Pharmacological characterization and molecular determinants of the activation of transient receptor potential V2 channel orthologs by 2-aminoethoxydiphenyl borate.

Abstract

Despite its expression in different cell types, transient receptor potential V2 (TRPV2) is still the most cryptic members of the TRPV channel family. 2-Aminoethoxydiphenyl borate (2APB) has been shown to be a common activator of TRPV1, TRPV2, and TRPV3, but 2APB-triggered TRPV2 activation remains to be thoroughly characterized. In this study, we have developed an assay based on cell lines stably expressing mouse TRPV2 channels and intracellular calcium measurements to perform a pharmacological profiling of the channel. Phenyl borate derivatives were found to activate mouse TRPV2 with similar potencies and thus were used to screen a panel of channel blockers. Besides the classic TRP inhibitors ruthenium red (RR) and 1-(beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF96365), two potassium channel blockers, tetraethylammonium (TEA) and 4-aminopyridine, and an inhibitor of capacitative calcium entry, 1-(2-(trifluoromethyl) phenyl) imidazole (TRIM), were found to inhibit TRPV2 activation by 100 microM 2APB. Activation by 300 microM 2APB, however, could only be inhibited by RR and TRIM. Electrophysiological recordings demonstrated that TEA inhibition was use-dependent, suggesting that high concentrations of 2APB might induce a progressive conformational change of the channel. Comparison of TRPV2 orthologs revealed that the human channel was insensitive to 2APB. Analysis of chimeric constructs of mouse and human TRPV2 channels showed that the molecular determinants of 2APB sensitivity could be localized to the intracellular amino and carboxyl domains. Finally, using lentiviral-driven expression, we demonstrate that hTRPV2 exerts a dominant-negative effect on 2APB activation of native rodent TRPV2 channels and thus may provide an interesting tool to investigate cellular functions of TRPV2 channels.

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Pharmacological Characterization and Molecular Determinants
of the Activation of Transient Receptor Potential V2 Channel
Orthologs by 2-Aminoethoxydiphenyl Borate□
S
Ve ´ronique Juvin, Aubin Penna, Jean Chemin, Yea-Lih Lin, and Franc ¸ois-A. Rassendren
Department of Molecular Pharmacology, Institut de Ge ´nomique Fonctionnelle, Montpellier, France (V.J., A.P., F-A.R.);
Department of Physiology, Institut de Ge ´nomique Fonctionnelle, Montpellier, France (J.C.); Centre National de la Recherche
Scientifique, Unite ´ Mixte de Recherche 5203, Montpellier, France (V.J., A.P., J.C., F-A.R.); Institut National de la Sante ´ et de la
Recherche Me ´dicale, U661, Montpellier, France (V.J., A.P., J.C., F.-A.R.); Universite ´ de Montpellier (IFR3), Montpellier, France
(V.J., A.P., J.C., F.-A.R.); Institut de Ge ´ne ´tique Humaine, Montpellier, France (Y.-L.L.); Centre National de la Recherche
Scientifique UPR1142, Montpellier, France (Y.-L.L.)
Received April 12, 2007; accepted August 2, 2007
ABSTRACT
Despite its expression in different cell types, transient receptor
potential V2 (TRPV2) is still the most cryptic members of the
TRPV channel family. 2-Aminoethoxydiphenyl borate (2APB)
has been shown to be a common activator of TRPV1, TRPV2,
and TRPV3, but 2APB-triggered TRPV2 activation remains to
be thoroughly characterized. In this study, we have developed
an assay based on cell lines stably expressing mouse TRPV2
channels and intracellular calcium measurements to perform a
pharmacological profiling of the channel. Phenyl borate deriv-
atives were found to activate mouse TRPV2 with similar poten-
cies and thus were used to screen a panel of channel blockers.
Besides the classic TRP inhibitors ruthenium red (RR) and
1-(?-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl)-1H-
imidazole hydrochloride (SKF96365), two potassium channel
blockers, tetraethylammonium (TEA) and 4-aminopyridine, and
an inhibitor of capacitative calcium entry, 1-(2-(trifluoromethyl)
phenyl) imidazole (TRIM), were found to inhibit TRPV2 activa-
tion by 100 ?M 2APB. Activation by 300 ?M 2APB, however,
could only be inhibited by RR and TRIM. Electrophysiological
recordings demonstrated that TEA inhibition was use-depen-
dent, suggesting that high concentrations of 2APB might in-
duce a progressive conformational change of the channel.
Comparison of TRPV2 orthologs revealed that the human chan-
nel was insensitive to 2APB. Analysis of chimeric constructs of
mouse and human TRPV2 channels showed that the molecular
determinants of 2APB sensitivity could be localized to the in-
tracellular amino and carboxyl domains. Finally, using lentiviral-
driven expression, we demonstrate that hTRPV2 exerts a dom-
inant-negative effect on 2APB activation of native rodent
TRPV2 channels and thus may provide an interesting tool to
investigate cellular functions of TRPV2 channels.
Transient receptor potential (TRP) proteins are cationic
channels that were first discovered in Drosophila melano-
gaster, in which they play a crucial role in the initial steps of
visual transduction (Hardie, 2001; Montell, 2005a). Since
then, more than 30 mammalian TRP channels have been
identified and are classified into seven families, which are
TRPC, TRPV, TRPM, TRPN, TRPA and the two distantly
related TRPP and TRPML (Montell, 2005b). All members of
the TRP superfamily share the same membrane topology as
voltage-gated potassium channels; they contain six hydro-
phobic domains spanning the plasma membrane and an ad-
ditional P-loop structure between the fifth and the sixth
transmembrane segments that participates in the pore-form-
ing domain of the channel. TRP proteins possess long cyto-
This work was supported in part by Centre National de la Recherche
Scientifique and Institut National de la Sante ´ et de la Recherche Me ´dicale.
J.V. and P.A. were supported by student fellowships from Association de la
Recherche sur le Cancer.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.107.037044.
□
S
The online version of this article (available at http://molpharm.
aspetjournals.org) contains supplemental material.
ABBREVIATIONS: TRP, transient receptor potential; 2APB, 2-aminoethoxydiphenyl borate; DPBA, diphenylboronic anhydride; RBL-2H3, rat
basophilic leukemia clone 2H3; RR, ruthenium red; TEA, tetraethlyammonium; 4-AP, 4-aminopyridine; TRIM, 1-(2-(trifluoromethyl)phenyl) imida-
zole; TMD, transmembrane domain; THC, ?9-tetrahydrocannabinol; SKF96365, 1-(?-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl)-1H-
imidazole hydrochloride; HA, hemagglutinin; GFP, green fluorescent protein; CHO, Chinese hamster ovary; HEK, human embryonic kidney; PBS,
phosphate-buffered saline; AM, acetoxymethyl ester; HBSS, Hanks’ balanced salt solution; BSA, bovine serum albumin; BK8644, 1,4-dihydro-
2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-3-pyridinecarboxylic acid, methyl ester; SKF96365, 1-(?-[3-(4-methoxyphenyl)propoxy]-4-
methoxyphenethyl)-1H-imidazole.
0026-895X/07/7205-1258–1268$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 72:1258–1268, 2007
Vol. 72, No. 5
37044/3263402
Printed in U.S.A.
1258

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plasmic amino and carboxyl termini, in which structural
motifs such as ankyrin repeats or coiled-coil domains are
often conserved. Although the structure of TRP channels is
still unresolved, they seem to assemble as tetramers, as do
potassium channels (Ramsey et al., 2006).
All TRP channels are permeable to cations, but ionic selec-
tivities and modes of gating are extremely diverse; to add to
this complexity, a given TRP can be activated by different
stimuli, depending on either the cell type in which it is
expressed or the subcellular-specific signaling complex asso-
ciated with the channel (Clapham, 2003). Hence, studying
TRP channel gating is still very challenging and prone to
controversy. One unifying view is that these channels are
tightly regulated by various cellular second messengers, such
as lipids, kinases, and calcium, but also by dynamic protein-
protein interactions; these regulations may underlie the abil-
ity of TRP channels to endorse a variety of cellular functions.
