European Journal of Pharmacology 756 (2015) 22–29

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

Carvacrol modulates voltage-gated sodium channels

kinetics in dorsal root ganglia
Humberto Cavalcante Joca a, Daiana Cardoso Oliveira Vieira a,b, Aliny Perreira Vasconcelos c,
Demetrius Antônio Machado Araújo c, Jader Santos Cruz a,n
a
Laboratório de Membranas Excitáveis e Biologia Cardiovascular, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade
Federal de Minas Gerais, Belo Horizonte, MG, Brazil
b
Laboratório de Eletrofisiologia, Instituto Superior de Ciências Biomédicas, Universidade Estadual do Ceará, Fortaleza, CE, Brazil
c
Laboratório de Biotecnologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal da Paraíba, João Pessoa, PB, Brazil

art ic l e i nf o a b s t r a c t

Article history: Recent studies have shown that many of plant-derived compounds interact with specific ion channels
Received 18 November 2014 and thereby modulate many sensing mechanisms, such as nociception. The monoterpenoid carvacrol
Received in revised form (5-isopropyl-2-methylphenol) has an anti-nociceptive effect related to a reduction in neuronal excit-
3 February 2015
ability and voltage-gated Na þ channels (NaV) inhibition in peripheral neurons. However, the detailed
Accepted 9 March 2015
Available online 17 March 2015
mechanisms of carvacrol-induced inhibition of neuronal NaV remain elusive. This study explores the
interaction between carvacrol and NaV in isolated dorsal root ganglia neurons. Carvacrol reduced the
Keywords: total voltage-gated Na þ current and tetrodotoxin-resistant (TTX-R) Na þ current component in a
Carvacrol concentration-dependent manner. Carvacrol accelerates current inactivation and induced a negative-
Peripheral neurons
shift in voltage-dependence of steady-state fast inactivation in total and TTX-R Na þ current. Further-
Dorsal root ganglia
more, carvacrol slowed the recovery from inactivation. Carvacrol provoked a leftward shift in both the
Voltage-gated sodium channels
Tetrodotoxin-resistant sodium current voltage-dependence of steady-state inactivation and activation of the TTX-R Na þ current component. In
addition, carvacrol-induced inhibition of TTX-R Na þ current was enhanced by an increase in stimulation
frequency and when neurons were pre-conditioned with long depolarization pulse (5 s at  50 mV).
Taken all results together, we herein demonstrated that carvacrol affects NaV gating properties. The
present findings would help to explain the mechanisms underlying the analgesic activity of carvacrol.
& 2015 Elsevier B.V. All rights reserved.

1. Introduction
Throughout human history, natural products have had an essen-
tial role in the treatment of a number of diseases (Chang and
Keasling, 2006; Koehn and Carter, 2005). Although the benefits are
sometimes obvious, traditional or herbal medicine is regarded with
skepticism, because the mechanism through which plant compounds
exert their powers are largely elusive. Recent studies have shown
however that many of these plant compounds interact with specific
ion channels and thereby modulate the sensing mechanism of the
human body (Alvarez-Collazo et al., 2014; Santos-Nascimento et al.,
2015; Straub et al., 2013).
Voltage-gated Na þ channels (NaV) underlie the generation of
action potentials that are extremely important for electrical commu-
nication between excitable cells. Modulation of NaV exerts important
impact on the control of neuronal excitability (Ahn et al., 2007).
n
Corresponding author. Tel.: þ 55 31 3409 2668; fax: þ 55 31 3409 2613.
E-mail address: jcruz@icb.ufmg.br (J.S. Cruz).
Importantly, Dorsal root ganglia (DRG) neurons were chosen to pursue
this study because their cell bodies or afferent fibers are carriers of
sensory information. With regard to cell size, cultured mammalian
DRG neurons have been classified into two major groups (1) large
neurons with short-lasting action potentials predominantly expressing
fast tetrodotoxin-sensitive Na þ currents (TTX-S); and (2) small neu-
rons with long-lasting action potentials, expressing a combination of
fast TTX-S and slow TTX-resistant (TTX-R) Na þ currents (Cummins
et al., 2007; Dib-Hajj et al., 2010; Roy and Narahashi, 1992). Recently,
single-cell analysis of Na þ channel transcripts indicated that TTX-S
and TTX-R are differentially expressed in large and small sensory
neurons and the authors provided evidence that NaV 1.8 is highly
expressed in a subpopulation of large myelinated DRG neurons (Ho
and O’Leary, 2011). These results support previous findings that have
identified NaV 1.8 expression in large-diameter DRG neurons by
immunolabelling (Amaya et al., 2000; Djouhri et al., 2003)
Bioprospection of new drugs that could be useful in the
treatment of pain is an increasingly active research field. Carvacrol,
5-isopropyl-2-methylphenol, is an organic compound naturally
occurring in essential oils of many plants. It is well known for its

