Selective Activation of hTRPV1 by N-Geranyl Cyclopropylcarboxamide, an Amiloride-Insensitive Salt Taste Enhancer
Salt Taste Enhancer. PLoS ONE 9(2): e89062. doi:10.1371/journal.pone.0089062
Selective Activation of hTRPV1 by N -Geranyl Cyclopropylcarboxamide, an Amiloride-Insensitive Salt Taste Enhancer
Min Jung Kim 0
Hee Jin Son 0
Yiseul Kim 0
Hae-Jin Kweon 0
Byung-Chang Suh 0
Vijay Lyall 0
Mee- Ra Rhyu 0
Yoshiro Ishimaru, University of Tokyo, Japan
0 1 Division of Metabolism and Functionality Research, Korea Food Research Institute , Bundang-gu, Sungnam-si, Gyeonggi-do , Republic of Korea, 2 Department of Brain Science, DaeguGyeongbuk Institute of Science and Technology (DGIST), Daegu, Republic of Korea, 3 Department of Physiology and Biophysics, Virginia Commonwealth University , Richmond, Virginia , United States of America
TRPV1t, a variant of the transient receptor potential vanilloid-1 (TRPV1) has been proposed as a constitutively active, nonselective cation channel as a putative amiloride-insensitive salt taste receptor and shares many properties with TRPV1. Based on our previous chorda tympani taste nerve recordings in rodents and human sensory evaluations, we proposed that Ngeranylcyclopropylcarboxamide (NGCC), a novel synthetic compound, acts as a salt taste enhancer by modulating the amiloride/benzamil-insensitive Na+ entry pathways. As an extension of this work, we investigated NGCC-induced human TRPV1 (hTRPV1) activation using a Ca2+-flux signaling assay in cultured cells. NGCC enhanced Ca2+ influx in hTRPV1expressing cells in a dose-dependent manner (EC50 = 115 mM). NGCC-induced Ca2+ influx was significantly attenuated by ruthenium red (RR; 30 mM), a non-specific blocker of TRP channels and capsazepine (CZP; 5 mM), a specific antagonist of TRPV1, implying that NGCC directly activates hTRPV1. TRPA1 is often co-expressed with TRPV1 in sensory neurons. Therefore, we also investigated the effects of NGCC on hTRPA1-expressing cells. Similar to hTRPV1, NGCC enhanced Ca2+ influx in hTRPA1-expressing cells (EC50 = 83.65 mM). The NGCC-induced Ca2+ influx in hTRPA1-expressing cells was blocked by RR (30 mM) and HC-030031 (100 mM), a specific antagonist of TRPA1. These results suggested that NGCC selectively activates TRPV1 and TRPA1 in cultured cells. These data may provide additional support for our previous hypothesis that NGCC interacts with TRPV1 variant cation channel, a putative amiloride/benzamil-insensitive salt taste pathway in the anterior taste receptive field.
Funding: This study was supported by Korea Food Research Institute (E0121201, E0131201), the National Research Foundation of Korea (NRF), funded by the
Ministry of Education, Science and Technology (No. 2012R1A1A2044699), and NIH/NIDCD grant DC011569. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Sodium ion (Na+) is the principal extracellular ion and is
essential for maintaining homeostasis in the body. At
concentrations below 250 mM Na+ is generally appetitive and helps to
enhance the flavor intensity of food. People in developed countries
ingest far more salt than is required to maintain a normal Na+
balance. In the United States, Na+ intakes in adults are around
3900 mg per day which is significantly greater than the maximum
recommended value of 2300 mg per day. Approximately 77% of
the Na+ consumed is derived from processed foods in USA .
The excessive salt consumption is associated with many diseases,
including hypertension, heart attack, stroke, fluid retention, weight
gain, Ca2+ deficiency and osteoporosis, stomach cancer .
Therefore, it is imperative that the food industry should make a
considerable effort in finding novel ways to reduce salt content in
their food products and shift their focus to search for salt taste
enhancers as one of the alternative approaches to lower salt intake
in the general population .
Currently, there is evidence for the presence of at least two salt
taste receptors in the tongue which are involved in appetitive salt
taste perception that demonstrate different sensitivity to amiloride
and benzamil (Bz). One salt taste receptor is the
amiloridesensitive epithelial Na+ channel (ENaC), the Na+ specific salt taste
receptor in fungiform papilla taste receptor cells (TRCs) in the
anterior tongue in mammals, including humans . In rats
and mice, ENaC consists of three subunits: a, b, and c. In humans,
an additional subunit, d is expressed, so the human TRC ENaC is
additionally composed of d, b and c subunits. ENaC is blocked by
amiloride and Bz, however, the dbc ENaC (IC50 around 2.7 mM)
is 10 times less sensitive to amiloride than the human abc ENaC
(IC50 around 0.11 mM) . However, amiloride at 100 mM
(approximately 50 times greater than the IC50 for human dbc
ENaC) reduced only 540% of lingual surface potential to salt
stimuli in humans measured by gustometer, while amiloride
inhibited about 70% of chorda tympani (CT) taste nerve response
to NaCl [12,13]. These results suggest that in both humans and
some rodents, besides ENaC, additional taste receptors must be
involved in salt taste transduction.