As yet, the precise cellular functions of most TRP channels
remain unresolved, because the lack of specific pharmacological
tools combined with their complex mode of gating represent a
considerable obstacle to physiological studies. However, it is
now clear that a subset of TRP channels is involved in sensory
transduction (Clapham, 2003). Indeed, different sensory stim-
uli, in particular heat or cold, activate TRPVs 1 to 4, TRPM8, or
TRPA proteins (Tominaga and Caterina, 2004). Several endog-
enous substances and pharmacologically active molecules are
TRPV channel agonists. In particular TRPV1, -3, and -4 are
gated by bioactive lipids such as phosphatidylinositol bisphos-
phate or fatty-acid derivatives (Benham et al., 2002). Although
TRPV1, -3, and -4 channels act unambiguously as cellular tem-
perature sensors and are gated or modulated by lipids, the
gating of TRPV2 (initially named VRL-1) is still a matter of
debate. Indeed, the temperature threshold for TRPV2 activa-
tion (?50°C) (Caterina et al., 1999) is well above physiological
range, and this channel has not been demonstrated to be sen-
sitive to lipids. In addition, in non-neuronal cells, other mech-
anisms have been reported to activate TRPV2, such as dynamic
insertion in the plasma membrane (Kanzaki et al., 1999), reg-
ulation by a PI3-kinase dependent pathway (Penna et al., 2006)
or mechano- and osmotic stimuli (Iwata et al., 2003; Muraki et
al., 2003). 2APB and its derivative diphenylboronic anhydride
(DPBA) have been shown to be potent activators of TRPV1,
TRPV2, and TRPV3 (Hu et al., 2004). Although activation of
TRPV1 and TRPV3 channels by 2APB has been characterized
in some detail (Chung et al., 2004), there are few data available
on its effect on TRPV2 channels. In this study, we provide a
characterization of the activation of TRPV2 channel orthologs
(mouse, rat, human) by 2APB. We show that 2APB activates
mouse and rat TRPV2 orthologs with different potencies,
whereas the human TRPV2 channel is remarkably insensitive
to 2APB. Through chimeric constructs of mouse and human
TRPV2 channels, we have identified the molecular determi-
nants of 2APB sensitivity. We also investigated the potential
cellular function of endogenous TRPV2 expressed in RBL-2H3
cells using a lentiviral-mediated expression of a dominant-neg-
ative human TRPV2 channel.
Materials and Methods
Reagents and Antibodies. All chemicals were purchased from
Sigma-Aldrich (St. Louis, MO), except for 2APB, which was from
Calbiochem (San Diego, CA). Culture media and reagents were ob-
tained from Invitrogen (Carlsbad, CA); endotoxin free-fetal calf se-
rum used for RBL-2H3 was from Biowest (Miami, FL).
Epitope Tagging, Mutagenesis, and Construction of Chime-
ras. Insertions of Flag and HA tags in hTRPV2 and site-directed
mutagenesis have been described previously (Penna et al., 2006).
Mutations introduced in the selectivity filter of hTRPV2 were
E599K, M607K, and E609K. Because of the high sequence homology
between human and mouse TRPV2 cDNAs, chimeras were con-
structed by introducing unique silent restriction sites by oligonucle-
otide-directed mutagenesis in both cDNAs. Sites inserted were the
following: SacII between Lys390 and Phe391, Bsu36I between
Arg537 and Phe538, EcoRI between Val649 and Asn650, BspeI be-
tween Ser726 and Gly727 for human TRPV2; SacII between Arg387
and Phe388, Bsu36I between Arg534 and Phe535, EcoRI between
Val644 and Asn645, and BspeI between Ser722 and Gly723 for
mouse TRPV2. After restriction digestion, human and mouse frag-
ments were swapped. All of the clones were verified by sequencing.
Lentiviral Vector Construction and Production. cDNAs cod-
ing for human flag-TRPV2HAand mutant human flag-TRPV2HA
channels were cloned in front of an IRES-GFP cassette into the
lentiviral expression vector pWPXL (Klages et al., 2000). To produce
GFP- and hTRPV2-harboring human immunodeficiency virus vec-
tors, pWPXL-GFP or pWPXL-hTRPV2 plasmids were cotransfected
in HEK 293T cells with the human immunodeficiency virus packag-
ing plasmid p8.2 and the plasmid pMD.G that encodes the vesicular
stomatitis virus glycoprotein envelope. Viruses were harvested and
concentrated, and titers were calculated as described previously
(Naldini et al., 1996).
Cell Culture, Transfection, and Viral Transduction. Chinese
hamster ovary cells (CHO-K1, American Type Culture Collection
number CCL-61) and rat basophilic leukemia cells (RBL-2H3; gift
from Dr U. Blank, Institut Pasteur, Paris, France) were grown in
Ham’s F-12 medium and Dulbecco’s modified Eagle’s medium, re-
spectively, supplemented with 10% fetal calf serum, 1% GlutaMAX,
100 IU/ml penicillin, and 100 ?g/ml streptomycin at 37°C in a hu-
midified 5% CO2incubator. For stable CHO cell lines expressing
mouse TRPV2 (clone IIE11), 200 ?g/ml G418 was added to the
culture medium but omitted for experiments. HEK-293 cells (Amer-
ican Type Culture Collection number CCL-1573) were grown in
Dulbecco’s modified Eagle’s medium supplemented with 10% fetal
calf serum, 1% GlutaMAX, 100 IU/ml penicillin, and 100 ?g/ml
streptomycin.
CHO and HEK-293 cells were transfected using AMAXA system
(AMAXA Inc., Gaithersburg, MD) or jetPEI (PolyPlus Transfection
Inc., New York, NY), respectively, according to the manufacturers’
instructions. Cells were used 48 h after transfection.
For viral transduction, CHO, clone IIE11, or RBL-2H3 cells were
plated at 2.5 ? 105cells/well (12-well plates). Lentiviral vectors were
then added to the cell cultures at a multiplicity of infection of 40,
along with 8 ?g/ml Polybrene (Sigma). Cell cultures were then cen-
trifuged at 300g for 60 min at 30°C, incubated for 18 h at 37°C in a
humidified 5% CO2incubator, and then washed twice in PBS. Ex-
periments were performed at least 5 days after transduction. In a
typical experiment, 95% of the cells were transduced.
Intracellular Calcium Measurements. For Fluo-4/AM calcium
measurements, CHO or RBL-2H3 cells were plated on poly(orni-
thine)-coated 96-well plates at a density of 5 ? 104cells/well. Plates
were washed three times in Hanks’ balanced salt solution (HBSS)
containing 142 mM NaCl, 5.6 mM KCl, 1 mM MgCl2, 2 mM CaCl2,
0.34 mM Na2HPO4, 0.44 mM KH2PO4, 10 mM HEPES, and 5.6 mM
glucose, 310 mOsM, pH 7.4. Cells were then incubated in HBSS
supplemented with 2.5 mM probenecid (Sigma), 100 ?g/ml pluronic
acid and 1 ?M Fluo-4/AM (both from Invitrogen) for 1 h at 37°C.
After the incubation, cells were washed twice with HBSS. Intracel-
lular calcium measurements were performed on a fluorescence plate
reader (FlexStation II; Molecular Devices, Sunnyvale, CA), and
drugs were applied at 2? using a fluid-handling integrated device.
2APB as a Tool to Study TRPV2 Pharmacology and Function
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All measurements were performed in triplicate at 30°C. For analysis,
the fluorescence signal was integrated over a 1-min period.
Immunocytochemistry.