http://dx.doi.org/10.1016/j.ejphar.2015.03.007
0014-2999/& 2015 Elsevier B.V. All rights reserved.
 H.C. Joca et al. / European Journal of Pharmacology 756 (2015) 22–29
wide pharmacological profile, especially analgesic (Guimarães
et al., 2010). Previous studies have demonstrated that carvacrol
elicited a significant inhibition of neuronal action potential
(Gonçalves et al., 2010; Joca et al., 2012).
Although previous results have indicated that carvacrol inhibits
voltage-dependent Na þ currents, the mechanisms underlying its
blocking effects remain elusive. In this study, we used whole-cell
patch-clamp to further investigate the effects of carvacrol on Na þ
channels. In addition, we studied the effects of carvacrol on TTX-R
Na þ currents carried by NaV 1.8 channels from DRG neurons.
2. Materials and methods
2.1. Isolation and cell culture of rat DRG neurons
DRG neurons were obtained from the lumbar segments of 10 to
14-w old male Wistar rats (220–250 g body weight) using an
enzymatic dissociation procedure as described previously (Joca
et al., 2012; Moraes et al., 2011). Rats were rendered unconscious
by exposure to CO2 and decapitated. Lumbar DRG were rapidly
dissected and placed in Ca2 þ - and Mg2 þ -free Hanks' balanced salt
solution (HBSS). After carefully removing the surrounding connective
tissues, each ganglion was incubated for 75 min, in a water bath at
37 1C, in HBSS containing type 1 collagenase 1 mg/ml (Sigma
Chemical Co., St. Louis, MO, United States) and further incubated
for 15 min at 37 1C in HBSS containing 2.5 mg/ml trypsin (Sigma
Chemical Co.). The enzyme digested ganglia were mechanically
agitated using a firepolished Pasteur pipette to disperse the neurons.
The dissociated neurons thus obtained were washed twice with
Dulbecco's Modified Eagle's Medium (Sigma Chemical Co.) supple-
mented with 10% fetal bovine serum and 1% penicillin (Cultilab,
Brazil). The neurons were then plated onto poly-D-lysine (0,1%, Sigma
Chemical Co.)-coated rectangular glass coverslips and were incubated
at 37 1C for 12–48 h in a humidified atmosphere of 95% air plus 5%
CO2 prior to the experiments. DRG neurons that appeared practically
spherical without neuronal processes were used for experiments. All
experimental procedures were reviewed and approved by the
institutional ethics committee of Universidade Federal da Paraíba
(CEUA-UFPB).
2.2. Electrophysiology
Sodium currents were recorded at room temperature (24–28 1C) in
the whole-cell mode of the patch-clamp technique (Hamill et al., 1981)
using a HEKA EPC-9/2 amplifier (HEKA Instruments, Germany).
Whole-cell currents were acquired at 20 kHz and filtered at 3 kHz.
Pulse generation and data acquisition were performed on a Windows-
based computer using the Patchmaster program (version 2.7; HEKA,
Lambrecht/Pfalz, Germany). Bath solution contained (in mM): NaCl
140, KCl 5, CaCl2 1.8, MgCl2 0.5, 4-(2-hydroxyethyl)-1-piperazineetha-
nesulfonic acid) (HEPES) 5, and glucose 5 with pH adjusted to 7.4.
Recording electrodes (1–2 MΩ) were fabricated from capillary glass
using a HEKA PIP6 pipette puller (HEKA, Lambrecht/Pfalz, Germany).
The standard pipette solution contained (in mM): NaCl 10, CsCl 100,
HEPES 10, ethylene glycol tetraacetic acid 11, tetraethylammonium-Cl
10, MgCl2 5, and pH adjusted to 7.2 with CsOH. Series resistance errors
were compensated using 60–85% series resistance compensation. DRG
neurons on glass coverslips were transferred into a recording chamber
containing standard bathing solution. After establishing the whole-cell
configuration, the bath solution was changed to one containing (in
mM): NaCl 40, KCl 3, HEPES 10, tetraethylammonium-Cl 20, CaCl2 1,
MgCl2 1, CdCl2 0.1, Choline-Cl 70, and glucose 10 with pH adjusted to
7.4. The extracellular Na þ concentration was maintained at 40 mM for
