THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 30, Issue of July 27, pp.

28613–28619, 2001
Printed in U.S.A.

Analysis of the Native Quaternary Structure of Vanilloid

Receptor 1*□ S

Received for publication, April 12, 2001, and in revised form, May 16, 2001
Published, JBC Papers in Press, May 17, 2001, DOI 10.1074/jbc.M103272200

Noemi Kedei‡, Tamas Szabo‡, Jack D. Lile§, James J. Treanor§, Zoltan Olah¶,
Michael J. Iadarola¶, and Peter M. Blumberg‡储
From the ‡Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, National Institutes of Health, Bethesda,
Maryland 20892, §Amgen, Thousand Oaks, California 91320, and the ¶Neuronal Gene Expression Unit, Pain and
Neurosensory Mechanisms Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892

Vanilloid receptor subtype 1 (VR1) is a ligand-gated to other noxious stimuli. This phenomenon underlies the seem-
channel that can be activated by capsaicin and other ingly paradoxical use of capsaicin as an analgesic agent in the
vanilloids as well as by protons and heat. In the present treatment of various painful disorders (4 – 6).
study, we have analyzed the oligomeric state of VR1. A functional vanilloid receptor termed vanilloid receptor
Co-immunoprecipitation of differently tagged VR1 mol- type 1 (VR1) has been cloned recently. Both when expressed
ecules indicated that VR1 can form oligomers. Using two endogenously in cultured dorsal root ganglion cells or when
different heterologous VR1 expression systems as well expressed heterologously in transfected human cells or frog
as endogenous VR1 expressed in dorsal root ganglion oocytes, VR1 can be activated by vanilloids and by noxious heat
cells, we analyzed oligomer formation using perfluoro- (⬎43 °C) and can be activated or potentiated by protons (extra-
octanoic acid polyacrylamide gel electrophoresis. Re- cellular pH ⬍6) (6 – 8). VR1 encodes a protein of 838 amino
sults were confirmed both with chemical cross-linking
acids with a predicted molecular mass of 92–95 kDa (9 –10). It
agents as well as through endogenous cross-linking me-
is a nonselective cation channel with high Ca2⫹ permeability
diated by transglutaminase. Our results clearly show
and belongs to the superfamily of cation channels with six
that VR1 forms multimers in each of the expression sys-
tems with a homotetramer as a predominant form. The transmembrane segments including the Shaker-related volt-
oligomeric structure of VR1 may contribute to the com- age-gated K⫹ channels, the hyperpolarization-activated cyclic
plexity of VR1 pharmacology. Finally, differences in gly- nucleotide-gated cation channels, the cyclic nucleotide-gated
cosylation between the systems were observed, indicat- cation channels, the polycystin channels and the transient
ing the need for caution in the use of the heterologous receptor potential (Trp) family of ion channels. These proteins
expression systems for analysis of VR1 properties. all contain six transmembrane domains, a pore-forming loop
between the fifth and sixth transmembrane domains, and cy-
toplasmic C- and N-terminal domains (6, 7, 11). Within this
Pain-producing stimuli are detected by specialized primary superfamily, VR1 is most closely related to the Trp family of ion
afferent neurons called nociceptors, which can be activated by channels. These channels include Drosophila Trp and TrpL,
different noxious chemical (acid, irritants, inflammatory medi- which play crucial roles in phototransduction, and the mam-
ators) or physical stimuli (heat, cold, pressure). Capsaicin, the malian homologues TrpC1–7, which are candidates for store-
pungent ingredient in hot peppers, and structurally related operated calcium channels.
vanilloids such as the ultrapotent irritant resiniferatoxin Understanding of the function, gating, regulation and
(RTX)1 bind to specific receptors on nociceptors (1–3), causing quaternary structure of Trp family members is emerging rap-
cation influx, triggering action potentials, and inducing the idly. The Drosophila Trp and TrpL have been shown to form
sensation of burning pain. Prolonged or repeated exposure to heteromultimeric channels associated in a signaling complex
capsaicin leads to pronounced tachyphylaxis and desensitiza- with a receptor (rhodopsin), an effector (phospholipase C), reg-
tion, in which nociceptors become insensitive to capsaicin and ulators (protein kinase C and calmodulin), and the scaffolding
protein INAD (12). Similarly, hTrp1 is localized in a caveolin-
This is an open access article under the CC BY license.
scaffolding lipid raft domain in plasma membrane, where it
* The costs of publication of this article were defrayed in part by the
interacts directly or indirectly with a complex of Ca2⫹ signaling
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to proteins such as inositol 1,4,5-trisphosphate receptor and
indicate this fact. G␣q/11 (13). Trp3 and Trp1 form hetero-oligomers with nonse-
□S The on-line version of this article (available at http://www.jbc.org) lective cation permeability (14, 15), and Trp4 coexpressed with
contains Supplementary Figs. 1– 6. Trp1 was proposed to form store-operated calcium channels in
储 To whom correspondence should be addressed: Laboratory of Cellu-
lar Carcinogenesis and Tumor Promotion, NCI, National Institutes of adrenocortical cells through homo- or heteromultimers (16).
Health, Bldg. 37, Rm. 3A01, 37 Convent Dr., MSC 4255, Bethesda, MD However, their actual subunit organization has not been deter-
20892-4255. Tel.: 301-496-3189; Fax: 301-496-8709; E-mail: blumberp@ mined. More distant members of the superfamily, e.g. voltage-
dc37a.nci.nih.gov.