Single unit recordings of the CT nerve [14,15] suggest that
additional salt taste transduction pathway must exist which
respond to a variety of cations, including Na+, K+, NH4+ and
Ca2+ that are amiloride-insensitive. In some mice the magnitude of
the amiloride-insensitive component is genetically determined
[10,16]. Lyall et al. first presented electrophysiological evidence
that modulators of the transient receptor potential vanilloid-1
channel (TRPV1), such as resiniferatoxin (RTX) and capsaicin,
modulate the amiloride- and Bz-insensitive NaCl CT response in a
biphasic manner, at low concentrations enhancing and high
concentrations inhibiting the Bz-insensitive NaCl CT response in
WT mice and rats [17,18]. The constitutive Bz-insensitive NaCl
CT response and the enhanced response in the presence of RTX
and capsaicin were inhibited by SB-366791, a specific TRPV1
blocker. In addition, other TRPV1 modulators, such as ethanol,
nicotine, temperature, and adenosine triphosphate (ATP) were
shown to modulate the Bz-insensitive NaCl CT response. These
data suggested that TRPV1-dependent pathway may be a major
component of the Bz-insensitive NaCl CT response in
SpragueDawley rats and WT mice. In addition, the SB-366791-sensitive
spontaneous Bz-insensitive NaCl CT response in the absence of
TRPV1 modulators, at room temperature, close to neutral pH
suggests that a variant of TRPV1 (TRPV1t) may be involved in
amiloride-insensitive salt taste [14,17,19,21]. Although studies
performed by Smith et al. (2012) did not support this hypothesis in
mice , a correlation between genetic variation in TRPV1 and
salt taste perception in human was observed by human salt taste
perception studies . The rs8065080 polymorphism from T
allele to C allele in TRPV1 gene significantly increased
suprathreshold salt taste sensitivity in human. These results
provide additional support for a link between TRPV1t and salt
taste in humans. At high concentration NaCl is aversive (.
500 mM) and is sensed by two aversive pathways localized in
bitter and sour sensing TRCs .
Among monovalent chloride salts, choline chloride is suggested
to be a salt taste enhancer and replacer for Na+ because choline
chloride has salt taste-enhancing properties in animal model .
Accordingly, several choline-containing compounds were
synthesized to develop as salt substitutes and/or enhancers . Based
on this report, N-geranylcyclopropylcarboxamide (NGCC),
Ngeranylisobutanamide, N-geranyl 2-methylbutanamide, allyl
Ngeranylcarbamate, and N-cyclopropyl E2,Z6-nonadienamide were
synthesized by International Flavors & Fragrances (IFF). The
effects of these compounds on the amiloride-sensitive and
amiloride-insensitive salt taste pathways havebeen investigated in
WT and TRPV1 KO mice by electrophysiological studies. Of
these compounds, only NGCC produced biphasic effects on the
amiloride-insensitive salt taste pathway, without altering the
amiloride-sensitive salt taste pathway . Most importantly,
NGCC at the concentrations around which it maximally
enhanced the Bz-insensitive NaCl CT response in rodents also
enhanced salt taste perception of NaCl solutions (6080 mM) in
Since NGCC enhances salt taste responses on both rodents and
humans, in this paper we performed Ca2+-flux signaling assay in
hTRPV1-transfected Human Embryonic Kidney (HEK293T)
cells to investigate whether NGCC directly activates hTRPV1,
and hence, the putative amiloride-insensitive salt taste receptor,
TRPV1t. To test whether NGCC specifically activates TRPV1 we
also tested its effects on the abc human ENaC (hENaC)-expressing
HEK293T cells by membrane potential assay. The effects of
NGCC on acid-sensing ion channels 1a (ASIC1a) were also
investigated because the proton-gated ASIC is
amiloride-insensitive and belongs to the ENaC/Degenerin superfamily. In addition,
we also investigated the effect of NGCC on hTRPA1-transfected
HEK293T cells because TRPA1 and TRPV1 are frequently
coexpressed in sensory neurons.
Materials and Methods
Capsaicin, capsazepine (CPZ), ruthenium red (RR),
HC030031, and benzamil (Bz) were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Allylisothiocyanate (AITC) was obtained
from Wako Pure Chemicals Industries Ltd. (Osaka, Japan). S3969
was synthesized as described previously  by professor Jeon at
Kwangwoon University. NGCC was kindly provided by Dr. Mark
Dewis at IFF (Union Beach, NJ, USA). S3969 and NGCC
structures are shown in Figure 1. All media used in cell cultures
were obtained from Life Technologies, Inc. (Grand Island, NY,
Cell Culture and Transfection
hTRPV1 was constructed by amplification of the hTRPV1
region (NCBI accession number: NG_029716.1) and cloned into
pEAK10 vector (Edge Biosystems, Gaithersburg, MD, USA). The
hTRPV1 construct was amplified by PCR and the nucleotide
sequence of the hTRPV1 gene was confirmed by sequencing with
an ABI 3130 DNA genetic analyzer (Applied Biosystems, Foster
City, CA, USA). The HEK293T cells which were cultured at
37uC in Dulbeccos Modified Eagles Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 1%penicillin/
streptomycin were seeded onto a 100-mm dish and transfected
with the hTRPV1 expression plasmid using Lipofectamine 2000
(Invitrogen). After 6 h, transfected cells were seeded onto 96-well
black-wall plate (BD Falcon Labware, Franklin Lakes, NJ, USA)
for 1826 h prior to their use in an experiment.