all experiments to reduce the size of Na þ current and thus improve
voltage-clamp conditions. Cd2þ (100 μM) was routinely included in
23
the bath solution to block Ca2þ channels. Although sub-milimolar
concentrations of Cd2 þ have been reported to block tetrodotoxin-
resistant Na þ channels (Ikeda and Schofield, 1987), its use in the
present study does not affect our conclusions because even in the
presence of Cd2 þ the size of tetrodotoxin-resistant Na þ currents were
sufficiently large to allow careful analysis (very large signal-to-noise
ratio). In a series of experiments, currents in DRG neurons were
measured in the presence (300 nM) of tetrodotoxin.
DRG neurons were held at  80 mV for 5 min before initiating the
experimental stimulation protocols to allow cell dialysis to complete
(Pusch and Neher, 1988). The DRG neuron under examination was
continuously perfused via a large-bore perfusion pipette positioned
with a mechanical micromanipulator in its vicinity. External test
solutions without (control) or with carvacrol were changed by an
electric command to a micro-solenoid valve (The Lee Co., Essex, CT,
USA) that controlled the bath perfusion.
Peak Na þ currents were measured using 100 ms pulses to
between 120 mV and 50 mV every 5 s from a holding potential of
 80 mV. The peak current was normalized for cell capacitance and
plotted against voltage to generate peak current density–voltage
relationships. Whole-cell conductance was calculated from the peak
current amplitude using the equation ðEm  Erev ÞnGmax =ð1 þ e^ðððV m 
Em Þ=ka Þ Þ Þ: Erev is the estimated Na þ current reversal potential, and
then normalized to the maximal conductance recorded between 5
and 25 mV. The normalized conductance as a function of voltage
curves was fit with the Boltzmann function, ðG  Gmax Þ=ð1 þ e^ðððEm 
V a Þ=ka Þ Þ Þ determine the voltage for half-maximal channel activation
(Va) and slope factor (ka). The voltage dependence of channel avail-
ability was assessed after a 300 ms prepulse to various potentials
followed by 100 ms pulse to 0 mV, the voltage at which peak Na þ
currents was measured. The normalized current was plotted against
the voltage, and steady-state channel availability curves were fit with
Boltzmann function “1=ð1 þ e^ðððEm  V h Þ=kh Þ Þ Þ” , to determine the
voltage for half-maximal channel inactivation (Vh) and slope factor (kh).
2.3. Chemicals and solutions
Carvacrol was obtained from Sigma Chemical Co. A 10 mM stock
solution was made up in dimethylsulfoxide (DMSO). All solutions
were prepared fresh daily. In a series of control experiments, 10 min
application of DMSO at 0.2% (v/v) had negligible effects on the
functional properties of TTX-S and TTX-R Na þ currents (data not
shown).
2.4. Data analysis
All data are expressed as means 7S.E.M., where n indicates the
number of experiments. The unpaired and paired Student's t-test
were used when appropriate, Po 0.05 is considered to indicate the
statistically significant difference. Mathematical curve fitting was
accomplished using SigmaPlot (11.0, Systat Software). All curve
fitting routines were performed using non-linear regression ana-
lysis (Levenberg–Marquadt algorithm).
3. Results
To test the hypothesis that ion channel antagonism may
underlie the anti-nociceptive effects of carvacrol we investigated
the blocking effect of carvacrol on voltage-gated Na þ channels in
isolated rat DRG neurons. We focused our attention on large DRG
neurons (estimated diameterZ30 μm based on membrane capa-
citance measurements) that has been reported to express TTX-S
and TTX-R voltage-dependent Na þ currents (Caffrey et al., 1992).
Carvacrol blocks all voltage-dependent Na þ currents in a
concentration-dependent fashion (10–2000 μM, n ¼50 cells) in
24 H.C. Joca et al. / European Journal of Pharmacology 756 (2015) 22–29

