TRPV1b overexpression negatively regulates ... - Wiley Online Library

Journal of Neurochemistry, 2006, 99, 1088–1102

doi:10.1111/j.1471-4159.2006.04145.x

TRPV1b overexpression negatively regulates TRPV1 responsiveness to capsaicin, heat and low pH in HEK293 cells Melissa H. Vos, Torben R. Neelands,1 Heath A. McDonald,1 Won Choi, Paul E. Kroeger, Pamela S. Puttfarcken, Connie R. Faltynek, Robert B. Moreland and Ping Han Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois, USA

Abstract Transient receptor potential channel type V (TRPV) 1 is a nonselective cation channel that can be activated by capsaicin, endogenous vanilloids, heat and protons. The human TRPV1 splice variant, TRPV1b, lacking exon 7, was cloned from human dorsal root ganglia (DRG) RNA. The expression profile and relative abundance of TRPV1b and TRPV1 in 35 different human tissues were determined by quantitative RT-PCR using isoform-specific probes. TRPV1b was most abundant in fetal brain, adult cerebellum and DRG. Functional studies using electrophysiological techniques showed that recombinant TRPV1b was not activated by capsaicin (1 lM), protons (pH 5.0) or heat (50C). However, recombinant TRPV1b did form multimeric complexes and was detected on the plasma

membrane of cells, demonstrating that the lack of channel function was not due to defects in complex formation or cell surface expression. These results demonstrate that exon 7, which encodes the third ankyrin domain and 44 amino acids thereafter, is required for normal channel function of human TRPV1. Moreover, when co-expressed with TRPV1, TRPV1b formed complexes with TRPV1, and inhibited TRPV1 channel function in response to capsaicin, acidic pH, heat and endogenous vanilloids, dose-dependently. Taken together, these data support the hypothesis that TRPV1b is a naturally existing inhibitory modulator of TRPV1. Keywords: dorsal root ganglion, splice variant, transient receptor potential channel type V, vanilloid receptor 1. J. Neurochem. (2006) 99, 1088–1102.

The vanilloid receptor (TRPV1/VR1) is a member of the transient receptor potential (TRP) cation channel superfamily consisting of seven subfamilies (TRPC, TRPM, TRPV, TRPA, TRPML, TRPP and TRPN) and 28 mammalian family members (Minke and Parnas 2006). TRPV1 is a nonselective cation channel that can be activated by ligands (capsaicin and endovanilloids), noxious heat and protons (Caterina et al. 1997). It is highly expressed on small diameter nociceptive primary afferents where it is proposed to integrate responses to multiple noxious stimuli, playing a potential role in pain sensation (Tominaga et al. 1998; Caterina et al. 2000; Davis et al. 2000; Ryu et al. 2003). TRPV1 has structural homology with other TRP family members (Minke and Parnas 2006), which all have six transmembrane domains, a pore region between the fifth and sixth transmembrane domain, a long N-terminal domain containing a variable number of ankyrin repeats, and a cytoplasmic C-terminus containing the TRP-like domain required for oligomerization (Kuzhikandathil et al. 2001; Jordt et al. 2003; Ferrer-Montiel et al. 2004; Garcia-Sanz et al. 2004). Tetrameric complex formation is required for

TRPV1 to be functional in response to vanilloids and protons (Kuzhikandathil et al. 2001; Garcia-Sanz et al. 2004). Alternative splicing has been reported in the TRP gene family, expanding the number of functionally distinct TRP proteins and potentially providing tissue-specific regulation. Several TRPV1 splice variants have been reported in mouse, rat and human (Suzuki et al. 1999; Schumacher et al. 2000;

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Received April 28, 2006; revised manuscript received June 9, 2006; accepted June 13, 2006. Address correspondence and reprint requests to Ping Han, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois 60064, USA. E-mail: [email protected] 1 Torben R. Neelands and Heath A. McDonald contributed equally to this work. Abbreviations used: BS3, bis[2(sulfosuccinimidyl)] suberate; CIP, calf intestinal alkaline phosphatase; DPBS, Dulbecco’s Phosphate Buffered Saline; DOC, deoxycholic acid; DRG, dorsal root ganglion; FLIPR, fluorometric imaging plate reader; HA, hemagglutinin; MES, 2-[Nmorpholino] ethanesulfonic acid; NADA, N-arachidonoyl-dopamine; NP-40, Nonidet P-40; TRPV, transient receptor potential channel type V; VR1, vanilloid receptor 1.