TRPV2
formed 48 h after transfection. Cells were fixed in 4% paraformalde-
hyde for 10 min, washed in 0.1 M glycine, blocked in PBS with 0.5%
BSA for 30 min, permeabilized using 0.05% Triton X-100 for 5 min,
and incubated with primary antibody in blocking solution overnight
at 4°C. After PBS with 0.5% BSA washes, cells were incubated with
fluorescent secondary antibody for 30 min at 37°C. Fluorescence was
visualized on a Leica DMRA2 epifluorescent microscope using a 63?
oil-immersion objective (Leica Microsystems, Inc., Deerfield, IL). Im-
ages were acquired using a cool-snap HQ (photometric) digital cam-
era. Image deconvolution was performed as described previously
(Penna et al., 2006). mTRPV2 was detected using anti-VRL-1 anti-
body (1/50) (EMD Biosciences, San Diego, CA), hTRPV2 was detected
using a specific rabbit antibody (1/250) produced in the laboratory
and Flag-tagged with anti-Flag (M2) monoclonal antibody (1/1000)
from Sigma. Because of sequence divergences between mouse and
human TRPV2, we checked that each antibody was species-specific
(data not shown). Secondary antibodies were Alexa Fluor 488 anti-
rabbit antibody (1/2000) from Invitrogen, and Cy3-conjugated anti-
mouse antibody (1/1000) was from Jackson ImmunoResearch Labo-
ratories Inc. (West Grove, PA).
Western Blotting. Cells were lysed in 200 ?l of lysis buffer [20
mM HEPES, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, and
protease inhibitor cocktail (Roche, Indianapolis, IN), pH 7.4] and
passed five times through a 26-gauge needle. After 30 min of solu-
bilization at 4°C under agitation, lysates were centrifuged (16,000g,
10 min, 4°C). Protein extracts were diluted in 6? Laemmli buffer,
resolved by SDS-polyacrylamide gel electrophoresis, and transferred
to nitrocellulose membranes. Blocking was performed using 5% non-
fat dry milk in Tris-buffered saline containing 0.05% Tween 20
followed by incubation with the primary antibody in the blocking
buffer at 4°C overnight. Antibody dilutions were anti-VRL-1, 1/250;
anti-hTRPV2, 1/500; anti-GFP, 1/2500 (Torrey Pines Biolabs, Hous-
ton, TX); horseradish peroxidase-conjugated anti-Flag (M2) anti-
body, 1/4000 (Sigma); and anti-tubulin, 1/2000 (Sigma). After a 30-
min wash with Tris-buffered saline containing 0.05% Tween 20,
membranes were incubated for 1 h in either anti-rabbit (1/10,000) or
anti-mouse (1/25,000) horseradish peroxidase-conjugated secondary
antibodies from GE Healthcare (Chalfont St. Giles, Buckingham-
shire, UK) and Pierce (Rockford, IL), respectively. Proteins were
detected using a chemoluminescence detection kit (Pierce).
Electrophysiological Recordings. HEK-293 cells were plated
at a density of 2 ? 105cells in individual 35-mm culture dishes.
Macroscopic currents were recorded at room temperature (?22°C) by
the whole-cell patch-clamp technique using an Axopatch 200B am-
plifier (Molecular Devices). The extracellular solution contained 150
mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 10 mM
HEPES, with pH adjusted to 7.35 with NaOH (330 mOsM). Borosili-
cate glass pipettes had a resistance of 1.5 to 2.5 M? when filled with
an internal solution containing 140 mM CsCl, 10 mM EGTA, 10 mM
HEPES, 3 mM magnesium-ATP, 0.6 mM GTP-sodium, and 2 mM
CaCl2, with pH adjusted to 7.2 with KOH (?315 mOsM). Drugs were
applied by a perfusion system controlled by solenoid valves allowing
a complete exchange of solution in less than 1 s. Recordings were
filtered at 2 kHz. Data were analyzed using pClamp 9 (Molecular
Devices) and Prism (GraphPad Software Inc., San Diego, CA) soft-
ware. One-way analysis of variance combined with a Newman-Keuls
post test were used to compare the different values and were consid-
ered significant at p ? 0.05. Results are presented as mean current
amplitudes (in picoamperes) or current densities (picoamperes per
picofarad) (i.e., amplitudes normalized relative to cell membrane
capacitance determined for each cell) ? S.E.M., and n is the number
of cells used.
?-Hexosaminidase Release Assays. ?-Hexosaminidase is a
granule-associated enzyme that degrades glucosamine residues
stored in secretion granules in mast cells. It is released in the
immunostainingwas per-
extracellular medium when degranulation is triggered by physiolog-
ical or chemical stimuli. Hence, a colorimetric assay of extracellular
?-hexosaminidase can provide an indirect measurement of mast cell
degranulation. RBL-2H3 cells were plated in 96-well plates at 5 ?
104cells/well and sensitized overnight at 37°C with anti-DNP IgE
antibody (1/1000) from Sigma. Cells were washed three times in PBS
and incubated for 30 min at 37°C in 50 ?l of Locke buffer (140 mM
NaCl, 1.2 mM KH2PO4, 5 mM KCl, 1.2 mM MgSO4, 10 mM glucose,
10 mM HEPES, 1.8 mM CaCl2, and 0.5% BSA; pH was adjusted to
7.4 using NaOH) containing either DNP-HSA or 2APB. PMA (50 nM)
or ionomycin (200 nM) was used as a positive control to induce
secretion. After stimulation, 20 ?l of supernatant was incubated for
90 min at 37°C in 50 ?l of citrate buffer (49.5% of 0.05 M citrate acid
and 50.5% of 0.05 M trisodium citrate, pH 4.5) containing 1.3 mM
p-nitrophenyl-N-acetyl-?-D-glucosamine. The reaction was stopped
by adding 150 ?l of glycine (0.2 M, pH 10.7). Total cellular content of
?-hexosaminidase was determined after cell lysis in 0.5% Triton
X-100 and directly assayed in the 96-well plates. Absorbance was
determined at 410 nm in a microtiter plate reader. Results were
expressed as a percentage of total ?-hexosaminidase cellular content
after correction for spontaneous release in unstimulated cultures.
Each measurement was made in triplicate.
Data Analysis. The dose-response curves for agonist and inhibi-
tors were fitted using GraphPad Prism. All data are mean ? S.E.M.
from at least three individual experiments performed in triplicate.
For dose-response curve experiments, EC50or IC50values were cal-
culated for each individual experiment using the sigmoid dose-re-
sponse equation with variable slope from PRISM, and values were
averaged.
Results
2APB and DPBA Induce Calcium Influx through
Mouse TRPV2 Channels. We reported previously that
transient transfection of TRPV2 cDNA can result in high
levels of protein expression, leading to cellular toxicity
(Penna et al., 2006). To avoid this problem, we generated
monoclonal stable CHO cell lines expressing mouse TRPV2
channels (mTRPV2). Two clones (IB11 and IIE11) were se-
lected based on moderate expression levels of TRPV2 and
correct localization of the protein at the plasma membrane
(Fig. 1). Because of the lower expression of TRPV2 in clone
IIE11 compared with clone IB11, clone IIE11 was selected for
all experiments in this study, unless indicated.
Phenyl-borate derivatives 2APB and DPBA have been
shown to activate TRPV1, TRPV2, and TRPV3 (Hu et al.,
2004). Here we used a fluorimetric calcium assay to perform
a more precise pharmacological characterization of the acti-
vation of mTRPV2 by 2APB and DPBA. Both molecules in-
duced a dose-dependent calcium increase in mTRPV2 stable
cell line. The threshold for activation was 30 and 10 ?M for
2APB and DPBA, respectively (Fig. 1B), and significant cal-
cium signals were obtained at 100 ?M; saturation was
reached at doses greater than 1 mM. A high background
signal was observed in parental cells for agonist concentra-
tions greater than 300 ?M. In these conditions, EC50values
were 160 ? 26 (n ? 4) and 105 ? 10 ?M (n ? 4) for 2APB and
DPBA, respectively. Because at 100 and 300 ?M, 2APB and
DPBA induced little if any calcium signals in nontransfected
cells, these two concentrations were used in all further ex-
periments, unless indicated.
Screening for mTRPV2 Channel Blockers. We used
our assay to screen a panel of small molecules that are known
to inhibit ion channels, including known TRP channel block-
ers, small inorganic cations, other cationic channel blockers,
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Juvin et al.

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and calcium channel antagonists (see Supplementary Fig.