1
gated K⫹ channels or cyclic nucleotide-gated channels, are
The abbreviations used are: RTX, resiniferatoxin; BS3, bis[sulfosuc-
cinimidyl]suberate; sulfo-BSOCOES, bis[2-(sulfosuccinimidooxycar- known to form tetramers of the same or different subunits (17).
bonyloxy)ethyl]sulfone; DRG, dorsal root ganglion; INAD, inactivation The first direct experiments evaluating the quaternary
no afterpotential D; PFO, perfluoro-octanoic acid; PKC, protein kinase structure of VR1 were performed by Szallasi and Blumberg (18)
C; PNGase F, peptide-N-glycosidase F; PPAHV, phorbol 12-phenylac- using radiation inactivation analysis. This technique, which
etate 13-acetate 20-homovanillate; PAGE, polyacrylamide gel electro-
phoresis; VR1, vanilloid receptor type 1; PBS, phosphate-buffered
gives the functional size of a receptor, yielded a value for the
saline; Trp, transient receptor potential; eGFP, enhanced green fluo- vanilloid receptor, detected by [3H]RTX binding, of 270 ⫾ 25
rescence protein. kDa; this value compares with a monomer mass for VR1 of 92

This paper is available on line at http://www.jbc.org 28613

28614 Subunit Structure of Capsaicin Receptor
kDa, strongly arguing for a functional size greater than the LipofectAMINE Plus (Life Technologies, Inc.) according to the proce-
monomer. Further evidence for oligomerization is the domi- dure recommended by the manufacturer. 48 h after transfection, 3 ml of
ice-cold radioimmune precipitation buffer without SDS (pH 7.5) con-
nant negative behavior of a mutant VR1 which does not re-
taining 50 mM Tris-HCl, 150 mM NaCl, 1% IGEPAL CA-630 (Sigma),
spond to capsaicin (19). Finally, a Hill-coefficient of ⬃2 is found 0.5% sodium deoxycholate (Sigma), and protease inhibitors (1 tablet/10
for VR1 interaction with different agonists, indicating positive ml of PBS) (Roche Molecular Biochemicals) was added to the cells and
cooperativity (6, 20 –22). Together with the similarities in incubated for 10 min on ice. The lysates were centrifuged at 3,000 ⫻ g
molecular structure between the members of the six transmem- for 10 min to remove cellular debris. Subsequently, 10 ␮l of anti-PKC⑀
brane domain channel superfamily, it thus seems highly prob- polyclonal antibody (Life Technologies, Inc.), 5 ␮l of anti-eGFP mono-
clonal antibody (Roche Molecular Biochemicals), or 10 ␮l of non-im-
able that the active VR1 channel is composed of more than one
mune serum was added to 1 ml of supernatant containing 0.4 mg of
subunit, forming homomultimeric or heteromultimeric ion protein and rotated for 4 h at 4 °C. Then, 20 ␮l of Protein A/G Plus-
channels (16, 23, 24). Potential partners include the VR1 ho- agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the
mologues SIC, VRL-1, and VR5’sv (25–27). Multimeric chan- immune complexes and rotated at 4 °C overnight. The immunoprecipi-
nels could contribute to the functional heterogeneity and tates were washed four times with ice-cold radioimmune precipitation
complex pharmacology of VR1 seen in binding or calcium up- buffer without SDS, eluted with 2⫻ SDS sample buffer, and analyzed
by Western blotting on 4 –12% SDS-polyacrylamide gels.
take experiments in DRGs and in different heterologous
Preparations of DRG Lysates—Cell membranes from rat DRGs were
systems (28 –31). prepared as described previously (29). Female Harlan Sprague-Dawley
The goal of the present paper is to evaluate the native qua- rats weighting 200 –250 g were decapitated under CO2 anesthesia. The
ternary structure of VR1, as the complex structure of the active spinal columns were opened, and DRGs from all levels were collected in
channel could provide insights into channel regulation and the ice-cold physiological saline. DRGs were homogenized in ice-cold buffer
design of new analgesic agents. Using three different biochem- (pH 7.4) containing 5 mM KCl, 5.8 mM NaCl, 0.75 mM CaCl2, 2 mM
MgCl2, 320 mM sucrose, and 10 mM HEPES using a tissue homogenizer
ical approaches, we clearly show that the VR1 channel forms
type PRO 200 (PRO Scientific Inc., Monroe, CT) followed by sonication.
multimers both in DRGs and in heterologous expression sys- Tissue homogenates were centrifuged for 10 min at 3,000 ⫻ g (4 °C) to
tems, with a tetramer as a predominant form. remove cellular debris and the supernatant was centrifuged again for
30 min at 35,000 ⫻ g (4 °C) to obtain a partially purified membrane
EXPERIMENTAL PROCEDURES fraction (pellet). After solubilization in PBS containing 2% n-dodecyl
Transfection of Cells and Cell Culture—Cos7 cells were cultured in ␤-D-maltoside (Sigma), the membrane proteins were separated on PFO-
Dulbecco’s modified Eagle’s medium (high glucose) supplemented with PAGE and analyzed by Western blotting.
10% fetal bovine serum, 50 units/ml penicillin, and 50 ␮g/ml strepto- PFO-PAGE—Proteins were fractionated by PFO-PAGE as described
mycin (Life Technologies, Inc.). Cos7 cells were transiently transfected by Ramjeesingh et al. (32). Freshly poured 6% Tris-glycine gels without
with VR1⑀ plasmid and/or VR1eGFP plasmid using LipofectAMINE SDS were used. Total cell lysates were prepared from Tet-Off induced
Plus (Life Technologies, Inc.) according to the procedure recommended CHO-VR1 cells 48 h after tetracycline withdrawal, from Cos7 cells
by the manufacturer. VR1⑀ and VR1eGFP plasmids were constructed as expressing VR1eGFP 48 h after transfection, and from rat DRGs as
described previously (22). described above. In some experiments cell lysates from CHO-VR1 cells
The selected stable CHO cell clone expressing pUHG102 VR1 and were treated with peptide-N-glycosidase F (New England Biolabs) for
pTet-Off Regulator plasmids (Tet-Off induced CHO-VR1 cells) is the 1 h at 37 °C. Twenty ␮g of lysates were mixed with doubly concentrated
generous gift of James E. Krause and Daniel N. Cortright (Neurogen sample buffer (100 mM Tris base, 2– 8% NaPFO (Oakwood Products
Corp., Branford, CT). These cells were cultured in F-12 medium sup- Inc., West Columbia, SC), 20% glycerol, 0.7 M ␤-mercaptoethanol, and
plemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 ␮g/ml 0.005% bromphenol blue, pH 8.0), the final PFO concentration being in
streptomycin, 500 ␮g/ml Geneticin, 10 mM HEPES (pH 7.4) (Life Tech- the range of 0.8 –3.2%. After 30 min of incubation at room temperature
nologies, Inc.), and 5 ␮g/ml tetracycline (Sigma). For the optimal level the samples were subjected to electrophoresis. The running buffer con-
of VR1 expression the cells were cultured in tetracycline-free medium tained 25 mM Tris, 192 mM glycine, 0.5% NaPFO, pH 8.5, adjusted with
containing 10 ␮M sodium butyrate (Calbiochem, San Diego, CA) for NaOH. The electrotransfer and immunostaining were performed as
36 – 48 h. described above. As molecular weight standard, cross-linked phospho-
Western Blot Analysis—The cell cultures were rinsed two times with rylase b (Sigma) was separated on the same gels, electroblotted, and
ice-cold phosphate-buffered saline (PBS), and then cells were harvested stained with Amido Black.