Fig. 1. Effects of carvacrol on total Na þ currents. (A) Na þ currents evoked by depolarization to potentials from  100 to þ 50 mV in 5 mV steps. Holding potential was set at
 100 mV. Control (Left panel); after addition of carvacrol 300 mM (Middle panel). Right panel illustrates carvacrol-sensitive Na þ currents obtained by digital subtraction of
the current with carvacrol from the control traces. (B) I–V relationship of total Na þ current in the presence of 300 mM carvacrol. Control (open circles) and carvacrol (filled
circles). Data are means 7 S.E.M. (n¼ 6 cells). (C) Means normalized fractional inhibition. Symbols represent means 7 S.E.M. (n¼ 6 cells). Data were obtained from (B).

rat DRG neurons (Supplemental Fig. 1), similar to previous report
(Joca et al., 2012). Hill plot giving an estimated IC50 of 267.7 μM,
then all subsequent experiments were performed using 300 μM
carvacrol.
To elicit total voltage-gated Na þ currents, DRG neurons were
submitted to a holding potential of  80 mV and a 100 ms pre-pulse
to  120 mV was applied and then depolarized to membrane
potentials ranging from  100 mV to 50 mV in 5 mV increments
every 5 s. A family of typical current traces is shown in Fig. 1A in
control conditions (i), after 2 min treatment with carvacrol (ii), and
after digital subtraction (iii) to unravel carvacrol-sensitive Na þ
currents (iii). Fig. 1B demonstrates the current–density vs. voltage
relationships for the total Na þ currents of DRG neurons in the
absence or presence of 300 μM carvacrol (n¼6). The activation
threshold potential was approximately  40 mV and carvacrol did
not change this parameter. The peak current density of total Na þ
current was  315.9745.7 A/F for control and 164.6728.4 A/F
after exposure to carvacrol. Carvacrol (300 μM at  10 mV) red-
uced the peak Na þ currents by 47.876.7%. The reversal potential
was not affected by the presence of carvacrol which indicates that ion
selectivity was preserved. In addition, carvacrol (300 μM) produced
an inhibition of the Na þ currents that, at the conditioning pre-pulse
of  120 mV, was independent of the test potential (Fig. 1C).
After establishing that carvacrol blocks Na þ currents in DRG
neurons, we proceeded with more detailed electrophysiological ana-
lysis to better characterize the interaction between carvacrol and Na þ
channels. To determine if the block of Na þ channels by carvacrol was
due to a depolarizing shift in the voltage-dependence of activation the
kinetics of activation of Na þ currents was calculated and fit with a
single Boltzmann equation as shown in Fig. 2A.
In contrast to the change in peak sodium current, normalized
conductance as a function of membrane potential curves (Fig. 2A)
shows that the voltage-dependence of activation was not changed in
the presence of carvacrol (300 μM, n¼6).
The reduction in peak current provoked by carvacrol without
change in the steady-state activation indicated that the voltage
dependence of inactivation of Na þ channels might be negatively
shifted by carvacrol, thereby contributing to the significant
decrease in peak Na þ current. After conditioning pre-pulses of
300 ms duration to membrane potentials more positive than
 90 mV, peak Na þ currents are progressively decreased by
steady-state inactivation (Fig. 2A). Exposure to carvacrol nega-
tively shifts the voltage dependence of inactivation from half-
inactivation voltage, Vh of  42.6 77.9 mV (n ¼6) in control to
 53.8 77.5 mV in the presence of carvacrol (Po 0.05; n ¼6). The
slope factors for the steady-state inactivation curves were 8.1 for
control and 6.9 in the presence of carvacrol, and the difference
between these values was not significant (n ¼6; P4 0.05). Steady-
state inactivation and activation curves intersect near  30 mV in
the control conditions (Fig. 2A).
The intersection of these curves at voltage range from  40 mV to
 10 mV predicts a substantial window current that could contribute
to control neuronal excitability. As carvacrol shifted the steady-state
inactivation curve to more hyperpolarized membrane potentials the
window current was reduced which could account for the decrease
in neuronal excitability reported by others (Gonçalves et al., 2010;
Joca et al., 2012).
Fig. 2B shows voltage-gated Na þ current examples recorded
from one DRG neuron in response to 0 mV test pulse. At this test
pulse potential, Na þ channels activate and inactivate within
several milliseconds. Carvacrol accelerates current inactivation,
such that the duration of the Na þ currents is shorter. We also
measured the inactivation time constants and determined that the
current decay exhibited two exponential components with a
predominant fast ( 80%) and a smaller (  20%) slow component.
Carvacrol (300 μM) did not change the contribution of each
component for current decay but it caused a significant decrease
in fast (1.64 70.37 ms to 0.99 70.20 ms, n¼ 6; P o0.05) and
 H.C. Joca et al. / European Journal of Pharmacology 756 (2015) 22–29 25