S1). In a first step, a single concentration of these molecules
was tested on calcium signals induced by 100 ?M 2APB. As
expected, the nonspecific TRP blockers ruthenium red (RR)
and SKF96365 (both 100 ?M) inhibited 2APB-mediated cal-
cium signals in TRPV2-expressing cells. Inhibition levels
were 96 ? 3% (n ? 3) and 78 ? 7% (n ? 3) for RR and
SKF96365, respectively. The small inorganic trivalent cat-
ions La3?and Gd3?(both 100 ?M), which block some TRP
channels, were unable to block 100 ?M 2APB-induced cal-
cium signals. Similar results were obtained with 100 ?M
Ni2?, Cd2?, Zn2?, hexamethonium, and the dihydropyridines
nifedipine or BK8644 (racemic mixture) (Fig. 2A). On the
contrary, the potassium channel blockers 4-aminopyridine
(4-AP) (5 mM) and tetraethylammonium (TEA) (10 mM) in-
duced inhibitions of 70 ? 6% (n ? 4) and 74 ? 6% (n ? 3),
respectively. Finally, we found that 500 ?M 1-(2-trifluorom-
ethylphenyl) imidazole (TRIM), an inhibitor of nitric-oxide
synthase and of capacitative calcium entry (Gibson et al.,
2001; Tobin et al., 2006) reduced 100 ?M 2APB-evoked re-
sponses by 62 ? 9% (n ? 3).
Complete inhibitory dose-response
SKF96365, TEA, 4-AP, and TRIM were determined on 100
?M 2APB-induced responses (Fig. 2B, Œ). IC50values were
RR, 7 ? 2 ?M (n ? 4); SKF96365, 21 ? 16 ?M (n ? 4); 4-AP,
2 ? 0.2 mM (n ? 3); and TEA, 1 ? 1 mM (n ? 3). However,
when similar inhibitory dose responses were performed using
300 ?M 2APB to activate TRPV2, a total loss of inhibition
was observed for most of the molecules tested. Only RR was
still effective (IC50? 12 ? 1 ?M, n ? 4) (Fig. 2B, f), as well
as TRIM, albeit with an apparently lower potency.
Because several of the molecules that inhibit activation of
TRPV2 by 2APB are traditionally known to be voltage-de-
pendent open-channel blockers of their primary targets, we
questioned whether their lack of effect on 300 ?M 2APB-
evoked responses could be related to such biophysical prop-
curves forRR,
erties. Indeed, high 2APB concentrations could depolarize
the membrane, thus reducing block of TRPV2 by open-chan-
nel blockers. On the other hand, 2APB could induce use-
dependent conformational changes that may reduce the ac-
cessibility of the antagonists to their binding site. Using
whole-cell recordings, we therefore analyzed whether the
inhibition of 2APB-induced currents by 3 mM TEA showed
any voltage-dependence and/or use-dependence. Because
TRPV2 currents are of small magnitude when evoked by
2APB concentrations lower than 1 mM (Hu et al., 2004),
these experiments were performed using HEK cells tran-
siently transfected with mTRPV2 to provide a robust expres-
sion of TRPV2, and 3 mM 2APB was used to activate the
channel to maximize current densities. Under these condi-
tions, 3 mM 2APB evoked robust currents only in transfected
cells, which displayed an activation time constant of 3.1 ?
0.3 s (n ? 20) and an inactivation time constant of 4.3 ? 0.4 s
(n ? 20) (Fig. 3A). 2APB-induced currents did not show
marked desensitization during agonist application or after
repeated stimulation (Fig. 3A). TEA blocking efficacy was
evaluated using two stimulation paradigms; in the first,
2APB was first applied alone and then coapplied with TEA.
In this situation, although TEA significantly decreased 2APB
currents, an important component of current remained un-
affected by TEA (Fig. 3, B and D), and sometimes no inhibi-
tion at all could be obtained (Fig. 3B). In the second stimu-
lation paradigm, TEA and 2APB were first coapplied, and
then 2APB was applied alone. When cells were exposed to
this sequence of application, 2APB-evoked currents were al-
most completely inhibited by TEA; current densities mea-
sured at ?50 mV were not significantly different from those
of control (Fig. 3, C and E). TEA inhibition of 2APB-evoked
currents occurred similarly over the entire voltage range
tested, indicating a lack of voltage dependence (Fig. 3C).
Taken together, these results show that TEA block is use-
dependent but not voltage-dependent, suggesting that 2APB
Fig. 1. 2APB and DPBA activate mouse TRPV2 stably
expressed in CHO cells. A, immunostaining (left) and West-
ern blot (right) showing expression of mTRPV2 in two
monoclonal CHO cell populations. Immunostaining and im-
age deconvolution (fourth on the right) clearly show the
membrane localization of mTRPV2 in clones IB11 and
IIE11. Western blot analysis confirmed the lack of expres-
sion of TRPV2 in parental CHO cells. B, dose-response
curve for 2APB- and DPBA-induced calcium signals in
clone IIE11 and parental CHO cells. Intracellular calcium
measurements were performed using the calcium-sensitive
probe Fluo-4/AM on a Flexstation II (Molecular Devices).
For comparison purposes, fluorescence signals were nor-
malized to the maximal response induced by 3 mM 2APB,
and independent experiments were averaged. Note that
both agonists induced intracellular calcium signals in pa-
rental cells at concentrations greater than 1 mM.
2APB as a Tool to Study TRPV2 Pharmacology and Function
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is likely to induce progressive conformational changes of the
channel that are responsible for the reduced block in the
presence of higher concentrations of 2APB.
TRPV2 Orthologs Display Different 2APB Sensitivi-
ties. The effects of 2APB were also investigated on calcium
responses mediated by rat and human TRPV2 channel or-
thologs. For comparison purposes, the three cDNAs were
cloned in the same expression vector and were transiently
transfected in CHO cells. As shown in Fig. 4A, rat, mouse,
and human TRPV2 channels display significant differences
in their apparent affinity for 2APB. Rat TRPV2 was the most
sensitive with an EC50value of 59 ? 13 ?M (n ? 4) followed
by mTRPV2, with an EC50of 187 ? 20 ?M (n ? 4) (Fig. 4A).
We were surprised to find that activation of the human
channel with 2APB could not be obtained, despite its correct
expression in the plasma membrane (Fig. 5). Similar differ-
ences in DPBA sensitivity were observed between TRPV2
orthologs and in transiently transfected HEK cells (data not
shown).
We next sought to determine whether the 2APB insensi-
tivity of hTRPV2 could affect 2APB activation of the mouse
channel when both subunits are associated within the chan-
nel complex. Different ratios of cDNA coding for both chan-
nels were cotransfected; the amount of mTRPV2 cDNA was
kept constant, whereas that of hTRPV2 was increased. Cal-
cium signals induced by 100 and 300 ?M 2APB were mea-
sured in each condition. As shown in Fig. 4B, increasing the
amount of hTRPV2 cDNA dose-dependently reduced the
2APB-mediated calcium signal. At 100 ?M 2APB, a 67 ? 2%
(n ? 4) reduction of the response was observed when the ratio
of m/h TRPV2 was 1:3, and maximal inhibition (90 ? 2%, n ?
4) was achieved for a ratio of 1:7. At 300 ?M 2APB, levels of
inhibition were lower (ratio 1:3, 37 ? 4%, n ? 4; ratio 1:7,
65 ? 2%, n ? 4; ratio 1:10, 72 ? 1%, n ? 4) but significantly
different from control (p ? 0.0001, unpaired Student’s t test).
These results show that both human and mouse TRPV2
subunits can form heteromeric channels and that the pres-
ence of a human subunit impairs the activation of the com-
posite channel by 2APB. Complete inhibition could not be
reached, presumably because of the presence of homomeric
mTRPV2 channels. Finally, recently ?9-tetrahydrocannabi-
nol (THC) was used to demonstrate that hTRPV2 was a
functional channel (Neeper et al., 2007). We confirm this
observation; THC activates hTRPV2 with an apparent affin-
ity close to that of mTRPV2 (Fig. 4C). These results clearly
demonstrate that hTRPV2 lacks 2APB sensitivity but is a
functional channel as initially shown (Neeper et al., 2007).
