak. Thus, neuronal Na+ channels expressed endogenously in GH3 cells, if kept open, are as sensitive to capsaicin block as muscle Nav1.4 open Na+ channels expressed heterologously in HEK cells.

4. Discussion

This report demonstrates for the first time that capsaicin induces a conspicuous time-dependent block of inactivation-deficient Na+ currents. This time-dependent block is concentration dependent; the higher the capsaicin concentration, the faster the open-channel block. Similar results were found for capsazepine. The steady-state block of inactivation-deficient Na+ currents by capsaicin yields an IC50 value of 6.8 μM. In addition, capsaicin at 30 μM elicits significant use-dependent block of inactivation-deficient Na+ currents by ~70% during repetitive pulses. Recovery from this use-dependent block by capsaicin is far slower than recovery from the time-dependent block during a single pulse. Such capsaicin-induced block in inactivation-deficient Na+ channels cannot be mediated by the TRPV1 receptor since HEK293 cells lack this receptor. Implications of these findings are discussed next.

4.1. Is there a specific capsaicin receptor responsible for the open Na+ channel block?

The resting and inactivated block induced by capsaicin is rather weak in rNav1.4 Na+ channels (Fig. 2). Nonetheless, our results are consistent with those reported by Lundbaek et al. (2005). Since capsaicin alters membrane properties significantly, these authors suggested that the block of resting and inactivated Na+ channel by capsaicin and capsazepine is primarily regulated by changes in bilayer elasticity.

Unlike resting and inactivated Na+ channel block, the capsaicin block of the open Na+ channel has a comparably high binding affinity, with an IC50 value of 6.8 μM (Fig. 3). Several pieces of evidence suggest that there is a non-TRPV1 receptor within rNav1.4 Na+ channel responsible for the open-channel block induced by capsaicin. First, the rapid time-dependent block by capsaicin occurs in the open state of Na+ channels. Such rapid open-channel block phenotype resembles those found for flecainide and lidocaine (Wang et al., 2003a; Ramos and O’Leary, 2004; Wang et al., 2004). The time-dependent block induced by flecainide and lidocaine is in fact through a specific receptor within the open Na+ channel. Second, the dose–response curve of capsaicin for the open-channel block has a Hill coefficient close to unity in rNav1.4-WCW mutant Na+ channels, suggesting that binding of one capsaicin molecule with its receptor likely blocks one open Na+ channel. Third, kinetic analyses of the time-dependent block yield the on- and off-rate constants that are also consistent with a one-to-one binding scheme (Fig. 4A). Fourth, the capsaicin open-channel block is minimal in BTX-modified Na+ channels. Resting-channel block by capsaicin found in the WCW mutant Na+ channels, however, persists in BTX-modified Na+ channels (Fig. 8A). This phenotype is consistent with a hypothesis that BTX selectively eliminates capsaicin binding to its receptor within the open Na+ channel. Taken together, our results suggest that there is a specific capsaicin receptor responsible for its open-channel block.

4.2. The capsaicin receptor within the open Na+ channel is distinct from the local anesthetic receptor

Despite the similarity in the open-channel block among capsaicin and local anesthetics, site-directed mutagenesis showed that the local anesthetic receptor is not responsible for capsaicin block of the open Na+ channel (Fig. 7; Table 1). In general, lysine substitutions of residues that are found critical for local anesthetic binding have only minimal or modest effects on capsaicin binding. Alanine substitution of F1579 residue also alters capsaicin binding only modestly with an IC50 value of 13.3 μM. The magnitude of this effect is far smaller than that reported for local anesthetic binding (e.g., Ragsdale et al., 1994).

Earlier studies have demonstrated that a number of Na+ channel blockers do not share the identical receptor site as local anesthetics. For example, prenylamine blocks the Na+ channel effectively via a receptor that is different from the local anesthetic receptor (Mujtaba et al., 2002). Furthermore, symmetrical N-linked lidocaine dimers appear to have a significantly higher affinity with the Na+ channel than lidocaine, indicating an additional receptor situated adjacent to the local anesthetic site (Smith et al., 2006). An adjacent binding site for capsaicin near the local anesthetic receptor is also consistent with the observation that batrachotoxin-modified Na+ channels are resistant to capsaicin block. Since batrachotoxin is likely bound within the inner cavity (Wang and Wang, 2003), it is feasible that the BTX receptor overlaps the capsaicin receptor. If bound within the inner cavity, capsaicin may occlude the Na+ channel permeation pathway directly.