The a, b, and c hENaC subunits were cloned from OriGene
(Rockville, MD; GenBank accession number NM_00001038 for
SCNN1A, NM_000336 for SCNN1B and NM_001039 for
SCNN1G). a, GFP-tagged b, and c hENaC were cloned into a
CMV promoter-based vector and expressed constitutively. The
HEK293T cells were seeded onto a 35 mm dish and transfected
with a, b, and c hENaC expression plasmid using Lipofectamine
2000 (Invitrogen). Then, transfected cells were seeded onto 96-well
black-wall plate for 1826 h prior to their use in an experiment.
Mouse ASIC1a (mASIC1a) was a kind gift from John A.
Wemmie (University of Iowa, IA, USA). For electrophysiological
experiments, HEK293T cells were cultured in DMEM
supplemented with 10% FBS and 0.2% penicillin/streptomycin and
transiently transfected using Lipofectamine 2000 (Invitrogen) with
various cDNAs. For TRPV1 channel expression, cells were
transfected with hTRPV1, for ASIC1a homomeric channel
expression, cells were transfected with mASIC1a. When needed,
0.10.2 mg of cDNA encoding tetrameric red fluorescence protein
(DsRed) was co-transfected with the cDNA as a marker for
successfully transfected cells. For non-transfected cells, cells were
transfected with 0.10.2 mg of cDNA encoding DsRed alone. The
next day, cells were plated onto poly-L-lysine-coated coverslip
chips, and fluorescent cells were studied within 12 days.
Flp-In 293 cell lines stably expressing hTRPA1constructed as
previously reported  was a gift from the professor Takumi
Misaka at University of Tokyo. The hTRPA1-expressing cells
were cultured in DMEM containing 10% FBS and 0.02%
hygromycin B (Invitrogen). Flp-In 293 cell lines were maintained
in DMEM containing 10% FBS. All cells were incubated at 37uC
in a humidified atmosphere of 5% CO2. Cultured
hTRPA1expressing cells and Flp-In 293 cell lines were seeded onto 96-well
black-wall plate for 24 h and used for Ca2+ responses to AITC and
Ca2+ Imaging Analysis of the Cellular Response of
hTRPV1- or hTRPA1-expressing Cells
Mock-transfected HEK293T cells, hTRPV1-expressing
HEK293T cells, non-hTRPA1-expressing Flp-In 293 cells, and
hTRPA1-expressing Flp-In 293 cells grown in 96-well black-wall
plates were rinsed with assay buffer (130 mM NaCl, 10 mM
glucose, 5 mM KCl, 2 mMCaCl2, 1.2 mM MgCl2, and 10 mM
HEPES, pH 7.4) and loaded with 5 mM Fura-2 AM (Invitrogen)
for 30 min at 27uC. The cells were washed with assay buffer and
treated with ligands. AITC (10 mM) and capsaicin (1 mM)
dissolved in assay buffer were used as the hTRPA1 and hTRPV1
ligands, respectively. hTRPA1 and hTRPV1 expressing cells were
treated with 30 mM NGCC dissolved in assay buffer. The
fluorescence intensities of Fura-2 excited at 340 and 380 nm were
simultaneously measured at 510 nm using a computer-controlled
filter changer (Lambda DG4; Sutter, San Rafael, CA, USA), an
Andor Luca CCD camera (Andor Technology, Belfast, UK), and
an inverted fluorescence microscope (IX-71; Olympus, Tokyo,
Japan). Intracellular calcium images were recorded every 3-s for
60-s and analyzed using MetaFluor software (Molecular Devices,
Sunnyvale, CA, USA). For the blocking assay, 30 mM RR or
1 mM CPZ was added with capsaicin or NGCC in
hTRPV1expressing cells and 30 mM RR or 100 mM HC-030031 was
added with AITC or NGCC in hTRPA1-expressing cells.
Measurement of Ca2+ Influx in hTRPV1- or
The ligand-induced changes on cytosolic Ca2+ level were
monitored by a FlexStation III microplate reader (Molecular
Devices). Fluo-4 AM (5 mM, Molecular Probes, Eugene, OR,
USA) in assay buffer was loaded to the cells in 96-well black-wall
plates for 30 min at 27uC. After dye treatment, each chemical was
added to each well at 17 s and changes on intracellular Ca2+ level
were monitored by relative fluorescence units (DRFU,
Ex = 485 nm; Em = 516 nm) for 120 s. The chemicals including
capsaicin (16102423610 mM) and NGCC (1610242
16103 mM) were treated to hTRPV1-expressing cells and those
including AITC (161024236102 mM) and NGCC (1610242
16103 mM) were treated to hTRPA1-expressing cells. The
responses from at least three wells receiving the same stimulus
were averaged. Plots of amplitude versus concentration were fitted
using the Hill equation. For the blocking assay, 30 mM RR or
1 mM CPZ was added with capsaicin or NGCC in
hTRPV1expressing cells and 30 mM RR or 100 mM HC-030031 was
added with AITC or NGCC in hTRPA1-expressing cells.