Fig. 2. Characterization of TRPV2 channel blockers. A,
inhibition levels of 2APB-induced calcium signals by differ-
ent chemicals. Chemicals were coapplied with 100 ?M
2APB at a concentration of 100 ?M except for TEA and
4-AP (1 mM) and TRIM (500 ?M). Results were normalized
to the fluorescent signal induced in clone IIE11 by 100 ?M
2APB applied alone. Each measurement was made in trip-
licate and averaged; results are mean ? S.E.M. of at least
three experiments. B, inhibitory dose-response experi-
ments for the five identified inhibitors. Increasing concen-
trations of inhibitor were coapplied with either 100 ?M (Œ)
or 300 ?M 2APB (f). Fluorescence was normalized to the
2APB-induced signal in the absence of inhibitor.
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Molecular Determinants of 2APB Sensitivity. We took
advantage of the sequence similarity between mouse and
human channels to identify the molecular determinants of
2APB sensitivity through chimera construction and analysis.
Four modules were defined in TRPV2: the cytoplasmic amino
terminus, transmembrane domains (TMD) from TM1 to
TM5, the pore region from TM5 to the end of TM6, and the
carboxyl terminus region (Fig. 5A). Modules from each chan-
nel were swapped individually or in combination. Proper
expression and membrane localization of all chimeras were
analyzed by immunostaining. We first investigated whether
the reciprocal transfer of the pore region affected the 2APB
sensitivity of the resulting chimera. As shown in Fig. 5B,
mTRPV2 channels comprising the human pore region (chi-
mera 1) are fully functional, whereas the mouse pore region
does not confer 2APB sensitivity to the human channel (chi-
mera A). This demonstrates that the 2APB binding site is not
localized to the pore-forming region. This also rules out the
possibility that the sequence differences in the TM5–TM6
linker of the human channel are responsible for a permeation
deficiency.
In a second series of chimeras, amino and carboxyl termini
were individually swapped. None of these chimeras was func-
tional, but none of the chimeras associating the carboxyl
terminus of hTRPV2 with the amino terminus of mTRPV2
were able to reach the plasma membrane (chimeras 3 and B).
We therefore swapped both cytoplasmic regions. In this case,
chimera D, which carries the human TMD and both mouse
cytoplasmic regions, was found to be sensitive to 2APB. The
mirror chimera 4 (mouse TMD with human cytoplasmic re-
gions) was not sensitive to 2APB, despite being correctly
localized to the plasma membrane.
The trafficking deficit of chimeras 3 and B prevented us
from examining the individual contribution of cytoplasmic
regions to 2APB sensitivity. However, we noticed that the
last 35 amino acids of the human and mouse TRPV2 are very
divergent (see Supplementary Fig. S2). We therefore tested
whether this stretch of amino acids could be involved either
in 2APB sensitivity and/or in the trafficking of the channels.
Swapping this minimal region between mouse and human
channels had no effect on 2APB sensitivity (Fig. 5B; chimeras
5 and F) but did restore membrane expression of the two
trafficking-deficient chimeras 3 and B (Fig. 5C; chimeras 6
and G). These data suggested that 2APB sensitivity may
involve both amino and carboxyl termini of the mouse chan-
nel. If so, coexpressing chimeras that individually contribute
either the N or the C terminus of mTRPV2 should lead to
partial 2APB sensitivity. Indeed, this is what was observed
upon coexpression of pairs of inactive chimeras (2 ? B), (3 ?
C), and (B ? C); each of these combinations led to functional,
2APB-sensitive channels, albeit with lower efficacy (Fig. 5D).
Fig. 3. 2APB-evoked mTRPV2 currents and inhibition by
TEA. A, mTRPV2 was transiently transfected in HEK cells
and experiments were performed 48 h later. Whole-cell
currents evoked by 3 mM 2APB were recorded at a holding
potential of ?80 mV. 3 mM 2APB was applied for 10 s
through a rapid perfusion system, every 20 to 30 s. Cur-
rents showed no desensitization either during 2APB appli-
cation or after repetitive stimulations. B to E, use-depen-
dence of TEA inhibition. Currents were recorded during
900-ms voltage ramps from ?100 to ?60 mV applied every
5 s. Cells were stimulated either first by 2APB and then by
2APB plus TEA (B and D) or first by 2APB plus TEA and
then by 2APB (C and E). B, and C, top, chart recording of
current intensities measured at ?50 mV; horizontal bars
indicate drug applications. Bottom, recordings of currents
elicited by voltage ramps before (a) or during drug applica-
tion (b and c) at times indicated in the top panel. D and E,
current densities induced by 2APB or 2APB plus TEA mea-
sured at ?50 mV in mTRPV2 transfected and control cells.
Note that when TEA is coapplied with 2APB before any
other stimulation (E), current densities are not different
from baseline. 2APB had no effect on non transfected cells.
One-way analysis of variance combined with a Newman-
Keuls post test were used to compare the different values.
n.s., not significant; ?, p ? 0.05; ??, p ? 0.01. Results are
presented as mean ? S.E.M.
2APB as a Tool to Study TRPV2 Pharmacology and Function
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In addition, these coexpressions also corrected the trafficking
deficit of chimeras 3 and B (Fig. 5E).
Use of hTRPV2 to Probe Rodent TRPV2 Functions.
The insensitivity of hTRPV2 to 2APB could provide a tool to
investigate cellular functions of TRPV2 channels of other
species. To test this possibility, we constructed lentiviral
vectors expressing either hTRPV2 or an hTRPV2[mut] cDNA
that carried three mutations (E599K, M607K, and E609K) in
the pore-selectivity filter. This mutated hTRPV2 was shown
previously to behave as a dominant-negative mutant on
mTRPV2 activity (Penna et al., 2006). We tested the ability of
both lentiviruses to inhibit 2APB activation of mTRPV2.
2APB dose-response experiments were performed on clone
IIE11 and CHO cells, transduced or not with hTRPV2 or
hTRPV2[mut]-expressing lentiviruses. As shown in Fig. 6,
overexpression of hTRPV2 induced a significant reduction of
2APB potency (Œ): 2APB EC50values were 110 ? 20 and
180 ? 25 ?M (n ? 3) for control and hTRPV2-transduced
cells, respectively (p ? 0.05, paired Student’s t test). Maximal
responses to 2APB were also affected, suggesting that 2APB
might act cooperatively to activate mTRPV2 and that full
activation can only be reached when all subunits of the chan-
nel are sensitive to 2APB. Although not significant compared
with hTRPV2-transduced cells, apparently stronger inhibi-
tory effects were observed in cells expressing hTRPV2[mut]
(Fig. 6, ?); the EC50for 2APB in hTRPV2[mut]-transduced
cells was 209 ? 17 ?M (n ? 3). A similar shift in potency was
observed for DPBA in cells transduced with wild-type and
mutant hTRPV2-expressing viruses (Fig. 6, right).
Activation of Native TRPV2 by 2APB. TRPV2 has been
reported to be expressed in the rat mast cell line RBL-2H3
(Stokes et al., 2004), and 2APB also has been shown to induce
cationic currents in these cells (Braun et al., 2003). To investi-
gate whether 2APB could activate native TRPV2 channels in
these cells, we compared 2APB-induced calcium signals in na-
ive RBL-2H3 cells and in cells transduced with lentiviruses
expressingeitherGFP,wild-typehTRPV2,orhTRPV2[mut].As
shown in Fig. 7A, native TRPV2 proteins in RBL-2H3 are
clearly localized at the plasma membrane and in punctated
intracellular compartments; a similar pattern was observed in
cells transduced with hTRPV2. Expression of endogenous
TRPV2 proteins was also confirmed by Western blotting.