2006 The Authors Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 1088–1102

TRPV1b, the native negative modulator of TRPV1 1089

Xue et al. 2001; Lyall et al. 2004; Wang et al. 2004; Lu et al. 2005; Tian et al. 2006). While the biological roles of these variants are unclear, the reported deletions lead to nonfunctional channels (mTRPV1b, VR5¢sv and TRPV1VAR) or channels with distinct properties (Lyall et al. 2004; Naeini et al. 2006) when examined in recombinant or native expression systems. TRPV1b, a splice variant of human TRPV1 cloned from brain cDNA library, showed no channel activity in response to capsaicin and protons, but was activated by thermal stimulus at 47C when characterized in Xenopus oocytes (Lu et al. 2005). Although TRPV1b mRNA was found to co-express with TRPV1 in rat trigeminal ganglia neurons, its effect on TRPV1 function remained unknown. The TRP channel tetrameric structure allows for formation of both homomers and heteromers (Hellwig et al. 2005; Rutter et al. 2005). Heteromeric structures have been shown to include closely related subfamily members (Strubing et al. 2001; Hofmann et al. 2002; Hellwig et al. 2005; Rutter et al. 2005) as well as splice variants of the same family member (Wang et al. 2004; Tian et al. 2005). The multiple family members, together with variable stoichiometric composition in the channel complex, could potentially generate an array of finely tuned sensors to noxious stimuli. In this study, we cloned human TRPV1 splice variant TRPV1b from dorsal root ganglion (DRG) total RNA, determined its tissue distribution, and characterized TRPV1b channel function and biological properties. Our studies demonstrate that TRPV1b is a non-functional splice variant of TRPV1. The recombinant human TRPV1b associates with TRPV1 and negatively regulates TRPV1 channel activity.

Materials and methods Antibodies and reagents Three TRPV1 antibodies were used in this study. TRPV1 N-terminal antibody ABRK-1 was described previously (McGaraughty et al. 2003). TRPV1 ABRK-4 antibody was raised in rabbit against the peptide CYTGSLKPEDAEVFK(NH2) corresponding to amino acids 817–830 of rat TRPV1 cysteine was added for chemical cross-linking (Anaspec, San Jose, CA, USA). TRPV1 P-19 antibody against the N-terminal region of rat TRPV1 from goat was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). a-Tubulin monoclonal antibody (Sigma, St. Louis, MO, USA), hemagglutinin (HA) monoclonal antibody 3F10 (Roche, Indianapolis, IN, USA) and glucose-regulated protein (GRP78) monoclonal antibody (BD Transduction Laboratories, San Diego, CA, USA) were obtained from the indicated sources. Cross-linking reagent bis[2(sulfosuccinimidyl)] suberate (BS3), cell surface biotinylatin reagent sulfoNHS-LC-Biotin and streptavidin beads were purchased from Pierce (Rockford, IL, USA). Metabolic labeling mix, EasyTag EXPRESS [35S] Protein Labeling Mix, was from PerkinElmer Life Science, Inc. (Boston, MA, USA). Transfection reagent lipofectamine 2000