Membrane Potential Assay on abc hENaC-expressing
hENaC-expressing cells were characterized using FLIPER
Membrane Potential (FMP) Assay Kit (Molecular Devices
Corporation, Sunnyvale, CA, USA). In this assay, changes in the
membrane potential were quantified via fluorescence change
caused by FMP dye. The hENaC-expressing cells were washed
with assay buffer (containing: 130 mM NaCl, 10 mM glucose,
5 mM KCl, 2 mM CaCl2, 1.2 mM MgCl2, and 10 mM HEPES,
pH adjusted to 7.4 with NaOH) and subsequently, FMP blue-dye
in assay buffer was loaded into the cells at room temperature for
30 min. After dye treatment, S3969 (0.03210 mM), an ENaC
agonist, S3969+Bz (ENaC antagonist, 0.01 mM), or NGCC
(0.01210 mM) was treated to hENaC-expressing cells. The plate
was assayed in a FlexStation III plate reader (Molecular devices
Corporation) by excitation at 530 nm and measuring the emission
at 560 nm.
Whole-cell Patch Clamp Recording
The whole-cell configuration was used to voltage-clamp at room
temperature (2225uC). Electrodes pulled from glass micropipette
capillaries (Sutter Instrument) had resistances of 22.5 MV, and
series resistance errors were compensated .60%. Fast and slow
capacitances were compensated before the application of
testpulse. Recordings were performed using aHEKAEPC-10
amplifier with pulse software (HEKA Elektronik). The pipette solution
contained: 140 mM KCl, 5 mM MgCl2, 10 mM HEPES,
0.1 mM 1,2-bis(2-aminophenoxy) ethane N,N,N,N-tetraacetic
acid(BAPTA), 3 mM Na2ATP, and 0.1 mM Na3GTP, adjusted
to pH 7.4 with KOH. The external Ringers solution used for
recording TRPV1 and TRPA1 currents contained: 150 mM
NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM EGTA, 10 mM Glucose,
and 10 mM HEPES, adjusted to pH 7.4 with NaOH. TRPV1
and TRPA1 currents were recorded by holding the cell at 2
80 mV. The external Ringers solution used for recording ASIC
current contained: 160 mM NaCl, 5 mM KCl, 1 mM MgCl2,
2 mM CaCl2, and 10 mM HEPES, adjusted to pH 7.4 with
tetramethylammonium hydroxide. For acidic solution of pH 6.0,
HEPES was replaced with MES. ASIC currents were recorded by
holding the cell at 270 mV. Reagents were obtained as follows:
BAPTA, Na2ATP, Na3GTP, EGTA, and tetramethylammonium
hydroxide (Sigma), HEPES (Calbiochem), MES (Alfa Aesar), and
other chemicals (Merck).
The statistical significance of the results was examined through
one-way analysis of variance (ANOVA) using Duncans multiple
range test. Doseresponse analyses were carried out with
GraphPad Prism software (GraphPad Software Inc., San Diego,
CA, USA). Data are presented as means 6 SEM.
NGCC-induced Activation of TRPV1t/TRPV1
The effects of capsaicin and NGCC on hTRPV1-expressing
cells or mock-transfected cells are shown in Figure 2A and
Figure 2B at the single concentration. Capsaicin, a specific agonist
of hTRPV1, was used as a positive control. As a result, NGCC
(30 mM) increased Ca2+ influx in hTRPV1-expressing cells, and
RR (30 mM) and CPZ (1 mM) blocked NGCC activity to baseline
levels (Figure 2A). NGCC induced no changes in fluorescence in
mock-transfected cells (Figure 2B). NGCC produced a
concentration-dependent increase in the intracellular Ca2+ in
hTRPV1expressing cells (Figure 2C). The EC50 values for capsaicin and
NGCC on hTRPV1-expressing cells were 0.527 mM and 115 mM,
respectively. CPZ (1 mM) blocked the effects of capsaicin and
NGCC, however, it did not completely inhibit the hTRPV1
activity at high concentrations of capsaicin ($30 mM) and NGCC
($103 mM). RR (30 mM) also inhibited capsaicin- and
NGCCinduced hTRPV1 activation, but was less effective than CPZ
(1 mM). In the presence of RR (30 mM) no increase in intracellular
Ca2+ was observed at low concentrations of the two agonists (both
less than 1 mM).
To verify whether hTRPV1 was activated by NGCC, we
patchclamped mock-transfected or hTRPV1-expressing cells (Figure 3).
We observed the effect of NGCC on cells transiently expressing
hTRPV1. As a positive control, we used capsaicin (100 nM) to
induce TRPV1 currents. Bath application of capsaicin for 10
seconds induced an inward current in the cell expressing
hTRPV1, but not in the non-transfected cell (Figure 3A).
Capsaicin-induced current was blocked by preincubation of the
cell with Ringers solution containing CPZ (1 mM) for 20 seconds
before the second application of capsaicin and the response
recovered after wash out of CPZ. Moreover, NGCC (103 mM) also
triggered an inward current in the same cell. Like capsaicin,
NGCC triggered no currents in non-transfected cells.