TRPV2 was identified as a double band (presumably a glycosy-
lated and unglycosylated forms) with a molecular mass of 85
kDa. Expressions of transduced hTRPV2 (either wild type or
mutant) or control virus were confirmed by immunostaining
and Western blotting using specific antibodies (Fig. 7A). These
different cell populations expressing endogenous TRPV2 chan-
nels or transduced proteins were exposed to 2APB, and calcium
signalsweremeasured.InnaiveorGFP-transducedcells,2APB
induced a dose-dependent calcium signal with an EC50value of
257 ? 16 ?M (n ? 4) and 269 ? 30 ?M (n ? 4), respectively. For
DPBA,EC50valueswere209?34?M(n?4)and188?35?M
(n ? 4) in naive and GFP-transduced cells, respectively. As
expected, transduction with hTRPV2 and hTRPV2[mut] caused
a reduction of both EC50values and the maximal response to
2APB (Fig. 7B, left). In hTRPV2-transduced cells, EC50values
for 2APB and DPBA were 812 ? 146 ?M (n ? 4) and 520 ? 120
?M (n ? 4), respectively. These values were significantly differ-
ent from those obtained in naive cells (p ? 0.005 and ? 0.05,
unpaired Student’s t test, for 2APB and DPBA, respectively).
Transduction with hTRPV2[mut] induced a significantly higher
inhibition of 2APB-evoked maximal responses than transduc-
tion with hTRPV2 (p ? 0.05, Student’s t test), further demon-
strating its dominant-negative behavior.
RBL-2H3 cells express high-affinity receptors (FceRI) for
Fig. 4. Lack of activation of hTRPV2 by 2APB. A, 2APB
dose-response curves for rat, mouse, and human TRPV2
transiently expressed in CHO cells. Fluorescent signals
were normalized to the maximal response obtained for each
channel. Note that there is no difference between nontrans-
fected and hTRPV2 transfected cells. B, hTRPV2 inhibits
2APB activation of mTRPV2 when both channels are coex-
pressed. mTRPV2 and hTRPV2 were transiently coex-
pressed at different cDNA ratios; the amount of mTRPV2
cDNA was kept constant, whereas that of hTRPV2 was
increased. Cells were stimulated with 100 ?M (u) or 300
?M 2APB (?). After subtraction of nonspecific signals mea-
sured in nontransfected cells, results were normalized to
2APB-induced responses in cells transfected with mTRPV2
alone. C, mouse and human TRPV2 are activated by THC.
Dose-response curves for THC in CHO cells transiently
transfected with either mouse or human TRPV2 cDNA.
??, p ? 0.01, ???, p ? 0.001, unpaired Student’s t test.
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IgE, which, when aggregated, cause the release of inflamma-
tory mediator-containing granules. It has been proposed that
in RBL-2H3 cells, TRPV2-mediated calcium influx could par-
ticipate to granule secretion and lead to serotonin release
(Stokes et al., 2004). We tested this hypothesis in RBL-2H3
cells transduced with either hTRPV2- or mutant hTRPV2-
coding viruses. After an overnight sensitization with an anti-
DNP IgE antibody, FceRI aggregation was triggered by in-
creasing DNP-HSA concentrations, and intracellular calcium
signals and exocytosis caused by antigen binding were mon-
itored using Fluo-4 AM and ?-hexosaminidase assay, respec-
tively. DNP-HSA-induced calcium signals were clearly not
different between naive, GFP-, hTRPV2-, or hTRPV2[mut]-
transduced cells (Fig. 7B, right), suggesting that calcium
influx through TRPV2 is not involved in mast cell degranu-
lation. The ability of 2APB to trigger degranulation from
RBL-2H3 cells was tested by monitoring the activity of ?-hex-
osaminidase, an enzyme stored in secretory granules of RBL-
2H3 cells. In control RBL-2H3 cells, at concentrations lower
than 1 mM, 2APB induced a dose-dependent increase of
secretion that seemed to saturate (Fig. 7C, left). This secre-
tion only reached 7% of total ?-hexosaminidase cellular con-
tent compared with the 35% that was observed using a sat-
urating concentrationof DNP-HSA
Nevertheless, 2APB-induced secretion was significantly im-
paired in cells transduced with hTRPV2[mut], suggesting
that activation of TRPV2 channels by 2APB could stimulate
mast cell degranulation, albeit at very low levels. When
DNP-HSA-triggered secretion was analyzed, overexpression
of hTRPV2[mut] did not affect ?-hexosaminidase release
(Fig. 7c, right). Taken together, these results suggest that, in
physiological conditions, calcium influx through TRPV2
channels does not participate to mast cell degranulation,
although its direct activation by 2APB can evoke low levels of
exocytosis.
(Fig. 7C, right).
Discussion
A subset of TRPV channels (TRPV1–V4) is known to be
involved in sensory transduction (Caterina, 2007). Among
them, TRPV2 still remains poorly characterized, mainly be-
cause of the lack of specific pharmacological and molecular
tools. TRPV2 is highly expressed in human blood cells, sug-
gesting that, in addition to its role as a noxious heat sensor
(Caterina et al., 1999), this channel certainly encompasses
other cellular functions (Saunders et al., 2007). Diphenyl
borate derivatives have been identified as chemical activa-
tors of TRPV1 to -3 channels (Hu et al., 2004); in the case of
TRPV3, these compounds have led to a better understanding
of channel gating and have also proved to be useful tools to
Fig. 5. Molecular determinants of 2APB sensitivity. A, diagram of the
different chimeras tested; gray and black shading corresponds to
mTRPV2 and hTRPV2 amino acid sequences, respectively; rectangles
represent transmembrane domains. PM, plasma membrane expression;
funct., function. B, 2APB-induced calcium responses of the different
active constructions tested. cDNAs were transiently transfected in CHO
cells, and experiments were performed 48 h later. Results were normal-
ized to responses induced by 100 or 300 ?M 2APB in mTRPV2-trans-
fected cells. C, for each chimera, membrane expression was monitored by
immunostaining. Note the clear intracellular localization of chimeras 3
and B compared with the others. D, partial 2APB sensitivity is restored
when mTRPV2 amino and carboxy termini are brought by two indepen-
dent subunits. Chimera cDNAs were transfected at the same ratio, and
responses to 100 ?M 2APB were measured 48 h later. Results were
normalized to the 2APB-induced response obtained in mTRPV2-trans-
fected cells. Note that coexpression of chimeras B and C lead to 2APB
sensitivity, although these chimeras only carry either the N or the C
terminus of mTRPV2. E, coexpression rescues the membrane expression
of trafficking-deficient chimera. Analysis of the membrane expression of
trafficking deficient chimeras 3 and B (white letters) was performed by
immunostaining. Specific immunostaining was made possible through
the use of species-specific antibodies directed against the divergent
C-terminal sequence of either human or rat channels.
Fig. 6. Lentivirus expression of hTRPV2 inhibits mTRPV2 activation by
2APB in stably expressing cells. Lentivirus-driven expression of hTRPV2
inhibits 2APB and DPBA activation of stably expressed mTRPV2. IIE11
and parental CHO cells were transduced with a lentivirus expressing
either hTRPV2 or hTRPV2[mut]; 5 days after transduction, dose-re-
sponse curves for 2APB (left) or DPBA (right) were performed. hTRPV2
or hTRPV2[mut] expression induced a rightward shift in the dose-re-
sponse curve for both agonists and a reduction of the maximal responses.
For comparison purposes, results were normalized to the maximal re-
sponse induced by 2APB or DPBA in nontransduced cells.
2APB as a Tool to Study TRPV2 Pharmacology and Function
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Page 9

activate native channels in primary keratinocytes (Chung et
al., 2004, 2005). In this study, we developed a calcium-based
screening assay to further characterize TRPV2 pharmacol-
ogy using 2APB and DPBA to activate the channel. Our
results show that both compounds activate TRPV2 with a
threshold in the range of 10 ?M. In nontransfected cells
(HEK or CHO), we observed a strong nonspecific calcium
signal for agonist concentrations greater than 1 mM that was
not additive with TRPV2-mediated calcium entry. We have
no explanation for this lack of additivity, although one pos-
sibility is that high concentrations of 2APB induce mem-
brane perturbations leading to calcium influx. Because of
these limitations it is difficult to calculate precise values of
the affinities of 2APB and DPBA for TRPV2 channels. How-
ever our EC50value estimations are consistent with previ-
ously reported values, indicating that 2APB activates TRPV2
and TRPV1 (Hu et al., 2004) within the same range of con-
centration. Activation of TRPV2 by other chemicals, includ-
ing known activators of TRPV channels such as capsaicin,
different bioactive lipids (arachidonic acid and its deriva-
tives), or phorbol esters, was also tested. However, none of
these molecules was active. In a recent report, THC was
proposed to activate both human and rat TRPV2 with affin-
ities in the micromolar range (Neeper et al., 2007). We here
confirm this observation, although we did not investigate it
further.