was from Invitrogen (Carlsbad, CA, USA). The enhanced chemiluminescence (ECL) plus western blot detection system was from Amersham Bioscience (Little Chalfont, UK). Cloning of human TRPV1b To identify possible splice variants of human TRPV1 at exon 7, RTPCR (Invitrogen) was performed on human DRG total RNA (BD Clontech, Palo Alto, CA, USA) using primers spanning the putative exon 7 splice sites and 5¢ untranslated region (UTR) primers, or the 3¢ end primer. Primers used are as follows: 5¢UTR1: 5¢-CAGAGTCACGCTGGCAACCACGAG-3¢; and 5¢UTR4: 5¢- AAGGCAACGCCGCTGACAAAGAAC-3¢; TRPV1b JKR: 5¢-AAGAGC ATGTCGTGGCGATTCCCG-3¢; TRPV1b JKF: 5¢-GAAGATCGGGAATCGCCACGACAT-3¢; TRPV1 R2520: 5¢-TCACTTCTCCCCGGAAGCGGCAGG-3¢; TRPV1b JKR¢: 5¢- CAAGAGCATGTCGTGGCGATTAGA-3¢; TRPV1b JKF¢:5¢- GCTGGGACCGGGAAGATCGGGTGC-3¢. Each of the 5¢UTRs combined with TRPV1b JKR generated PCR products that translated from the first methionine of TRPV1. TRPV1b JKF and R2520 generated one product that was identical to TRPV1 but lacked exon 7. The full-length splice variant was obtained by combining the two overlapping PCR fragments as templates and amplifying with the 5¢ UTR4 and 3¢ end TRPV1 R2520 primers. TRPV1b JKF¢ and TRPV1b JKR¢ are negative control primers that span the splice sites, but the last three nucleotides hybridized 14 and 26 nucleotides further down the junction sites. Real-time quantitative PCR (qRT-PCR) The copy numbers of TRPV1 and TRPV1b mRNAs were assessed by qRT-PCR (TaqMan) technology (BD Bioscience, San Diego, CA, USA and Ambion, Inc., Austin, TX, USA). All TaqMan probes were 5¢-labeled with the reporter, fluorescein, and 3¢-labeled with the quencher, tetramethylrhodamine. The sequences for forward and reverse primers of TRPV1 were 5¢-GTGCACTCCTCGCTGTACGA-3¢ and 5¢-CACCTCCAGCACCGAGTTCT-3¢, respectively. The TaqMan probe used was 5¢-FAM-TGTCCTGCATCGACACCTGCGAG-TAMRA-3¢. The sequences for forward and reverse primers of TRPV1b were 5¢-GAATGACGCCGCTGGCT-3¢ and 5¢-CAGCGGCTCCACCAAGAG-3¢, respectively. The TaqMan probe used was 5¢-FAM-GGGAAGATCGGGAATCGCCACGA-TAMRA-3¢. The plasmids containing each variant insert were quantified and serially diluted to generate a series of standard curves. The standard curve for each variant was then used to determine the copy number in the tissues tested. To ensure specific amplification for each variant, each qRT-PCR set was cross-validated on plasmid controls containing the other spliced sequences. For qRT-PCR analysis, 50 ng total RNAs, commercially available from Ambion, Inc. and BD Bioscience, were DNase1-treated as described by the manufacturer (Invitrogen). qRT-PCR reaction was prepared in duplicate in a final volume of 25 lL containing: 1· platinum quantitative PCR thermoscript buffer (Invitrogen), 5.5 mM MgCl2, 400 nM gene-specific (GS) primers/100 nM GS TaqMan probe and 0.5 U thermoscript polymerase mix (Invitrogen). Each sample was normalized using 100 nM 28S rRNA primers/150 nM 28S rRNA TaqMan probe. Cycling was 30 min at 50C, followed by 5 s at 95C, and then 40 cycles of 15 s at 95C, 1 min at 60C. Data were collected during each extension phase of the PCR reaction and analyzed with the ABI-7700 SDS software package (Applied Biosystems, Foster City, CA, USA).


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Rat DRG isolation DRGs were dissected from male Sprague–Dawley rats (200–350 g) (Charles River, Wilmington, MA, USA). Animals were housed five per cage and given tap water and certified food ad libitum. The room was kept at a constant temperature (20–21C) with a 12 h alternating light/dark cycle. All protocols were from the Institutional Animal Care and Use Committee (IACUC, Abbott Laboratories), and were in accordance with the ethical principles for pain-related animal research of the American Pain Society. After rats had been killed with CO2, the spinal cords were immediately removed and cut open. Twenty pairs of DRG were isolated from each rat and snap frozen on dry ice. Tissues were stored at )80C until protein extraction. Transfection and immunodetection of TRPV1 and TRPV1b TRPV1 and TRPV1b expression plasmids cloned into pCIneo were transfected into HEK293 cells using lipofectamine 2000. At 30 h post-transfection, cells were harvested and then lysed in phosphatebuffered saline (PBS) buffer, containing 0.5% deoxycholic acid (DOC) and 0.5% Nonidet P-40 (NP-40), on ice for 10 min. Then, 200–400 lg cell lysate were diluted to 1 mL with PBS for each immunoprecipitation assay. ABRK-1 or ABRK-4 antibody (4 lL) was added to the diluted cell lysate, followed by continuous rotation for more than 3 h at 4C. A 30 lL volume of protein A agarose beads was subsequently added to capture the immune complex. Following centrifugation, the beads were washed with buffer containing 37.5 mM NaCl, 2.5 mM Tris pH 8.3, 1.25 mM EDTA, 0.025% Triton X-100 and 1· complete protease inhibitors cocktail (Roche) for 4 · 10 min at 4C. Immune complexes were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on 4–12% Nu-PAGE gels (Invitrogen) and visualized by western blotting using the TRPV1 P-19 antibody. Co-immunoprecipation of TRPV1b with HA-tagged TRPV1 was performed under the same conditions, except that the HA antibody 3F10 and protein G agarose beads were used for the immunoprecipitation. Calcium influx assay using fluorometric imaging plate reader Capsaicin-evoked calcium flux was measured by a fluorometric imaging plate reader (FLIPR; Molecular Devices, Sunnyvale, CA, USA) as described previously (Witte et al. 2002). Briefly, cells were plated into 96-well black-wall Biocoat tissue culture plates (Becton Dickinson Labware, Bedford MA, USA) at the density of 50 000 cells/well 24 h post-transfection. At 48 h post-transfection, the cells were pre-loaded with 1.14 lM fluo-4/AM Invitrogen for 2 h, then washed with 0.25 mL DPBS (with calcium and magnesium; Invitrogen) twice to remove extracellular fluo-4/AM. Fluorescent readings were made over a 4 min period at 1–5 s intervals following addition of agonist to the cells. The peak increase in relative fluorescent units over the baseline fluorescence was calculated and analyzed with GRAPHPAD PRISM (GraphPad Software, San Diego, CA, USA). Electrophysiology Patch-clamp recordings were performed 48 h post-transfection. Biocoated coverslips (Becton Dickenson Labware) plated with transfected HEK293 cells were placed in a perfusion chamber containing the extracellular recording solution (pH 7.4, 325 mOsm) consisting of (mM): 155 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 12 glucose. When the external recording solutions were lowered to