in ice-cold PBS containing protease inhibitors (1 tablet/10 ml of PBS) Chemical Cross-linking—The following water-soluble, homobifunc-
(Roche Molecular Biochemicals), and sonicated. Protein content was tional N-hydroxysuccimide esters were used for protein cross-linking
measured by a micromethod using the Bio-Rad protein assay. SDS- studies: the non-cleavable bis[sulfosuccinimidyl]suberate (BS3) and the
PAGE was performed on a mini-slab apparatus using precast 4 –12% base-cleavable bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone
SDS-polyacrylamide gels (Novex, San Diego, CA) by the method of (sulfo-BSOCOES; Pierce). Both reagents were dissolved in PBS imme-
Laemmli. Twenty ␮g of lysates were mixed to equal volumes of 2⫻ SDS diately before use. The lysates prepared from Tet-Off induced CHO-
sample buffer (125 mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 0.7 M VR1 cells 48 h after tetracycline withdrawal and from Cos7 cells ex-
␤-mercaptoethanol, and bromphenol blue), boiled for 5 min, and sub- pressing VR1eGFP 48 h after transfection were incubated with
jected to SDS-PAGE, followed by electrotransfer onto nitrocellulose different concentrations of cross-linkers (1 ␮M to 10 mM) or without any
membranes (Schleicher & Schuell, Keene, NH). For immunostaining, cross-linker for 30 min at room temperature. To stop the reaction, an
anti-eGFP monoclonal antibody (Roche Molecular Biochemicals) was equal volume of 1 M glycine was added to the mixtures and incubated for
used at 0.4 ␮g/ml concentration, anti-VR1 polyclonal antibody was an additional 20 min. The quenched samples were subsequently mixed
diluted 1:1000 and anti-PKC⑀ polyclonal antibody (Life Technologies, with 4⫻ concentrated Nu-PAGE sample buffer (564 mM Tris base, 424
Inc.) was diluted 1:1000. The anti-VR1 polyclonal antibody was gener- mM HCl, 292 mM lithium dodecyl sulfate, 2 mM EDTA, 0.7 M ␤-mercap-
ated in rabbits against the extreme C terminus of rat VR1, namely toethanol, 0.88 mM Serva Blue G250, 0.70 mM Phenol Red, pH 8.5)
peptide E824-K838 (Amgen, Thousand Oaks, CA) and showed specific according to the description provided by the manufacturer (Novex, San
recognition of VR1. Nonspecific binding of antibodies to the membranes Diego, CA), incubated at 70 °C for 15 min, and then subjected to elec-
was blocked by a 60-min incubation with 5% milk in PBS. The mem- trophoresis. The samples were separated on 3– 8% gradient Nu-PAGE
branes were probed overnight at 4 °C with the different antibodies Tris acetate gels and analyzed by Western blotting. Cross-linking ex-
diluted in 5% milk in PBS. Following the incubation, the membranes periments were also performed on living Cos7 cells transiently express-
were washed three times for 10 min in PBS containing 0.05% Tween 20 ing VR1eGFP. The cells were incubated for 30 min at 37 °C in PBS
and then incubated for 60 min with horseradish peroxidase-conjugated containing different concentrations of BS3 (0.01–10 mM) followed by
anti-mouse or anti-rabbit IgG (Bio-Rad). After the membranes were preparation of cell lysates and their Western blot analysis.
rinsed three times for 10 min in PBS containing 0.05% Tween 20,
immunostaining was visualized by ECL (Amersham Pharmacia Bio- RESULTS
tech). BenchMark prestained Protein Ladder (Life Technologies Inc.)
was used as molecular size marker. Co-immunoprecipitation—To determine if two or more VR1
Co-immunoprecipitation—Plasmids encoding VR1⑀ and VR1eGFP subunits form a complex, VR1⑀ and VR1eGFP were coex-
were transfected into Cos7 cells either together or individually using pressed in Cos7 cells. These differently tagged VR1 proteins
 Subunit Structure of Capsaicin Receptor 28615
the protein. On SDS-PAGE, VR1 migrated as a double band
without and as a single band after PNGase F treatment (Fig.