NGCCinduced currents were also inhibited by CPZ, and then, partially
recovered after wash out of CPZ similar to capsaicin-evoked
currents in cells expressing hTRPV1 (Figure 3B). Normalized
current density was decreased to 0.1260.05 (n = 4) by treatment of
CPZ and recovered to 0.6160.12 (n = 4) after wash out of CPZ
(Figure 3C). NGCC activated inward currents in cells expressing
hTRPV1 in a concentration-dependent manner (Figure 3D).
These results suggest that NGCC is an agonist for TRPV1.
Effects of NGCC on Amiloride/Benzamil-sensitive Salt
Taste Receptor, ENaC
Prior to investigating the effects of NGCC on the
amiloridesensitive salt taste receptor, the efficiency of abc
hENaCexpressing cells was evaluated using S3969 (Figure 4). Between
0.03 and 10 mM, S3969 depolarized the membrane potential in
hENaC expressing cells in a concentration-dependent manner
with an EC50 value of 1.26260.290 mM. No effect of S3969 was
observed in the continuous presence of Bz. Following this, we
investigated the effect of NGCC on abc hENaC-expressing cells
using same system. Although we tested a wide range of NGCC
concentrations, no significant changes in membrane potential were
observed in abc hENaC-expressing cells relative to cells stimulated
by buffer alone. Thus, similar to our earlier findings , NGCC
did not affect amiloride/benzamil-sensitive salt taste receptor.
Effects of NGCC on Amiloride-sensitive Acid-sensing Ion
To determine whether NGCC also activates amiloride-sensitive
acid-sensing ion channels (ASICs) which belong to ENaC/DEG
superfamily of ion channels , we also tested the effect of
NGCC on ASIC1a homomeric channels in HEK293T cells.
Previous study showed that ASIC1 subunit is expressed in taste
receptor cells and involved in taste transduction [29,30]. Rapid
change of extracellular pH from 7.4 to 6.0 for 10 seconds evoked
inward currents in cells expressing homomeric ASIC1a channels
(Figure 5A). Although HEK293 cells are reported to express
endogenous human ASIC1a , the current density of pH
6.0induced currents in non-transfected HEK293T cells was
5.5261.33 pA/pF (n = 3), while the current density of pH
6.0induced currents in mASIC1a-expressing HEK293T cells was
118.1633.0 pA/pF (n = 3). Therefore, we regarded pH
6.0induced currents as mASIC1a-mediated currents in cells
transiently expressing mASIC1a. For the purpose of this study, we
applied pH 6.0 solution or NGCC on the extracellular site of cells
expressing mASIC1a to determine whether NGCC has potential
for activating ASIC1a homomeric channels. As a result,
mASIC1a-mediated currents were activated by extracellular pH
change from 7.4 to 6.0, but not by application of NGCC
Activation of hTRPA1 by NGCC
Finally, the effects of AITC and NGCC on hTRPA1-expressing
cells were investigated in a single dose. AITC, a TRPA1 agonist
was used as a positive control. Figure 6A showed that the
intracellular Ca2+ concentration was increased after 10 mM AITC
or 30 mM NGCC treatment and the Ca2+ influx induced by
10 mM AITC and 30 mM NGCC was significantly inhibited by
RR (30 mM) and HC-030031 (100 mM). Comparison of NGCC
activity in non-hTRPA1-expressing Flp-In 293 cells showed that
NGCC reacts specifically with hTRPA1 (Figure 6B).
Subsequently, AITC- and NGCC-induced Ca2+ influx was monitored in a
concentration-dependent manner (Figure 6C). As a result, AITC
and NGCC activated Ca2+ influx in hTRPA1-expressing cells in a
concentration-dependent manner. The EC50 values for AITC and
NGCC on hTRPA1-expressing cells were 5.852 mM and
83.65 mM, respectively. RR (30 mM) and HC-030031 (100 mM)
almost blocked NGCC activity on hTRPA1-expressing cells.
However, RR (30 mM) and HC-030031 (100 mM) partially
blocked AITC activity below 30 mM and 10 mM, respectively.
These results were also confirmed using patch clamp technique.
We tested the effect of NGCC on cells stably expressing hTRPA1.
Application of AITC (100 mM) which is well-known as an agonist
of TRPA1 for 10 seconds, evoked an inward current in the
hTRPA1-expressing cell, but not in the non-transfected cell
(Figure 7A). AITC-induced current was partially blocked by
preincubating the cells with Ringers solution containing
HC030031 (100 mM) for 30 seconds before the second application of
AITC and the response recovered after wash out of HC-030031.
In the same cell, NGCC (103 mM) also induced an inward current.