Fig. 2. Effects of carvacrol on functional properties of Na þ currents. (A) Overlay of steady-state activation (circles) and inactivation (squares) curves before (open symbols)
and after application of carvacrol (closed symbols). Continuous lines are representing the best fit to a Boltzmann distribution (see Section 2 for more details). (B) Normalized
Na þ currents at 0 mV (from  100 mV holding potential) recorded from the same cell in control conditions and after carvacrol addition to the bath. The current decay in
carvacrol is accelerated. Time constants for current inactivation were calculated by a fit with double exponential function (superimposed continuous lines). (C) Composite
data to compare fitting parameters of current inactivation in control and after carvacrol effect reaches steady-state. *Po 0.05. Paired Student's t-test. Bar graphs represent
means 7 S.E.M. (n ¼6 cells). (D) Recovery from inactivation. After a depolarizing test pulse, channels were allowed to recover from fast inactivation at  120 mV for varying
intervals before testing for fractional recovered current at 0 mV. Averaged data were fit with a double exponential function.

slow (6.61 70.66 ms to 4.37 70.87 ms, n ¼6; Po 0.05) time
constants (Fig. 2C).
To examine the effect of carvacrol on the repriming of Na þ
currents from the inactivated state, a double-pulse protocol was used
by applying a test pulse to 0 mV for 100 ms and then a second test
pulse followed by a recovery pulse (120 mV) to 0 mV for varying
durations. Recovery from inactivation (Fig. 2D) in the absence or
presence of carvacrol was well described by a sum of two exponential
functions. The fast time constant was 3.4070.75 ms and the slow
time constant 156.60719.89 ms in control, while after carvacrol
exposure these values increase to 4.3970.90 and 265.80720.36 ms,
respectively.
Having demonstrated that exposure of DRG neurons to carva-
crol was capable of inhibiting voltage-gated Na þ currents, we next
asked the question whether carvacrol would preferentially block
TTX-resistant Na þ currents in rat DRG neurons.
To do so, DRG neurons were incubated with tetrodotoxin
(300 nM) which blocks TTX-sensitive Na þ currents and allowed
TTX-resistant Na þ currents to be isolated. Fig. 3A shows TTX-
resistant Na þ current examples recorded from DRG neurons in
response to 0 mV test pulse. We found that TTX-resistant Na þ
currents were significantly inhibited by carvacrol at two different
concentrations (Top panels). Fig. 3B demonstrates the comparison of
current inhibition elicited by carvacrol at 300 μM ( 30%, n¼7) and
at 1000 μM (82.4477.45%, n¼3) for TTX-resistant Na þ currents.
We further investigated the effects of carvacrol on TTX-resistant
Na þ currents by looking at its effects on the time course of inactiva-
tion. Specifically, the inactivation time-course determined at 0 mV
exhibited biexponential decay (Fig. 3C). At this membrane potential,
both the slow and fast exponential time constants were significantly
decreased by the presence of carvacrol (300 μM, n¼7). There is no
significant change in the relative contributions for both components
(Fig. 3C). These findings indicated that changes in the inactivation
kinetics may be responsible for carvacrol inhibitory effects on TTX-
resistant Na þ currents.
To better investigate the effect of carvacrol on TTX-resistant
Na þ channels activation we recorded voltage-dependent inward
currents with slow inactivation by holding the membrane poten-
tial at  80 mV and applying 100 ms test pulses to potentials
between  100 to þ55 mV in 5 mV increments. Typical whole-cell
currents are depicted in Fig. 4A (Top panels). When normalized for
cell capacitance the average peak TTX-resistant Na þ current
density of carvacrol at 300 μM (  63.00 78.12 A/F) was statisti-
cally different from control (  93.37 716.58 A/F, n ¼7, Po 0.05).
As represented in Fig. 4B, TTX-resistant current density–voltage
curve in the presence of carvacrol was shifted by 10 mV more
hyperpolarized than control DRG neurons. The reversal potential is
unaffected by the presence of carvacrol.
The voltage dependence of activation and steady-state inactivation
for control and in the presence of carvacrol is illustrated in Fig. 4C. As
compared with control, the mid-point of activation was shifted 8.9 mV
more negative for neurons exposed to 300 μM carvacrol (control:
 8.0170.6 mV, n¼7; carvacrol:  16.971.1 mV, n¼7; paired t-test,
Po0.05), confirming that carvacrol shifts activation of TTX-resistant
Na þ channels in a hyperpolarizing direction. There is no change in the
slope factor.
Steady-state inactivation (Fig. 4C, squares) was assessed at
0 mV after applying a conditioning pre-pulse ranging from  100 to
26 H.C. Joca et al. / European Journal of Pharmacology 756 (2015) 22–29