2F). On PFO-PAGE the upper band of VR1 monomer and some
upper bands of the dimers and trimers were markedly reduced
with PNGase F treatment, and there also was a shift in the
mobility of VR1 tetramer (Fig. 2B). Based on these observa-
tions, we assume that the multiple bands corresponding to VR1
multimers are due to the different combinations of the glyco-
sylated and non-glycosylated monomers, with predicted appar-
ent molecular masses ranging from 190 to 220 kDa for dimers
(three possible bands of 190, 205, and 220 kDa) and from 285 to
330 kDa for trimers (four possible bands of 285, 300, 315, and
330 kDa). The failure to resolve multiple bands of VR1 tetram-
ers (five possible bands with predicted apparent molecular
FIG. 1. Co-immunoprecipitation of VR1⑀ and VR1eGFP. Plas- masses of 380, 395, 410, 425, and 440 kDa) can be explained by
mids encoding VR1⑀ and VR1eGFP were transfected into Cos7 cells the decreased resolution of the gel in the higher molecular
either together or individually. Cell lysates were processed for Western
analysis or used for immunoprecipitation. The result shown is from one
weight region; the increased mobility of VR1 tetramer after
of two independently performed experiments. A, cell lysates were im- glycosidase treatment is still apparent. In the treated sample,
munoprecipitated with anti-PKC⑀ polyclonal antibodies (IP-PKC⑀) or a band with higher molecular mass and some protein aggre-
non-immune serum (IP-NIS) and Western blots of total cell lysates gates stacked at the top of the separating gel are also seen.
(lysate), and the immune complexes were probed with anti-eGFP anti-
bodies. B, cell lysates were immunoprecipitated with anti-eGFP mono-
Based on our present studies, their nature and importance
clonal antibodies (IP-eGFP) or non-immune serum (IP-NIS) and West- cannot be determined.
ern blots of total cell lysates (lysate) and the immune complexes were In the other heterologous system used, VR1eGFP migrated
probed with anti-PKC⑀ antibodies. as four distinct bands on 6% PFO gels (Fig. 2C) with estimated
molecular masses of 119, 236, 345, and 464 kDa, respectively
were immunoprecipitated with anti-eGFP or anti-⑀ tag anti-
(Fig. 2D). Molecular mass determination of the observed pro-
bodies and Western blots containing the immune complexes
tein species was based on the linear relationship of the loga-
were probed with the reciprocal antibodies. Transfection with
rithm of the molecular masses of cross-linked phosphorylase b
just one construct as well as immunoprecipitation with non-
subunits used as molecular weight standards versus their mi-
immune serum were used as controls. The results shown in Fig.
gration distance. Based on the obtained molecular weights, we
1 demonstrate that VR1eGFP co-immunoprecipitated with
assume that these four bands represent the VR1eGFP mono-
VR1⑀ and vice versa, suggesting the existence of at least two
mers, dimers, trimers, and tetramers (predicted molecular
VR1 molecules in a protein complex.
mass for VR1eGFP of 124, 248, 372, and 496 kDa). VR1eGFP
PFO-PAGE—The first method used to determine the nature
migrated as a single band on SDS-PAGE as shown on Fig. 2F,
of the VR1 channel complex was PFO-PAGE. In this method,
although some glycosylation (10 –15%) was seen in some exper-
PFO is used in place of SDS in gel electrophoresis, as it is less
iments, resulting in the appearance of a double band when
disruptive of interactions within protein oligomers and thus
permits molecular mass determination of multimeric proteins. higher exposure times were used for ECL development.
VR1eGFP-transfected Cos7 cell lysates, Tet-Off induced CHO- After treating the cells expressing VR1eGFP for 30 min with
VR1 cell lysates, and partially purified DRG membranes were the VR1 agonist RTX at doses activating the channel,
separated on PFO-PAGE and analyzed by Western blotting VR1eGFP was separated as monomer, dimer, trimer, and tet-
(Fig. 2). The pattern of protein migration depended on time and ramer on PFO-PAGE (Fig. 2C) and as monomer, dimer, and
temperature of incubation with sample buffer containing PFO; multimer on SDS-PAGE (Fig. 3A), showing that some covalent
a longer incubation time and higher temperature resulted in a bonds are formed between the subunits of the complex. Some
greater degree of subunit dissociation from the protein complex higher molecular weight bands running in the PFO-stacking
(see Supplementary Figs. 1– 6, available in the on-line version gels could be visualized with higher exposure times (see sup-
of this article). Subunit dissociation was also highly dependent plemental material, available on line) possibly because some
on the ratio of PFO to protein and/or lipid (Fig. 2A). CHO-VR1 other proteins or another preformed complex are covalently
cell lysates were incubated with different PFO concentrations cross-linked to VR1 tetramer.
(0.4 –3.2%) for 30 min at room temperature and separated on When partially purified DRG membranes were subjected to
6% PFO-PAGE. Using 3.2% and 1.6% PFO, VR1 migrated as PFO-PAGE using optimized conditions (solubilization of mem-
monomers and some dimers. With 0.8% PFO, VR1 was sepa- brane proteins with 2% n-dodecyl ␤-D-maltoside and incubation
rated into multiple bands representing VR1 monomers, dimers, with sample buffer containing PFO at a final concentration of
trimers, and tetramers; using 0.4% PFO, VR1 migrated mainly 0.8% for 30 min room temperature), VR1 migrated as a major
as tetramer, but higher molecular weight protein aggregates band representing VR1 tetramer and as a minor band
were also seen, probably due to insufficient solubilization of representing VR1 monomer (Fig. 2E). When higher PFO con-
membrane proteins (Fig. 2A). Based on these results in each centrations were used, VR1 monomers, dimers, trimers, and
system, we varied the PFO concentration to find the appropri- tetramers could be also seen (see supplemental material, avail-
ate conditions for detecting oligomers. able on line). VR1 monomer migrated as a single band both on
In CHO-VR1 cells VR1 monomer is present in two different PFO and SDS-PAGE showing the lack of the glycosylated form
forms: a non-glycosylated form with apparent molecular mass of VR1 in DRG (Fig. 2, E and F). In accordance with this
of 95 kDa and a glycosylated form with calculated molecular observation, VR1 tetramer from DRG had a slightly higher
mass of 114 kDa as shown in Fig. 2 (A, B, and F). Treatment for mobility than the VR1 tetramer from CHO-VR1 cells, as the
1 h at 37 °C with peptide-N-glycosidase F, an enzyme that tetramer of the latter is a combination of the glycosylated and
cleaves N-glycosidic bonds between the sugar portion and as- non-glycosylated form of VR1 resulting in a complex with
paragine of glycoproteins, reduced or eliminated the upper higher molecular weight.