Consistent with Ca2+ imaging data, the amplitude of current
induced by NGCC (103 mM) was bigger than that of AITC
(100 mM)-induced current. However, NGCC triggered no current
in non-transfected cells as like AITC. NGCC-induced currents
were almost completely inhibited by HC-030031 and the response
recovered after wash out of HC-030031 in hTRPA1-expressing
cells (Figure 7B). Normalized current density was decreased to
Figure 2. Calcium responses in hTRPV1-expressing cells stimulated with capsaicin (Cap) and NGCC. (A) Cells expressing hTRPV1 were
loaded with Fura-2 AM and Ca2+ images were obtained at 0 and 60 s after stimulation with Cap or NGCC. The non-specific blocker of TRP channels,
RR (30 mM), and the specific TRPV1 antagonist, CPZ (5 mM), were added to test the selectivity of Cap and NGCC. Representative ratiometric images are
shown after treatment with Cap and NGCC. (B) As a control, the Ca2+ response was monitored in non-hTRPV1-expressing HEK 293T cells treated with
Cap or NGCC. (C) The effects of Cap or NGCC treatment were quantified using Calcium-4 in a cell-based assay in the presence or absence of 30 mM RR
or 5 mM CPZ. Experiments were repeated in triplicate and data points represent the means 6 SEM (n = 3).
0.2560.03 (n = 4) by addition of HC-030031 and recovered to
1.3760.07 (n = 4) (Figure 7C). NGCC triggered inward currents
in cells stably expressing hTRPA1 in a dose-dependent manner
The sense of taste relies on Na+ for sensing salty taste. Because
excessive salt consumption is associated with numerous diseases,
there is a great incentive to develop Na+ substitutes and salt taste
enhancers that can help in reducing salt intake. Previously, we
have shown that NGCC is a salt taste modifier. Specifically, using
whole CT nerve recordings in WT and TRPV1 KO mice and
stimulating the tongue with NaCl+Bz (an ENaC inhibitor), NGCC
produced biphasic effects on the Bz-insensitive NaCl CT response.
At low concentrations NGCC enhanced and at high
concentrations inhibited the Bz-insensitive NaCl CT response . In
addition, at the concentrations at which NGCC produced the
maximum increase in the CT response in rodents, it produced a
synergistic salt taste-enhancing effect in human psychophysical
tests. At the concentrations that NGCC inhibited the
Bzinsensitive NaCl CT response in rodents, it produced a salt
masking effect in human psychophysical tests. NGCC did not
enhance the Bz-sensitive NaCl CT response . In our earlier
paper we did not have direct evidence that NGCC modulates the
activity of TRPV1 and ENaC is insensitive to it. In this paper we
provide direct evidence that NGCC modulates the activity of
hTRPV1 by monitoring NGCC-induced change in intracellular
Ca2+ levels in hTRPV1-expressing cells.
As shown in Fig. 2, capsaicin, a TRPV1 agonist, produced a
dose dependent increase in [Ca2+]i in HEK 293 cells expressing
hTRPV1, the maximum increase in [Ca2+]i was observed at
around 100 mM capsaicin. The EC50 for capsaicin was around
0.5 mM. In our rat CT recordings, capsaicin produced a biphasic
effect on the Bz-insensitive NaCl CT response . The
maximum increase in the NaCl+Bz CT response was obtained
at 40 mM capsaicin. At concentrations .40 mM capsaicin
inhibited the NaCl+Bz CT response and at 200 mM capsaicin
completely inhibited the NaCl+Bz CT response to the rinse
baseline level. Relative to capsaicin, NGCC produced a
significantly smaller increase in [Ca2+]i and the dose-response
relationship was shifted to the right to higher agonist concentration. The
EC50 for NGCC was around 115 mM. In contrast to the effects of
NGCC on the expressed hTRPV1, in our earlier human sensory
studies NGCC produced a significant enhancement in NaCl taste
Figure 3. Activation of TRPV1 by capsaicin and NGCC. (A) TRPV1 current was activated by capsaicin (100 nM) and reversibly blocked by TRPV1
antagonist, CPZ (1 mM) in HEK293T cells transiently transfected with hTRPV1. NGCC (1 mM) induced an inward current in a capsaicin-sensitive cell.
Neither capsaicin nor NGCC evoked the currents in non-transfected cells (n = 3). Dashed lines indicate zero current. (B) NGCC-induced currents were
blocked by CPZ in hTRPV1-expressing HEK293T cells. (C) Summary of normalized current density in cells expressing hTRPV1 (n = 4). (D) Current
density of the first pulse was normalized to 1.0. NGCC triggered inward currents in a dose-dependent manner in hTRPV1-expressing HEK293T cells
(n = 3). Results are presented as the mean 6 SEM.
Figure 4. Effects of S3969 or NGCC on abc hENaC-expressing cells. Cells expressing abc-hENaC were loaded with FMP blue-dye and the
effects of S3969 or NGCC were quantitatively evaluated using membrane potential assay. S3969 depolarized membrane potential on abc
hENaCexpressing cells and benzamil (Bz) effectively inhibited S3969 activity. NGCC showed no effect on abchENaC-expressing cells. Experiments were
repeated in triplicate and data points represent the means 6 SEM (n = 34).