Fig. 3. Carvacrol inhibits TTX-resistant voltage-gated Na þ current (TTX-R Na þ current) of dissociated dorsal root ganglia neurons. (A) Individual examples of TTX-R Na þ
current records for control (Left, black trace), after 120 s of exposure to 300 mM (Middle, dark gray trace) and 1000 mM of carvacrol (Middle, superimposed light gray trace).
Right panel depicts carvacrol-sensitive current traces. (B) The concentration-dependent effects of carvacrol on TTX-R Na þ current are shown for 300 μM (closed bar) and
1000 μM (open bar). Data are plotted as means 7S.E.M. (n ¼16 cells). (C) Composite data to compare fitting parameters of TTX-R Na þ current inactivation in control and after
carvacrol effect reaches steady-state. Amplitude and time constants (fast and slow components) for current inactivation were calculated by a fit with double exponential
function. *Po 0.05. Paired Student's t-test. Bar graphs represent means 7 S.E.M. (n¼7 cells).

Fig. 4. Effects of carvacrol on TTX-R Na þ currents. (A) TTX-R Na þ currents evoked by depolarization to potentials from  100 to þ 55 mV in 5 mV steps. Holding potential
was set at  100 mV. Control (Left panel); after addition of carvacrol 300 mM (Middle panel). Right panel illustrates carvacrol-sensitive TTX-R Na þ currents obtained by
obtained by digital subtraction of the current with carvacrol from the control traces. (B) I–V relationship of TTX-R Na þ current in the presence of 300 mM carvacrol. Control
(open circles) and carvacrol (filled circles). Data are means 7S.E.M. (n ¼7 cells). (C) Voltage-dependence for steady-state activation (circles) and inactivation (squares) of TTX-
R Na þ current in the absence (open symbols) and in presence of carvacrol at 300 μM (filled symbols). Carvacrol induced a negative-shift in steady-state activation and
inactivation. Continuous lines are representing the best fit to a Boltzmann distribution (see Section 2 for more details). (n¼ 7 cells in each experimental condition).

þ10 mV in 5 mV increments and maintaining the holding potential at to 300 μM carvacrol (control: –24.270.8 mV, n¼7; carvacrol: 40.4
80 mV. The mid-point of inactivation was shifted 16.1 mV towards 71.5 mV, n¼7; paired t-test, Po0.05). We did not observe any
more hyperpolarized membrane potentials for DRG neurons exposed significant difference in the slope factor.
 H.C. Joca et al. / European Journal of Pharmacology 756 (2015) 22–29 27

Fig. 5. Recovery from inactivation, use-dependence, and state-dependence: effects of carvacrol. (A) The recovery time from inactivation. Neurons were depolarized by
paired-pulses with different time intervals (n¼ 10). (B) Expanded version of recovery graph to depict the early (first 3 ms) time points. (C) Increase in stimulation frequency
(from 0.2 Hz to 1 Hz) enhanced the carvacrol-induced inhibition of TTX-R Na þ current. (D) Carvacrol preferentially blocks TTX-R Na þ channels that are in the inactivated
state. *Po 0.05. One Way Anova followed by Tukey test was used for statistical analyses.