band of VR1 monomer representing the glycosylated form of Endogenous Cross-linking of VR1 by Transglutaminases
28616 Subunit Structure of Capsaicin Receptor

FIG. 2. Determination of VR1 oligomeric structure using PFO-PAGE. Total cell lysates prepared from VR1eGFP-transfected Cos7 cells,
Tet-Off induced CHO-VR1 cells treated or not with PNGase F, and partially purified DRG membranes solubilized with 2% n-dodecyl ␤-D-maltoside
were separated on 6% PFO-PAGE, electrotransferred to nitrocellulose membranes, and immunostained with appropriate antibody. In parallel,
cross-linked phosphorylase b was separated on the same gel, electrotransferred, and visualized by Amido Black staining for molecular weight
determination purposes. These results are representative of at least three independent experiments. A, CHO-VR1 lysates were incubated with
sample buffer containing different PFO concentrations for 30 min at room temperature before electrophoresis; the Western blot was probed using
anti-VR1 antibody. B, CHO-VR1 cell lysates were treated or not with peptide-N-glycosidase F for 1 h at 37 °C; the Western blot was probed using
anti-VR1 antibody. C, separation of VR1eGFP (with or without agonist activation) on 6% PFO gel. Anti-eGFP monoclonal antibody was used for
probing. D, the molecular weights of the different VR1eGFP protein species were calculated using the linear relationship between the distance of
migration (x axis) and the logarithm of molecular weight (y axis) of cross-linked phosphorylase b. E, separation of VR1 from DRG and CHO-VR1
on 6% PFO-PAGE; anti-VR1 antibody was used for probing. F, separation of VR1eGFP, and VR1 from induced CHO cells and DRG on 4 –12%
SDS-PAGE. Anti-eGFP monoclonal and anti-VR1 polyclonal antibody were used for probing.

upon Vanilloid Treatment of Cells—Upon VR1 activation by treated with other VR1 agonists (capsaicin, olvanil, PPAHV) at
agonists such as capsaicin or its ultrapotent analog RTX, VR1 doses known to activate the VR1 channel but was not observed
became covalently cross-linked as detected by SDS-PAGE. The with the competitive inhibitor capsazepine at concentrations
cross-linking depended on time, on ligand concentration, and up to 30 ␮M. Likewise, pretreatment with 10 ␮M capsazepine
on the presence of extracellular calcium. Treatment of Cos7 could prevent the covalent bond formation between VR1 mono-
cells expressing VR1eGFP with different doses of RTX (0.001–1 mers (Fig. 3B). Treatment of cells with 1–10 ␮M ionomycin, an
nM) for 30 – 60 min resulted in formation of VR1 dimers and agent that increases intracellular Ca2⫹, did not result in the
multimers; the cross-linking was not seen when 1 mM EGTA same degree of cross-linking, although some formation of
was added to the extracellular fluid (Fig. 3A). The lowest RTX dimers and a very low amount of higher oligomers could be
concentration causing cross-linking was 0.01 nM. This ligand seen (see supplemental information, available on line). We
concentration is sufficient to activate the channel and induce a surmise that covalent cross-linking of VR1 reflects the Ca2⫹-
sustained increase in intracellular Ca2⫹ concentration (see dependent activation of endogenous transglutaminases. Con-
supplemental information, available on line). Cross-linked sistent with this interpretation, pretreatment with 20 mM
VR1eGFP showed a pattern similar to control VR1eGFP with- cysteamine or 250 ␮M mono-dansylcadaverine, two transglu-
out agonist treatment when separated on PFO-PAGE (Fig. 2C). taminase inhibitors, prevented the covalent cross-linking (Fig.
The same multimerization was observed when cells were 3B). Similar cross-linking of VR1 in response to VR1 agonists
 Subunit Structure of Capsaicin Receptor 28617
partly because dimers, trimers, and tetramers with different
molecular weights can be formed from glycosylated and non-
glycosylated VR1 monomers. To prevent this phenomenon,
PNGase F-treated VR1 was used. Using different BS3 concen-
trations up to 1 mM, formation of VR1 dimers, trimers, and
tetramers was seen (Fig. 4B). At higher cross-linker concentra-
tions, multimers with calculated molecular weights corre-
sponding to VR1 hexamers and octamers were also formed. The
molecular weight determination of the higher molecular weight
complexes is problematic, however, because of the decreased
resolution of Nu-PAGE in this range as well as the lack of good
high molecular weight markers. When sulfo-BSOCOES was
used as a cross-linker, similar cross-linking patterns were ob-
served, although sulfo-BSOCOES was somewhat less efficient
than was BS3 (see supplemental information, available on
line).
Cross-linking experiments were also performed on living
Cos7 cells expressing VR1eGFP, when the chemical cross-
linker can form covalent bonds only between accessible pri-
mary amines sitting on the outer surface of cell membranes.
The cells were incubated with different concentrations of BS3
for 30 min at 37 °C; cell lysates were prepared and analyzed by
Western blotting. As shown on Fig. 4C, at higher BS3 concen-
trations (1–10 mM), VR1eGFP migrated as four sets of bands
representing monomers, dimers, trimers, and tetramers with-
FIG. 3. Cross-linking of VR1 by transglutaminase upon VR1 out any other, intermediate molecular weight or high molecu-
channel activation. Cos7 cells expressing VR1eGFP were treated lar weight complexes being present except some highly cross-
with different doses of RTX for 30 min in the absence or presence of 1 linked polymers not able to enter the gel. The VR1eGFP dimers
mM EGTA, or after pretreatment with the competitive VR1 antagonist
capsazepine or inhibitors of transglutaminase. The total cell lysates run as double or multiple bands; we assume that dimers are
were analyzed on 4 –12% SDS-PAGE, followed by electrotransfer and formed between the glycosylated and non-glycosylated forms of
immunostaining with anti-eGFP monoclonal antibodies. Similar results VR1eGFP, which can be observed with the higher exposure
were obtained in three to five independent experiments for each agent. times needed to visualize trimers and tetramers present at
A, immunoblot of cross-linked VR1 upon treatment with different doses
of RTX in the absence or presence of EGTA. B, 1-h pretreatment with lower amount.