The screening for potential blockers of TRPV2 confirmed
the inhibitory effect of known TRP channel blockers such as
ruthenium red and SKF96365. We also found that two po-
tassium channel blockers, TEA and 4-AP, inhibited TRPV2
with apparent affinities in the millimolar range. This result
is surprising because these compounds are considered spe-
cific blockers of potassium channels. TEA and 4-AP binding
sites have been localized in the Kvfamily potassium channel
pore-forming region, which shares significant sequence ho-
mology with that of TRPV proteins (Voets and Nilius, 2003).
It is conceivable that TEA and 4-AP can block TRPV2 and
potassium channels through similar mechanisms. One novel
finding is the inhibitory effect of TRIM on 2APB-mediated
activation of TRPV2. TRIM is described as an inhibitor of NO
synthase (Handy et al., 1995) but also has an effect on store-
operated calcium entry (Gibson et al., 2001). It is noteworthy
Fig. 7. Lentiviral-mediated hTRPV2 expression inhibits
2APB activation of endogenous TRPV2 in RBL-2H3 cells
but not DNP-HSA-induced secretion. All hTRPV2 cDNAs
were modified to carry an N-terminal flag and an extracel-
lular HA epitope. A, left, immunostaining of endogenous
rTRPV2 and of transduced Flag-hTRPV2HAin RBL-2H3
cells. Endogenous rTRPV2 was detected using the commer-
cial VRL-1 antibody, whereas hTRPV2 was detected using
anti-HA tag antibody. Right, Western blotting of endoge-
nousrTRPV2, lentiviral-transduced
hTRPV2, and hTRPV2[mut] in RBL-2H3 cells. Note the
clear molecular mass difference between rat and human
TRPV2 channels and the lack of cross-reactivity between
VRL-1 and hTRPV2 antibodies. Tubulin was used as load-
ing control. B, analysis of intracellular calcium increase
induced by 2APB and DNP-HSA in transduced and non-
transduced RBL-2H3 cells. 2APB-induced intracellular cal-
cium increase is inhibited by overexpression of hTRPV2 or
hTRPV2 carrying three mutations in the pore-selectivity
filter. Note that GFP transduction does not affect 2APB-
mediated calcium increase. After sensitization, DNP-HSA
triggered an intracellular calcium increase that was not
inhibited by hTRPV2 or hTRPV2[mut], indicating that
TRPV2 is not involved in DNP-HSA-mediated calcium in-
crease. C, 2APB-mediated TRPV2 activation induced low
levels of ?-hexosaminidase secretion. Left, low concentra-
tions of 2APB induced a small dose-dependent increase of
?-hexosaminidase secretion that is inhibited by overexpres-
sion of hTRPV2[mut]. Right, DNP-HSA-induced secretion
is not inhibited by hTRPV2[mut] expression. Note that the
secretion induced by DNP-HSA reaches 35% compared
with 7% with 2APB. Results were normalized to intracel-
lular ?-hexosaminidase contents after substraction of basal
secretion.
GFP, wild-type
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that TRIM inhibits oxytocin-induced neuronal firing in the
supraoptic nucleus of the hypothalamus (Tobin et al., 2006),
in which TRPV2 is highly expressed and localized on oxytocin
neurons (Wainwright et al., 2004).
Another unexpected finding was the reduced effect of most
of these blockers when TRPV2 was activated by 300 ?M
2APB. One possible explanation is that these blockers act as
competitive antagonists. However, our results suggest that
the 2APB binding site is located intracellularly, implying
that all competitive inhibitors would need to cross the plasma
membrane to target the same site as 2APB. Although we
cannot completely rule out this possibility, it is clearly not
supported in the case of TEA, which is weakly membrane-
permeant. Another interpretation could be that these inhib-
itory effects strongly depend on the cellular membrane po-
tential and that activation by 300 ?M 2APB induces a
membrane depolarization strong enough to reduce channel
inhibition. This is unlikely, because a depolarization induced
by increasing the extracellular potassium concentration from
5 to 30 mM did not affect inhibition of either 100 or 300 ?M
2APB-evoked calcium signals by SKF96365, TEA, or 4-AP
(data not shown). In addition, patch-clamp recordings clearly
show that the inhibitory effect of TEA does not depend on
membrane potential but rather displays a strong use-depen-
dence; indeed, TEA inhibition of 2APB-evoked currents con-
sistently decreased upon repetitive stimulations. This raises
the possibility that high concentrations of 2APB may induce
progressive conformational changes of the channel that re-
duce the accessibility of the blockers to their binding site. On
the other hand, these conformational changes could affect the
pore selectivity of TRPV2 channels. Such selectivity changes
induced by 2APB have been demonstrated in the case of
TRPV3 gating (Chung et al., 2005). Further biophysical stud-
ies are necessary to understand the mechanism underlying
this use-dependence.
One main result of this study is the lack of sensitivity of
hTRPV2 to 2APB. This confirms previously published work
showing that hTRPV2 did not respond to 2APB, whereas
TRPV2 from rat is 2APB-responsive (Neeper et al., 2007).
How could this lack of sensitivity be explained? The most
likely hypothesis is that hTRPV2 is not activated by 2APB
because of intrinsic properties of the protein. This is sup-
ported by the chimera experiments that clearly demonstrate
that 2APB sensitivity can be transferred from the mouse to
the human channel and is determined by intracytoplasmic
regions. hTRPV2 shows the most divergence in a comparison
of the sequences of the three TRPV2 homologs (21.2 and
24.8% against mouse and rat, respectively, whereas rat
shows 7.5% divergence against mice). These amino acid dif-
ferences are mostly clustered in the distal parts of both
amino and carboxyl cytoplasmic regions (see Supplementary
Fig. S2) and are likely to underlie 2APB sensitivity. Although
extracellular TM1–TM2, TM3–TM4, and TM5–TM6 linkers
also display sequence divergences, several chimeras contain-
ing the mouse TMD do not show any 2APB sensitivity, ruling
out a possible contribution of TMD to 2APB sensitivity. Sim-
ilar findings were discovered through chimeric studies using
human-rat hybrid channels (Neeper et al., 2007). Our re-
sults further extend these observations to mouse-human
chimeras.
Our results provide some insight on the mechanism of
action of 2APB. First, they demonstrate that the 2APB bind-
ing site is unlikely to be extracellular, as proposed previously
(Hu et al., 2004), but rather located intracellularly, because
2APB gating is only observed when both intracytoplasmic
regions of mTRPV2 are present. This is compatible with the
hydrophobic nature of 2APB, which is known to be able to
reach intracellular targets such as inositol 1,4,5-triphosphate
receptors (Missiaen et al., 2001). Our data also show that
hTRPV2 exerts a dominant-negative inhibitory effect on
2APB gating. Indeed, lentiviral expression of hTRPV2 and
the mixing experiments induce both a reduction of both max-
imal response and apparent affinity of 2APB for mTRPV2.
This raises the possibility that conformational changes of
mTRPV2 subunits induced by 2APB lead to the opening of
the channel, and these changes are impaired when an
hTRPV2 subunit is present in the protein complex. These
conformational changes might be a direct consequence of
2APB binding to the channel. On the other hand, 2APB
might act indirectly via intracellular proteins that specifi-
cally interact with rodent TRPV2 channels or through spe-
cific post-translational modifications of the channels. Assum-
ing that 2APB gates TRPV1, -V2, and -V3 through similar
mechanisms, it is easier to interpret our results by a direct
binding of 2APB to the channels rather than through a com-
mon interacting protein or post-translational modifications.