To further establish whether carvacrol altered TTX-resistant Na þ
channels repriming kinetics we decided to perform experiments to
evaluate the recovery from inactivation of the TTX-resistant Na þ
current in isolated DRG neurons. Fig. 5A illustrates the observed
changes in the recovery from inactivation that occur when TTX-
resistant Na þ channels are exposed to carvacrol. The ratio of current
amplitude was plotted against the repriming interval at  120 mV. The
repriming process was well described by the sum of two exponential
functions. For DRG control neurons (n¼6), recovery was biexponential
and included a large fast component (contribution of  75%). This
initial phase represents Na þ channels recovering from fast-inactivated
states in sub-millisecond time scale (τfast ¼0.6770.05 ms) and, the
remaining channels are recovering from slow-inactivated states
(τslow ¼77.2715.3 ms). In the presence of carvacrol (n¼ 6), the con-
tribution (75%) of the rapid component of recovery was not altered,
but the rate of recovery from fast-inactivated states was significantly
delayed (τfast ¼ 1.3770.07 ms). The calculated time-constant (τslow) for
the slow component of recovery was 298.8756.1 ms. These results
support the notion that carvacrol by interacting with TTX-resistant
Na þ channels elicits modifications in the repriming kinetics. The
average repriming kinetics at  120 mV for the first 3 ms of recovery
are illustrated in Fig. 5B.
At this point we considered to be of interest to determine if the
stimulation frequency (use-dependent blocking) and the membrane
potential influence the effects of carvacrol on TTX-resistant Na þ
channels. Fig. 5C depicts frequency-response of carvacrol at 300 μM.
The data obtained in the presence of carvacrol revealed evidence of
frequency-dependent TTX-resistant Na þ channel block. Increasing
stimulation frequency from 0.2 Hz to 1 Hz an approximately 20%
additional TTX-resistant Na þ current is inhibited by the drug (Fig. 5C).
The same not occurs to total voltage-dependent Na þ current (Supple-
mental Fig. 2A). The use-dependence of carvacrol on TTX-resistant
Na þ channels was indicative of state-dependent binding interactions
which could arise from voltage-dependent changes in the conforma-
tion of the drug binding site (Hille, 1977). We, therefore, evaluated
carvacrol block at different membrane holding potentials. Fig. 5D
shows that greater block was observed when DRG neurons were held
at  50 mV than 120 mV. Also, total voltage-dependent Na þ current
shows similar pattern (Supplemental Fig. 2B).
4. Discussion
Our results demonstrate that carvacrol is a Na þ channel blocker.
We propose that carvacrol causes a decrease in membrane excit-
ability via inhibition of voltage-gated Na þ channels in DRG neurons.
Decreases in Na þ current density impairs action potential generation
by DRG neurons ultimately resulting in decrease in cell excitability.
The major findings that support this conclusion are as follows:
(1) Carvacrol causes a robust decrease in voltage-dependent Na þ
currents; (2) Carvacrol elicits a leftward shift in the voltage depen-
dence of steady-state inactivation; (3) Carvacrol accelerates time-
dependent inactivation; (4) Carvacrol blocks indistinctively TTX-R
and TTX-S Na þ currents; and (5) Carvacrol stabilizes inactivated
states of the TTX-resistant voltage-dependent Na þ channels. These
findings are consistent with results from previous studies implicating
carvacrol as an action potential blocker (Gonçalves et al., 2010; Joca
et al., 2012).
In a preceding study we have demonstrated that carvacrol inhibits
voltage-gated Na þ currents in isolated DRG neurons. However, the
detailed mechanism was not investigated. We made, therefore, an
attempt to characterize how carvacrol-induced changes in Na þ gating
could explain its pharmacological effects.
Firstly, we will discuss the main results obtained by measuring
what we called total voltage-dependent Na þ current which represents
the contribution of TTX-sensitive and TTX-resistant Na þ channels,
28 H.C. Joca et al. / European Journal of Pharmacology 756 (2015) 22–29
since large DRG neurons express TTX-S Na þ channels NaV 1.1, NaV
1.6 and NaV 1.7, and the TTX-R Na þ channels NaV 1.8 and NaV 1.9
(Dib-hajj et al., 2010).
Carvacrol had negligible effect on the voltage-dependent char-
acteristics of Na þ currents activation but produced a clear hyper-
polarization shift in the steady-state inactivation relationship. This
profile is quite consistent with the possibility that carvacrol bind
selectively to the inactivated state of the channels. State depen-
dent affinity has been described previously for other Na þ channel
blockers, notably the local anesthetics (like lidocaine, Balser et al.,
1996) and a variety of anticonvulsants (phenytoin, carbamazepine,
and lamotrigine; Catterall, 1999).
As suggested by the results shown in Fig. 2B, carvacrol enhanced
current inactivation indicating that carvacrol could be an open-
channel blocker. If we add this observation on top of the state-
dependent inhibitory effect, carvacrol has basically all basic features
to be considered a local anesthetic. The slow dissociation observed