pH 5.0, MES was used in place of HEPES in the external solution. Following establishment of whole-cell recording conditions, the cells were voltage-clamped to )60 mV and bath perfusion (approximately 2 mL/min) was initiated. Patch-pipettes composed of borosilicate glass (1B150F-3; World Precision Instruments, Inc., Sarasota, FL, USA) were pulled and fire-polished using a DMZUniversal micropipette puller (Zeitz Instrumente GmbH, Munich, Germany). Pipettes (2–6 MW) were filled with an internal solution (pH 7.3, 295 mOsm) consisting of (mM): 122.5 K-aspartate, 20 KCl, 1 MgCl2, 10 EGTA, 5 HEPES, 2 ATPÆMg. Standard whole-cell recording techniques were used in an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). Application of control bath solution through a multibarrel application device with a common 360 lm polyimide tip (Cell Microcontrols, Norfolk, VA, USA), positioned approximately 100 lm from the cell, was continued throughout the recording except during drug application. Each drug reservoir was connected to solenoid Teflon valves that were controlled by a ValveLink16 system (AutoMate Scientific, San Francisco, CA, USA). Drug application protocols were established using pCLAMP (Axon instruments) software that controlled rapid valve switching through the ValveLink system. Drugs were applied by gravity feed for 5 s and each drug application sequence was followed by a washout period of 90 s. In studies involving heated solutions, the control bath solution was not applied during washout periods but rather, only bath perfusion was maintained. In these experiments, heated solutions were applied by manual control of the solenoid valves (1–5 s), using a temperature-calibrated MPRE8 multitube with the temperature at the tip of the multitube controlled by a TC2PKG (Cell MicroControls). For studies investigating voltage dependence, cells were voltage-clamped at )80 mV. Following a 2 s application of either control solution, pH 5.0 solution, 1 lM capsaicin solution, or 50C solution, the membrane potential was ramped to + 80 mV over 1600 ms in the continued presence of activators. Data acquisition and analysis were performed using pCLAMP 9.0, and subsequent graphs were plotted using GRAPHPAD PRISM. Dephosphorylation and deglycosylation reactions Calf intestinal alkaline phosphatase (CIP; New England Biolabs, Inc., MA, USA) was used for dephosphorylation reaction. A 250 lg aliquot of rat DRG total cell lysate, diluted to 200 lL with buffer containing 0.5· PBS, 50 mM Tris 9.4, 1 mM dithiothreitol (DTT) and 1 mM ZnCl2, was incubated with 1 lL (10 U) CIP enzyme at 30C for 30 min. After the reaction, 10% (25 lg) of total protein lysates was fractionated on SDS–PAGE. The effect of dephosphorylation was checked by Pro-Q Diamond phosphoprotein gel staining solution (Invitrogen). The rest of the lysates were diluted with 1· PBS to 1 mL for immunoprecipitation and western blot analysis of TRPV1 protein. Peptide N-glycosidase F (PNGase F) (New England Biolabs) was used for TRPV1 deglycosylation. Immunoprecipitated TRPV1 was denatured by incubation in 50 lL 1· glycoprotein denature buffer (New England BioLabs) at 100C for 10 min. Then, 0.5 M sodium phosphate (pH 7.5) and 10% NP-40 were added to neutralize the denatured reagents. The deglycosylation reaction was carried out at 37C for 1 h, incubating with PNGase F. The reaction was terminated with SDS sample buffer and heating at 100C for 3 min. TRPV1 deglycosylation was observed by western blot analysis using P-19 antibody.