capsazepine (10 ␮M), with cysteamine (20 mM) or with mono-dansylca-
DISCUSSION
daverine (250 ␮M) prevented the formation of VR1 multimers in re-
sponse to 0.1 nM RTX treatment. Analysis of the native, multimeric structure of membrane
proteins is problematic. SDS-PAGE, although a powerful tech-
was seen in CHO-VR1 cells as well (see supplemental informa- nique for determination of monomer molecular weights, dis-
tion, available on line). rupts protein-protein interactions. Sucrose density gradient
Chemical Cross-linking—A third method to explore VR1 oli- centrifugation and gel filtration after solubilization of mem-
gomerization and to potentially reveal associated proteins in brane complexes with relatively mild detergents can be used,
complex with VR1 was cross-linking with chemical cross-link- assuming that adequate conditions for solubilization can be
ing agents. Two water-soluble cross-linkers were examined in established (32–34); however, these methods require large
detail: the non-cleavable cross-linker BS3 with a spacer arm of amounts of recombinant or purified proteins and lack the re-
1.14 nm and the base-cleavable cross-linker sulfo-BSOCOES solving power of PAGE.
with a spacer arm of 1.3 nm. Total cell lysates from VR1eGFP- To evaluate the quaternary structure of VR1, we used three
transfected Cos7 cells and from induced CHO-VR1 cells, different approaches. First, we coexpressed two differently
treated or not treated with PNGase F, were incubated with tagged versions of VR1 in Cos7 cells. Immunoprecipitation of
different concentrations of the cross-linkers for different times either caused co-immunoprecipitation of the other, implying
at room temperature. The formed complexes were separated on the presence of at least two subunits of VR1 in the receptor
Nu-PAGE Tris acetate gels for better separation of high molec- complex. These results complement the findings of Oxford et al.
ular protein complexes and were analyzed by Western blotting. (19), who demonstrated that a mutant of VR1 could have dom-
Fig. 4 (A and B) shows the immunoblot of the reaction products inant negative activity on channel function.
when different concentrations of BS3 were used as cross-linkers To characterize the composition of the VR1 receptor complex,
with a 30-min incubation time at room temperature. As shown we used PFO-PAGE, a novel method for quaternary structure
in Fig. 4A, the amount of VR1eGFP monomers decreased with determination of membrane proteins (32). We analyzed VR1 in
increasing concentration of BS3, whereas the amount of the two different heterologous expression systems, Cos7 cells tran-
oligomeric forms increased concomitantly; first dimers, then siently expressing VR1eGFP and a Tet-Off inducible CHO-VR1
trimers and tetramers were formed. Incubation with 300 ␮M cell line, and we examined native VR1 in rat dorsal root gan-
BS3 resulted in formation of VR1eGFP dimers, trimers, and glion membranes. In the two heterologous systems, we detected
tetramers, but no other protein species of intermediate molec- VR1 monomers, dimers, trimers, and tetramers, although by
ular weights, indicating the presence of other proteins in the lowering the PFO concentration we were able to decrease the
VR1 complex, were found in this system. At higher cross-linker relative ratio of the lower oligomers and find conditions where
concentrations, multimers with calculated molecular weights tetramer was the major form of VR1. In each system the opti-
corresponding to VR1 hexamers were also formed. When the mal protein/PFO ratio had to be determined to yield a PFO
cross-linking experiments were performed on CHO-VR1 cell percentage that did not result in disaggregation of subunits
lysates, a more complex pattern of protein separation was seen, from the protein complex but did give adequate solubilization.
28618 Subunit Structure of Capsaicin Receptor

FIG. 4. Chemical cross-linking of

VR1 by BS3. Total cell lysates of Cos7
cells expressing VR1eGFP and of induced
CHO-VR1 cells treated or not with
PNGase F were incubated with different
concentrations of BS3 (1 ␮M-10 mM) for 30
min at room temperature. After quench-
ing the reaction, the cross-linked proteins
were separated on 3– 8% Nu-PAGE, elec-
troblotted, and immunostained with ap-
propriate antibodies. In living cell exper-
iments, Cos7 cells expressing VR1eGFP
were treated with different concentra-
tions of BS3 for 30 min at 37 °C followed
by Western blot analysis of the lysates.
The molecular mass of the protein com-
plexes was determined by cross-linked
phosphorylase b separated on the same
gels, electroblotted and stained with
Amido Black and by SeeBlue pre-stained
protein standard. A and B are from a rep-
resentative experiment repeated an addi-
tional two times; the experiment illus-
trated in C was repeated once with
similar results. A, BS3 cross-linked
VR1eGFP probed with anti-eGFP mono-
clonal antibody (asterisks indicate oli-
gomers higher than tetramer). B, BS3
cross-linked CHO-VR1 after PNGase F
treatment probed with anti-VR1 poly-
clonal antibody (asterisks indicate hexam-
ers and octamers). C, cross-linking exper-
iments on living cells. The Western blot
was probed with anti-eGFP monoclonal
antibody.