2APB is an interesting tool to apprehend the physiological
function of native TRPV2 channels. TRPV2 is expressed in
the RBL-2H3 mast cell line, where it has been proposed to be
involved in the exocytosis of granule contents. 2APB also
induces cationic channel activity in these cells (Braun et al.,
2003). Our results clearly show that in RBL-2H3 cells, 2APB
induces an intracellular calcium increase that can be reduced
by overexpression of hTRPV2. However, 2APB induces min-
imal degranulation of sensitized RBL-2H3 cells compared
with a physiologically triggered secretion. Furthermore,
DNP-HSA-induced secretion was not affected by transduc-
tion of cells with lentiviruses encoding hTRPV2 or inhibited
by overexpression of a pore mutant channel with known
dominant-negative effects (Garcı ´a-Martı ´nez et al., 2000;
Penna et al., 2006). This strongly argues against the involve-
ment of TRPV2-mediated calcium entry in degranulation or
secretion mechanisms in mast cells. Further investigations
are undoubtedly required to understand the cellular func-
tions of TRPV2.
Thus, TRPV2 remains a channel with enigmatic functions,
highly expressed in immune cells and in neurons. In this
study, we provided new insights on the pharmacology of
TRPV2 as well as molecular tools that should help decipher
these functions. In addition, this work confirms that hTRPV2
has a specific pharmocological signature (Neeper et al.,
2007). As such, these findings greatly oppose the widely held
belief that TRPV2 responses are universal across mamma-
lian species.
Acknowledgments
This work was made possible thanks to the pharmacology-screen-
ing facility of Institut Fe ´de ´ratif de Recherche 3 and to the Montpel-
lier RIO Imaging facility. RBL-2H3 cells were provided by Dr. U.
Blank (Institut Pasteur, Paris, France). We thank Drs. T. Durroux
and I. A. Lefevre for their comments on the manuscript and P. Atger
for help with figures.
2APB as a Tool to Study TRPV2 Pharmacology and Function
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References
Benham CD, Davis JB, and Randall AD (2002) Vanilloid and TRP channels: a family
of lipid-gated cation channels. Neuropharmacology 42:873–888.
Braun FJ, Aziz O, and Putney JW Jr (2003) 2-aminoethoxydiphenyl borane activates
a novel calcium-permeable cation channel. Mol Pharmacol 63:1304–1311.
Caterina MJ (2007) Transient receptor potential ion channels as participants in
thermosensation and thermoregulation. Am J Physiol Regul Integr Comp Physiol
292:R64–R76.
Caterina MJ, Rosen TA, Tominaga M, Brake AJ, and Julius D (1999) A capsaicin-
receptor homologue with a high threshold for noxious heat. Nature 398:436–441.
Chung MK, Guler AD, and Caterina MJ (2005) Biphasic currents evoked by chemical
or thermal activation of the heat-gated ion channel, TRPV3. J Biol Chem 280:
15928–15941.
Chung MK, Lee H, Mizuno A, Suzuki M, and Caterina MJ (2004) 2-aminoethoxydi-
phenyl borate activates and sensitizes the heat-gated ion channel TRPV3. J Neu-
rosci 24:5177–5182.
Clapham DE (2003) TRP channels as cellular sensors. Nature 426:517–524.
Garcı ´a-Martı ´nez C, Morenilla-Palao C, Planells-Cases R, Merino JM, and Ferrer-
Montiel A (2000) Identification of an aspartic residue in the P-loop of the vanilloid
receptor that modulates pore properties. J Biol Chem 275:32552–32558.
Gibson A, Fernandes F, Wallace P, and McFadzean I (2001) Selective inhibition of
thapsigargin-induced contraction and capacitative calcium entry in mouse anococ-
cygeus by trifluoromethylphenylimidazole (TRIM). Br J Pharmacol 134:233–236.
Handy RL, Wallace P, Gaffen ZA, Whitehead KJ, and Moore PK (1995) The antino-
ciceptive effect of 1-(2-trifluoromethylphenyl) imidazole (TRIM), a potent inhibitor
of neuronal nitric oxide synthase in vitro, in the mouse. Br J Pharmacol 116:2349–
2350.
Hardie RC (2001) Phototransduction in Drosophila melanogaster. J Exp Biol 204:
3403–3409.
Hu HZ, Gu Q, Wang C, Colton CK, Tang J, Kinoshita-Kawada M, Lee LY, Wood JD,
and Zhu MX (2004) 2-Aminoethoxydiphenyl borate is a common activator of
TRPV1, TRPV2, and TRPV3. J Biol Chem 279:35741–35748.
Iwata Y, Katanosaka Y, Arai Y, Komamura K, Miyatake K, and Shigekawa M (2003)
A novel mechanism of myocyte degeneration involving the Ca2?-permeable growth
factor-regulated channel. J Cell Biol 161:957–967.
Kanzaki M, Zhang YQ, Mashima H, Li L, Shibata H, and Kojima I (1999) Translo-
cation of a calcium-permeable cation channel induced by insulin-like growth fac-
tor-I. Nat Cell Biol 1:165–170.
Klages N, Zufferey R, and Trono D (2000) A stable system for the high-titer produc-
tion of multiply attenuated lentiviral vectors. Mol Ther 2:170–176.
Missiaen L, Callewaert G, De Smedt H, and Parys JB (2001) 2-Aminoethoxydiphenyl
borate affects the inositol 1,4,5-trisphosphate receptor, the intracellular Ca2?
pump and the non-specific Ca2?leak from the non-mitochondrial Ca2?stores in
permeabilized A7r5 cells. Cell Calcium 29:111–116.
Montell C (2005a) TRP channels in Drosophila photoreceptor cells. J Physiol 567:
45–51.
Montell C (2005b) The TRP superfamily of cation channels. Sci STKE 2005:re3.
Muraki K, Iwata Y, Katanosaka Y, Ito T, Ohya S, Shigekawa M, and Imaizumi Y
(2003) TRPV2 is a component of osmotically sensitive cation channels in murine
aortic myocytes. Circ Res 93:829–838.
Naldini L, Blomer U, Gage FH, Trono D, and Verma IM (1996) Efficient transfer,
integration, and sustained long-term expression of the transgene in adult rat
brains injected with a lentiviral vector. Proc Natl Acad Sci U S A 93:11382–11388.
Neeper MP, Liu Y, Hutchinson TL, Wang Y, Flores CM, and Qin N (2007) Activation
properties of heterologously expressed mammalian TRPV2: evidence for species
dependence. J Biol Chem 282:15894–15902.
Penna A, Juvin V, Chemin J, Compan V, Monet M, and Rassendren FA (2006)
PI3-kinase promotes TRPV2 activity independently of channel translocation to the
plasma membrane. Cell Calcium 39:495–507.
Ramsey IS, Delling M, and Clapham DE (2006) An introduction to TRP channels.
Annu Rev Physiol 68:619–647.
Saunders CI, Kunde DA, Crawford A, and Geraghty DP (2007) Expression of tran-
sient receptor potential vanilloid 1 (TRPV1) and 2 (TRPV2) in human peripheral
blood. Mol Immunol 44:1429–1435.
Stokes AJ, Shimoda LM, Koblan-Huberson M, Adra CN, and Turner H (2004) A
TRPV2-PKA signaling module for transduction of physical stimuli in mast cells. J
Exp Med 200:137–147.
Tobin V, Gouty LA, Moos FC, and Desarmenien MG (2006) A store-operated current
(SOC) mediates oxytocin autocontrol in the developing rat hypothalamus. Eur J
Neurosci 24:400–404.
Tominaga M and Caterina MJ (2004) Thermosensation and pain. J Neurobiol 61:3–
12.
Voets T and Nilius B (2003) The pore of TRP channels: trivial or neglected? Cell
Calcium 33:299–302.
Wainwright A, Rutter AR, Seabrook GR, Reilly K, and Oliver KR (2004) Discrete
expression of TRPV2 within the hypothalamo-neurohypophysial system: implica-
tions for regulatory activity within the hypothalamic-pituitary-adrenal axis. J
Comp Neurol 474:24–42.
Address correspondence to: Dr. Franc ¸ois-A Rassendren, Department of
Molecular Pharmacology, Institut de Ge ´nomique Fonctionnelle, 141 rue de la
Cardonille, 34094 Montpellier, France. Email: far@igh.cnrs.fr
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