4.3. Recovery from the use-dependent block of capsaicin is slow in rNav1.4-WCW inactivation-deficient Na+ channels

Capsaicin at 30 μM elicits additional use-dependent block of remaining inactivation-deficient Na+ currents by ~70% during repetitive pulses. Recovery from this use-dependent block is rather slow compared with recovery from the fast time-dependent block. This unique phenotype is similar to that of the use-dependent block of acetyl-KIFMK-amide in its open-channel block (Eaholtz et al., 1999). This peptide elicits fast time-dependent block of inactivation-deficient Na+ currents. Repetitive pulses produce additional use-dependent block, which however recovers slowly at the holding potential (−140 mV). According to these authors, the additional use-dependent block induced by acetyl-KIFMK-amide is due to sequential binding reactions of the ligand to an adjacent binding site(s) deep within the open pore, which “traps” the ligand when the channel is closed by the activation gate. This proposed mechanism also readily explains the use-dependent phenotype induced by capsaicin in rNav1.4-WCW Na+ channels.

A possible alternative interpretation of this slow recovery phenotype following the use-dependent block is that capsaicin enhances slow inactivation gating of the Na+ channel. An enhancement in slow inactivation will render the channel unavailable for a long period of time, consistent with the slow recovery after the use-dependent block (Fig. 6D). Additional experiments are needed in order to distinguish these two possibilities.

4.4. The non-TRPV1 capsaicin receptor site in the open Na+ channel as a potential target for the treatment of neuropathic pain?

It is well documented that drug-induced analgesia may occur via the block of ectopic high-frequency discharges at the injured nerve (Devor and Seltzer, 1999). Such an analgesic pathway has been observed clinically and experimentally after i.v. lidocaine injection (Boas et al., 1982; Devor et al., 1992). Our results shown in Fig. 9 indicate that neuronal open Na+ channels expressed endogenously in GH3 cells are as sensitive to capsaicin block as the open rNav1.4 counterparts. Thus, capsaicin as an effective open-channel blocker with an IC50 value of ~4 μM will in theory help silence the ectopic high-frequency discharges that may be perceived as spontaneous pain. This mode of μM-capsaicin action at the open Na+ channel may operate as a secondary pathway in conjunction with its dominant sub-μM action at the TRPV1 receptor near the injured region. Additional use-dependent block of open Na+ channels during repetitive pulses (Fig. 6B) and the slow recovery from such block (Fig. 6D) will also likely enhance the capsaicin efficacy significantly during high-frequency discharges. Normal infrequent impulses will not be sensitive to capsaicin given that the drug has relatively low affinity toward resting and inactivated Na+ channels. Nonetheless, since these compounds may block open Na+ channels effectively, the potential liability of capsaicin analogues for systemic toxicity should be considered. Accidental overdose of the Na+ channel blocker can be fatal as shown in the intravascular injection of local anesthetics.

An alternative mechanism through the Na+ channel pathway was suggested by Blair and Bean (2003) who showed that small-diameter dorsal root ganglion neurons quickly adapted their action potential firing when treated with 0.5 μM capsaicin. Slow inactivation of Na+ channels built up during capsaicin-induced high-frequency firings presumably contributes to desensitization of action potential firing in these DRG neurons. This interpretation does not necessarily require direct capsaicin binding with neuronal Na+ channels. Nonetheless, direct enhancement of slow inactivation by capsaicin may also account for the strong use-dependent phenotype during repetitive pulses as discussed above. Interestingly, Su et al. (1999) reported that certain afferent neurons express TTX-resistant Na+ channel isoforms that are potently inhibited by capsaicin, with an IC50 value of ~0.5 μM, a potency that is ~10 times higher than the open-channel block of rNav1.4 TTX-sensitive Na+ channels. If confirmed, this high selectivity of TTX-resistant Na+ channel isoforms toward capsaicin represents the third option for the Na+ channel pathway in capsaicin-induced analgesia. Regardless of the mechanisms of the capsaicin actions on Na+ channels, the presence of a non-TRPV1 receptor within open Na+ channels will have therapeutic implications for pain management. Development of potent analogues that target this specific capsaicin receptor within the open Na+ channel may also benefit patients with neuropathic pain.

Acknowledgments

This work was supported by grants from NIH (GM48090). We thank Drs. Igor Kissin and Gary Strichartz for their invaluable comments. We are grateful to Dr. John Daly for providing us batrachotoxin and to Dr. Edward Moczydlowski for the pcDNA3-rNav1.4 clone and a HEK293 cell line expressing wild-type rNav1.4 channels.

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