Figure 5. Effects of NGCC in amiloride-sensitive ASIC1a. (A) Rapid change of extracellular pH from 7.4 to 6.0 for 10 seconds induced an
inward current in HEK293T cells transiently transfected with mASIC1a. Current density of pH 6.0-evoked inward currents is 5.5261.33 pA/pF (n = 3)
and 118.1633.0 pA/pF (n = 3) in non-transfected cells and mASIC1a-expressing cells, respectively. (B) mASIC1a currents were triggered by
extracellular pH drop from 7.4 to 6.0. However, ASIC1a channels are not sensitive to NGCC (1 mM). The time interval between each stimulation is 120
seconds. Normalized current density was measured in cells expressing mASIC1a (n = 5). Results are presented as the mean 6 SEM.
at 5 and 10 mM . These studies suggest that the threshold
concentration of NGCC for producing enhancement in the NaCl
taste perception is significantly lower than the concentration that
It is important to note that unlike our studies with expressed
hTRPV1 (Figure 2), in taste cells NGCC  and capsaicin ,
ethanol , nicotine , NGCC  and naturally occurring
glycol-conjugated peptides  produced biphasic effects on the
Bz-insensitive neural responses and in human salt taste sensory
evaluation studies [27,34]. The biphasic effects of the above
TRPV1 modulators are most likely related to TRPV1 regulation
by changes in [Ca2+]i in TRCs that in turn modulates the
phosphorylation state of the channel via activation of protein
kinase C (PKC) and calcineurin . In addition, changes in
membrane phosphatidylinositol 4,5-bisphosphate (PIP2) in taste
cells modulate the biphasic effects of RTX on the Bz-insensitive
NaCl CT response . Thus differences in the NGCC response
of the expressed TRPV1 channel and the salt sensory perception
in human subjects may reflect differences in the phosphorylation
and regulation of the TRPV1/TRPV1t channel in the anterior
taste receptive field.
In our studies abc hENaC was expressed in HEK293T cells
and the activity of abc hENaC was confirmed by S3969. The
EC50 value for S3969 in abc hENaC-expressing cells was
1.26260.290 mM. This value is very close to the theoretical value
(1.260.1 mM) obtained by patch-clamp . Thus, abc
hENaCexpressing cells were used to investigate the effect of NGCC on
amiloride/benzamil-sensitive salt taste receptor. NGCC did not
alter the membrane potential in abc hENaC-expressing cells,
indicating that NGCC does not stimulate hENaC. This is
consistent with our neural data in rodents in which Bz-sensitive
component of the CT response remained unchanged after
stimulating the tongue with salt solutions containing NGCC
. These results indicate that in both rodents and humans
NGCC alters salt taste by specifically modulating only the
Bzinsensitive component of the CT response in rodents. The
NGCCinduced activation of hTRPV1 was confirmed by blocking
hTRPV1 using RR or CPZ. These findings provide support for
the role of NGCC in the activation of hTRPV1, as suggested
previously . Consistent with these in vitro studies, Bz-insensitive
NaCl CT responses in the absence and presence of TRPV1
modulators were blocked by RR, CZP and SB-366719 in rats and
WT mice [17,18,27,34].
In addition, the effects of NGCC on ASIC was also evaluated
because ASIC is amiloride-sensitive ion channel and a subfamily of
the ENaC/Deg superfamily of ion channels. ASIC is expressed in
mouse taste buds and proton-gated cation channel, therefore
ASIC may be related to sour taste due to its activation by proton
(H+) . ASIC is activated by pH 5.96.5 of half maximal
activation (pH0.5) and directly modulates PKA . Although
responding to protons, ASIC1-expressing cells were not activated
by NGCC. That is, NGCC is not associated with sour taste via
ASIC1-dependent pathway. In an earlier study , the
Bzinsensitive CT response in rodents showed many properties that
are similar to those observed with the cloned TRPV1 expressed in
heterologous cells. The following common properties were
Figure 6. Calcium responses in hTRPA1-expressing cells stimulated with AITC and NGCC. (A) hTRPA1-expressing cells were loaded with
Fura-2 AM and Ca2+ images were obtained at 0 and 60 s after stimulation with AITC or NGCC. The selectivity of AITC and NGCC was tested by adding
the non-specific blocker of TRP channels, RR (30 mM), or a specific TRPA1 antagonist, HC-030031 (100 mM). Representative ratiometric images are
shown after treatment with AITC and NGCC. (B) As a control, the Ca2+ response to AITC or NGCC treatment was monitored in mock-transfected Flp-In
293 cells. (C) AITC and NGCC treatment showed dose-dependent effects in hTRPA1-expressing cells. The effects of AITC or NGCC treatment in the
presence or absence of 30 mM RR or 100 mM HC-030031 were quantified using Calcium-4 in a cell-based assay. Experiments were repeated in
triplicate and data points represent the means 6 SEM (n = 3).
observed between the Bz-insensitive NaCl CT responses and
TRPV1: (i) activation by resiniferatoxin (RTX), capsaicinand
elevated temperature; (ii) additive effects of temperature and
vanilloids on the CT response; (iii) inhibition by TRPV1 blockers,
RR, CZP and SB-366791; (iv) RTX produced biphasic changes in
CT response to NaCl, KCl, NH4Cl and CaCl2 that were inhibited
by SB-366791. This suggests that the Bz-insensitive CT responses
to the above cations are dependent upon their influx through a
non-selective cation channel; and (v) the absence of the constitutive
Bz-insensitive NaCl CT response and insensitivity to vanilloids and
temperature in TRPV1 KO mice. Moreover, spontaneous
Bzinsensitive NaCl CT response was also observed at room
temperature, in the absence of vallinoids and at the physiological
pH. Finally, in the absence of vanilloids, the Bz-insensitive NaCl
CT response was not affected by changes in the stimulus pH.