When partially purified DRG membranes were analyzed by tetramer and, to a much lower extent, some higher polymers
PFO-PAGE using optimized conditions, VR1 was present but with no bands indicative of the formation of hetero-oli-
mainly as tetramer and some monomer but without evident gomers between VR1 and protein subunits of a different size.
dimers, trimers, or higher molecular weight protein aggregates The VR1 agonist dependence, the inhibition by the VR1 com-
being present, although at higher PFO concentrations the oli- petitive antagonist capsazepine, and the inhibition in the ab-
gomers smaller than tetramer became detectable. VR1 in DRG sence of extracellular calcium all fit with the model that the
was non-glycosylated in contrast to VR1 in CHO cells, which covalent cross-linking reflects elevated intracellular calcium in
were highly glycosylated (⬃ 60%). We cannot distinguish response to VR1 activation. The probable mechanism respon-
whether the existence of oligomers smaller than the tetramers sible to this protein cross-linking is through transglutaminase,
represent artifacts of disaggregation by the PFO or are present since pretreatment of the cells with known transglutaminase
in the native membranes. Kir 2.1, an inwardly rectifying po- inhibitors could prevent VR1 oligomerization. Transglutami-
tassium channel known to exist as tetramers in membranes, nases are a family of structurally and functionally related
was shown to migrate as a single band with a molecular weight Ca2⫹-dependent enzymes that catalyze cross-linking reactions
corresponding to the tetramer on PFO-PAGE. On the other of proteins by transferring the ␥-carboxy group from protein-
hand, the GABA-A receptor complex, which is thought to be a bound glutamine to the ⑀-amino group of protein-bound lysine
heteropentameric oligomer formed from three different sub- residues or other primary amines (35, 36). The cross-linking of
units, was found to migrate as trimers and pentamers on PFO- proteins by transglutaminases has been shown to play a vari-
PAGE, the trimeric form being the predominant band (32). We ety of physiologically important functions in different systems.
conclude that VR1 is indeed a multimer, most possibly a ho- For example, transglutaminases are involved in cross-linking
motetramer. We observe modest differences in post-transla- of cornified envelope proteins during terminal differentiation of
tional modification between native VR1 and VR1 expressed in keratinocytes (36 –39) and in programmed cell death (40, 41).
heterologous systems, just as somewhat different behavior of In the current study, we have explored the endogenous cross-
endogenous and heterologously expressed VR1 has been seen linking of VR1 as a reporter for its state of association. Studies
in calcium uptake, binding, and other experiments (28 –31). are in progress to evaluate its possible contribution to VR1
These differences emphasize the need for caution in interpre- desensitization or DRG toxicity in response to vanilloid
tation of results using heterologous expression systems. treatment.
In the course of our studies, we found that VR1 becomes Our third approach to characterize VR1 subunit structure
covalently cross-linked spontaneously in the presence of VR1 was to use chemical cross-linking agents. This method has been
agonists. The pattern of cross-linking, which was analyzed widely used in the nearest neighbor analysis of membrane
mostly by SDS-PAGE, was similar to that found for VR1 by proteins. The bifunctional chemical cross-linkers used in the
PFO-PAGE in the absence of agonists, namely multimers up to present study produce covalent bonds between primary amines
 Subunit Structure of Capsaicin Receptor 28619
of proteins resulting in intra- or intermolecular cross-linking. 21, 531–543
8. Jordt, S.-E., Tominaga, M., and Julius, D. (2000) Proc. Natl. Acad. Sci. U. S. A.
The cross-linked oligomers subsequently can be separated on 97, 8134 – 8139
SDS-PAGE and their structures deduced from the sizes of the 9. Guo, O., Vulchanova, L., Wang, J., Li, X., and Elde, R. (1999) Eur. J. Neurosci.
11, 946 –958
complexes. This technique has helped demonstrate the hexam- 10. Jerman, J. C., Brough, S. J., Prinjha, R., Harries, M. H., Davis, J. B., and
eric nature of bacterophage T7 primase/helicase (42), the oli- Smart, D. (2000) Br. J. Pharmacol. 130, 916 –922
gomeric structure of human immunodeficiency virus-1 reverse 11. Harteneck, C., Plant, T. D., and Schultz, G. (2000) Trends Neurosci. 23,
159 –166
transcriptase (43), and the stoichiometry of subunits in the 12. Xu, X.-Z. S., Choudhury, A., Li, X., and Montell, C. (1998) J. Cell Biol. 142,
native atrial G-protein-gated K⫹ channel, IKACh (44). Using 545–555
13. Lockwick, T. P., Liu, X., Singh, B. B., Jadlowiec, J., Weiland, S., and
BS3 or sulfo-BSOCOES, a non-cleavable or a base-cleavable
Ambudkar, I. S. (2000) J. Biol. Chem. 275, 11934 –11942
water soluble cross-linking agent, we have found that 14. Xu, X.-Z. S., Li, H.-S., Guggino, W. B., and Montell, C. (1997) Cell 89,
VR1eGFP expressed in Cos7 cells can be cross-linked to form 1155–1164
15. Lintschinger, B., Balzer-Geldstezer, M., Baskaran, T., Graier, W. F., Romanin,
dimers, trimers, and tetramers, as well as higher molecular C., Zhu, M. X., and Groschner, K. (2000) J. Biol. Chem. 275, 27799 –27805
weight polymers. We did not observe other associated proteins. 16. Philipp, S., Trost, C., Warnat, J., Rautmann, J., Himmerkus, N., Schrots, G.,
The higher molecular weight polymers (more than tetramer) Kretz, O., Nastainczyk, W., Cavalie, A., Hoth, M., and Florckerzi, V. (2000)
J. Biol. Chem. 275, 23965–23972
were not observed when the cross-linking experiments were 17. Catterall, W. A. (1988) Science 242, 50 – 61
performed on living cells, conditions where the water-soluble 18. Szallasi, A., and Blumberg, P. M. (1991) Life Sci. 48, 1863–1869
19. Oxford, G. S., Kuzhikandathil, E. V., Szabo, T., Wang, H., and Blumberg, P. M.
agents can form covalent bonds only between the accessible (2000) Abstracts of the 2000 Society for Neuroscience Annual Meeting,
primary amines sitting on the outer surface of cell membranes. November 4 –9, New Orleans, LA, Abstract 632.11, p. 1689, Society for
In CHO-VR1 cells the same pattern of oligomerization was Neuroscience, Washington, D. C.