for carvacrol-induced block of total Na þ currents is likely because of
its high lipophilicity (octanol:water partition coefficient approxi-
mately 4,370). However, we cannot totally rule out the possibility
that carvacrol-induced acceleration of Na þ current decay may be
related to drug-induced inactivation (Hille, 1977).
In the present study, we demonstrated that carvacrol blocked
total voltage-gated Na þ currents in DRG neurons with an IC50 of
300 μM at a holding potential of  100 mV. At this point, we may
conclude that carvacrol inhibits voltage-dependent Na þ channels in
the resting state in a way that is similar to the other essential oil
constituents. Sheets et al., 2008 stated that possible side effects
related mainly to the central nervous system may significantly limit
the use of Na þ channel blockers in attenuating pain; therefore, they
concluded that drugs which are better able to differentiate normal
and abnormal neuronal activity are desirable because these drugs
will present a much better safety profile and tolerability.
As we mentioned above two distinct types of TTX-resistant Na þ
currents (NaV 1.8- and NaV 1.9-type) have been identified in DRG
neurons (Cummins et al., 1999; Dib-Hajj et al., 1999; Sheets et al.,
2008). Akopian et al. (1999) demonstrated that NaV 1.8 channels are
implicated in inflammatory pain. Lai et al. (2002) provided evidence
that NaV 1.8 channels are involved in neuropathic pain. The most
important is the fact that selective block of NaV 1.8 currents allev-
iates both of these types of pain (Jarvis et al., 2007). Therefore, NaV
1.8 currents are an important target when investigating new anti-
nociceptive substances.
Ho and O’Leary (2011) in a study using single-cell analysis of NaV
expression in DRG neurons came to the conclusion that NaV 1.8 chan-
nels actually underlie the TTX-R Na þ current as measured by whole-
cell recordings. We decided to block TTX-sensitive Na þ current with
tetrodotoxin (300 nM) to isolate NaV 1.8 current and conduct a better
characterization in terms of changes in gating kinetics. We found that
carvacrol at 300 μM inhibited TTX-resistant Na þ currents to a lesser
extent (approximately 25%) when compared to total Na þ currents
which at the same concentration elicited 50% blockade. This observa-
tion suggests that NaV 1.8 channels are less sensitive to carvacrol than
TTX-S component (Fig. 3).
Our present experiments reveal that when carvacrol (300 μM) is
present, a pronounced shift towards more hyperpolarized membrane
potentials ( 9 mV) was observed in the NaV 1.8 steady-state activa-
tion. To our knowledge, this is the first report to document the effect
on NaV 1.8 activation gating induced by a terpene. This result was
particularly surprising given that carvacrol is assumed to have anti-
nociceptive effects. At this point, we do not have a clear mechanistic
explanation for this effect.
It is well accepted that drugs which act on ion channels could
exhibit preferential block of one or more channel states over
others. For the NaV 1.8 channels, closed state block by carvacrol
was rather weak but when we changed frequency stimulation by a
factor of 5 (0.2–1.0 Hz) a 25% additional block could be achieved.
Taken together these results suggest that NaV 1.8 channel states
visited during high frequency stimulation uncovered higher affinity
binding sites compared with the closed state in which channels stay at
more negative potentials.
The phenomenon of use-dependence relies on the repetitive
and brief membrane depolarizations that force Na þ channels to
cycle through activated, inactivated and repriming (rate of recov-
ery from inactivation) states. The use-dependent block exerted by
carvacrol is probably due to slow rate of dissociation from blocked
NaV 1.8 channels during membrane repolarization.
Furthermore, membrane depolarization increased carvacrol affi-
nity by approximately 17-fold (to estimate the projected IC50 for
both holding potentials,  120 mV (IC50  900 μM) and  50 mV
(IC50  53 μM), we used the equation as described by Sheets et al.
(2008)). These data indicate that carvacrol is much more effective at
inhibiting inactivated NaV 1.8 channels than resting channels. One
would argue that carvacrol, as a potential analgesic drug, would
benefit from its higher affinity towards inactivated NaV 1.8 channels
because nociceptor hyperexcitability and increased pain sensations
are likely to be associated with depolarized membrane potential in
neurons.
5. Conclusion
In summary, the present data clearly demonstrate that carva-
crol is capable of blocking NaV 1.8 channels in isolated DRG
neurons at concentrations that may be useful as a pharmacological
tool to further explore the actual role of NaV 1.8 current in
physiological and pathophysiological conditions.
Acknowledgments
This work was supported by research grants from CNPq, Brazil
(DAM Araújo, #479764/2012-3; JS Cruz, # 476411/2013-0 and
308007/2013-1) and FAPEMIG, Brazil (JS Cruz, #APQ-00460-12).
HC Joca held a scholarship from CAPES. JS Cruz and DAM Araújo
are CNPq research fellows.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.ejphar.2015.03.007.
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