These results led Lyall et al. to propose that the Bz-insensitive
responses may be derived from a variant of TRPV1. Although
TRPV1 and TRPV1t are not exactly identical, the experimental
conditions used in this study (27uC, neutral pH, moderate ATP
concentration) suggested that ligand-stimulated hTRPV1
activation could represent the activation of the putative
amilorideinsensitive salt taste receptor.
TRPV1 is often co-expressed with the transient receptor
potential channel ankyrin 1 (TRPA1), a member of the TRP
channel family, in sensory nerve endings . In sensory neurons,
97% of TRPA1-expressing cells co-express TRPV1, and 30% of
TRPV1-expressing cells co-express TRPA1 . Several
compounds, such as 6-shogaol and 6-paradol, stimulate both TRPV1
and TRPA1 . TRPA1 is a nonselective cation channel with
high Ca2+ permeability . Similar to TRPV1, TRPA1 is
associated with somatosensation in response to environmental
irritants, cold, and pain . Using immunohistochemical studies,
the expression of TRPA1 has been demonstrated in the human
lingual trigeminal nerve and the nerve bundles of the mouse
Treatment of hTRPA1-expressing cells with NGCC stimulated
hTRPA1 and caused an increase in the intracellular Ca2+
concentration. The EC50 value for NGCC in hTRPA1-expressing
cells was 83.65 mM. The response of hTRPA1-expressing cells to
NGCC treatment was inhibited by RR and a specific hTRPA1
Figure 7. Activation of hTRPA1 by AITC and NGCC. (A) AITC (100 mM) induced an inward current in hTRPA1-expressing cells. AITC-induced
currents were partially inhibited by TRPA1 antagonist, HC-030031 (100 mM) and recovered after wash out. NGCC triggered an inward current in a
AITC-sensitive cell. Neither AITC nor NGCC evoked the currents in non-transfected cells (n = 3). (B) NGCC-induced currents were blocked by
HC030031 in hTRPA1-expressing cells. (C) Summary of normalized current density in cells expressing hTRPA1 (n = 4). (D) NGCC triggered inward currents
in a dose-dependent manner in hTRPA1-expressing cells (n = 4). Results are presented as the mean 6 SEM.
antagonist, HC-030031. It is likely that at high concentrations, the
trigeminal effects of NGCC are due to its interactions with
Interactions between hTRPA1 and salty tasting compounds
have not been investigated. However, the similarity between
hTRPA1 and hTRPV1 suggest that salty tasting compounds may
interact with hTRPA1. hTRPA1 and hTRPV1 are both members
of the TRP superfamily, Ca2+-permeable, and co-expressed in the
same lingual nerve endings . Human TRPA1 does not form a
heterotetramer channel with hTRPV1; however, the formation of
a complex between hTRPA1 and hTRPV1 may occur in the
plasma membranes of sensory neurons. In addition, TRPV1 may
influence the activity of the TRPA1 channel. In sensory neurons,
capsaicin and mustard oil, a TRPA1 agonist, pharmacologically
desensitized TRPA1 via Ca2+-dependent and independent
pathways, respectively . Both TRPA1 and TRPV1 channels could
control transmission of inflammatory stimuli through nociceptors
[45,46]. Taken together, these results suggest that hTRPA1
channels may be activated by NGCC.
In our studies the constitutive Bz-insensitive NaCl CT response
in the absence of TRPV1 modulators was not affected by changes
extracellular pH between pH 2 and 10 . Similarly, at constant
external pH, changes in intracellular pH induced by exposing the
TRCs in vivo to organic acids (e.g. CO2) did not alter the
magnitude of the NaCl+Bz CT response. However, the NaCl+Bz
CT response in the presence of several TRPV1 agonists (RTX,
ethanol, nicotine, and MRPs) varied as a function of extracellular
pH. The relationship between pH and the magnitude of the CT
response was bell shaped. The maximum increase in the CT
response was observed at a pH between 6.0 and 6.5 [17,18,34].
TRPA1 is activated by changes in intracellular pH only . This
suggests that TRPA1 is most likely not involved in the
Bzinsensitive response in the absence and presence of TRPV1
modulators. Rather these data suggest that a variant (TRPV1t)
rather than TRPV1 is involved in Bz-insensitive salt responses
In conclusion, the results presented in this paper indicate that
NGCC modulates the activity of hTRPV1 and hTRPA1 in a
concentration dependent manner. The interaction between
NGCC and hTRPV1 are most likely related to the observed
alteration in the salty taste in humans in the presence of
NGCC. The interactions of NGCC with TRPA1 most likely
contribute to its trigeminal effects at high concentrations. This
study suggested that NGCC or related compounds can be used
to reduce salt consumption, which could prevent the adverse
effects of excessive salt consumption, such as hypertension, heart
attack, and stroke.
We would like to thank the professor Heung BaeJeon for offering S3969,
the professor Takumi Misaka for TRPV1 gene and TRPA1-expressing
cells, and Dr. Mark L. Dewis for NGCC used in this article.
Conceived and designed the experiments: MRR. Performed the
experiments: HJS MJK HJK BCS. Analyzed the data: MJK. Contributed
reagents/materials/analysis tools: YK. Wrote the paper: MJK MRR VL.
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