20. Szallasi, A., Conte, B., Goso, C., Blumberg, P. M., and Manzini, S. (1993) Life
found, but occasionally some bands with intermediate molecu- Sci. 52, 221–226
lar weight were also seen, albeit at a much lower level. This 21. Oh, U., Hwang, S. W., and Kim, D. (1996) J. Neurosci. 16, 1659 –1667
22. Olah, Z., Szabo, T., Karai, L., Hough, C., Fields, R. D., Caudle, R. M.,
suggests the possibility that proteins can be associated with Blumberg, P. M., and Iadarola, M. J. (2001) J. Biol. Chem. 276,
VR1. The Drosophila Trp and TrpL proteins are known to be 11021–11030
associated with proteins such as phospholipase C or protein 23. MacKinnon, R. (1991) Nature 350, 232–235
24. Zhu, X., and Birnbaumer, L. (1998) News Physiol. Sci. 13, 211–217
kinase C via the scaffolding protein INAD (12). Likewise, 25. Suzuki, M., Sato, J., Kutsuwada, K., Ooki, G., and Imai, M. (1998) J. Biol.
hTRP1 was shown to be associated with G-proteins and caveo- Chem. 274, 6330 – 6335
lin (13). On the other hand, some other members of the super- 26. Caterina, M. J., Rosen, T. A., Tominaga, M., Brake, A. J., and Julius, D. (1999)
Nature 398, 436 – 441
family are known to form homo- or heterotetramers without 27. Schumacher, M. A., Moff, I., Sudanagunta, S. P., and Levine, J. D. (2000)
being in tight complexes with other proteins (17, 44). An unre- J. Biol. Chem. 275, 2756 –2762
28. Acs, G., Biro, T., Acs, P., Modarres, S., and Blumberg, P. M. (1997) J. Neurosci.
solved issue is the origin of the bands with mobilities corre- 17, 5622–5628
sponding to a hexamer or octamer. Based on the expectation of 29. Szallasi, A., Blumberg, P. M., Annicelli, L. L., Krause, J. E., and Cortright,
a tetrameric structure, we surmise that we have cross-linked D. N. (1999) Mol. Pharmacol. 56, 581–587
30. Caterina, M J, Leffler, A., Malmberg, A. B., Martin, W. J., Trafton, J.,
subunits of adjacent tetrameric channels. Drosophila Trp and Petersen-Zeitz, K. R., Koltzenburg, M., Basbaum, A. I., and Julius, D.
TrpL proteins are known to be clustered in multichannel com- (2000) Science 288, 306 –313
31. Davis, J. B., Gray, J., Gunthorpe, M. J., Hatcher, J. P., Davey, P. T., Overend,
plexes (12). In any case, the role of such clustering in the P., Harries, M. H., Latcham, J., Clapham, C., Atkinson, K., Hughes, S. A.,
regulation of VR1 and its pharmacology remains to be assessed. Rance, K., Grau, E., Harper, A. J., Pugh, P. L., Rogers, D. C., Bingham, S.,
In summary, the experiments presented here using multiple Randall, A., and Sheardown, S. A. (2000) Nature 405, 183–187
32. Ramjeesingh, M., Huan, L. J., Garami, E., and Bear, C. E. (1999) Biochem. J.
independent methods demonstrate that the VR1 channel is a 342, 119 –123
multimer and that the VR1 channel is most likely made up of 33. Vince, J. W., Sarabia, V. E., and Reithmeier, R. A. (1997) Biochim. Biophys.
Acta 1326, 295–306
four identical subunits. Variation in the individual subunits of 34. Lagree, V., Froger, A., Deschamps, S., Pellerin, I., Delamarche, C., Bonnec, G.,
the tetramer, whether through splice variants or through post- Gouranton, J., Thomas, D., and Hebert, J.-F. (1998) J. Biol. Chem. 273,
translational modification, could provide a mechanism to gen- 33949 –33953
35. Folk, J. E. (1980) Annu. Rev. Biochem. 49, 517–531
erate complexity. 36. Greenberg, C. S., Birckbichler, P. J., and Rice, R. H. (1992) FASEB J. 5,
3071–3077
REFERENCES 37. Aeschlimann, D., Wetterwald, A., Felisch, H., and Paulsson, M. (1993) J. Cell
1. Szolcsanyi, J., and Jancso-Gabor, A. (1975) Drug Res. 25, 1877–1881 Biol. 120, 1461–1470
2. Bevan, S., and Szolcsanyi, J. (1990) Trends Pharmacol. Sci. 11, 330 –333 38. Rice, R. H., Mehrpouyan, M., O’Callahan, W., Parenteau, N. L., and Rubin,
3. Szallasi, A., Blumberg, P. M. (1990) Brain Res. 524, 106 –111 A. L. (1992) Epithelial Cell Biol. 1, 128 –137
4. Szolcsanyi, J., Anton, F., Reeh, P. W., and Handwerker, H. O. (1988) Brain 39. Dlugosz, A. A., and Yuspa, S. H. (1994) J. Invest. Dermatol. 102, 409 – 414
Res. 446, 262–268 40. Fesus, L. (1993) FEBS Lett. 328, 1–5
5. Szallasi, A., and Blumberg, P. M. (1996) Pain 68, 195–208 41. Fesus, L., and Thomazy, V. (1988) Adv. Exp. Med. Biol. 231, 119 –134
6. Caterina, M. J., Schumacher, M. A., Tominaga, M., Rosen, T. A., Levine, J. D., 42. Patel, S. S., and Hingorani, M. M. (1993) J. Biol. Chem. 268, 10668 –10675
and Julius, D. (1997) Nature 389, 816 – 824 43. Debyser, Z., and De Clercq, E. (1996) Protein Sci. 5, 278 –286
7. Tominaga, M., Caterina, M. J., Malmberg, A. B., Tosen, T. A., Gilvert, H., 44. Corey, S., Krapivinsky, G., Krapivinsky, L., Clapham, D. E. (1998) J. Biol.
Skinner, K., Raumann, B. E., Basbaum, A. I., and Julius, D. (1998) Neuron Chem. 273, 5271–5278