Pro-Q Diamond phosphoprotein gel staining Pro-Q Diamond phosphoprotein staining solution was obtained from Invitrogen. SDS–PAGE staining was performed following the manufacturer’s protocol. Briefly, after fractionation of CIP-treated lysates on 4–12% Bis-Tris NuPAGE gel, the gel was fixed with 50% methanol and 10% acetic acid at room temperature (22–24C) overnight. Following intensive washing with an ample amount of water for 3 · 10 min, the gel was incubated with Pro-Q Diamond phosphoprotein staining solution for 90 min in the dark. The gel was subsequently de-stained with 100 mL buffer containing 50 mM sodium acetate, pH 4.0, and 20% acetonitrile for 3 · 30 min. Finally, the gel was rinsed in ultrapure water and observed using a FLA-5000 laser scanner with filters for excitation at 532 nm and emission at 580 nm (longpass filter).

was added to the supernatant fluid and incubated for 3 h at 4C with rotation. Then, 40 lL protein A agarose beads were added for an additional 2 h. The immune complex beads were washed five times (10 min each) at 4C with buffer containing 37.5 mM NaCl, 2.5 mM Tris 8.0, 1.25 mM EDTA. 0.025% Triton X-100, 1.25 mM methionine, 1.25 mM cysteine, 0.25 mg/mL BSA and 1· complete protease inhibitor cocktail. After the last wash, the proteins were eluted from the protein A agarose beads by boiling in SDS sample buffer for 3 min. The immune precipitates were fractionated by 4–12% Bis-Tris SDS–PAGE and were then dried and exposed onto X-ray film (Kodak, NY, USA).

Cell-surface labeling Cell-surface protein biotinylation was performed as previously described (Yan et al. 2001). Briefly, TRPV1- or TRPV1b-transfected HEK293 cells were placed on ice and rinsed with ice-cold DPBS twice before sulfo-NHS-LC-Biotin reagent (0.5 mg/mL in DPBS) was added. The biotinylation reaction was performed twice on ice for 20 min and was stopped by replacing the medium with 50 mM NH4Cl and incubating for an additional 10 min. Cells were washed with PBS and collected into tubes for protein extraction. Strepavidin beads were used to capture the cell-surface biotin-labeled proteins for further analysis.

Determination of TRPV1 and TRPV1b mRNA expression levels in human tissues by qRT-PCR The cDNA of TRPV1b, a splice variant of full-length TRPV1 lacking exon 7, was amplified by RT-PCR using total human DRG RNA. Primer TRPV1b JKR, together with either 5¢UTR1 or 5¢UTR4, generated one product starting from the initiation methionine with deletion in exon 7. Similarly, primers TRPV1b JKF and TRPV1 R2520 only generated one product with deletion in exon 7. In order to demonstrate that exon 7 deletion was not introduced as an artifact by RT-PCR, TRPV1b JKR¢ and JKF¢ primers, which span the splice sites but the last three nucleotides hybridized 14 and 26 nucleotides further down the junction sites, were used in RT-PCR reactions. None of these primer pairs was able to generate deletions (data not shown), suggesting that the exon 7 deletion was naturally existing. The human TRPV1b cDNA sequence obtained was identical to the one reported by Lu et al. (2005) except for two previously described TRPV1 polymorphisms (TRPV1b amino acid positions), I315M and T409I (Cortright et al. 2001). This sequence was submitted to GenBank and was assigned the number AY986821. The expression profile and abundance of human TRPV1 and TRPV1b were determined in 35 different human tissues using qRT-PCR with isoform-specific TaqMan probes that have been cross-tested on TRPV1 and TRPV1b plasmids (Fig. 1a). The specificities of the primers in qRTPCR were further examined using conventional PCR reactions. Forward and reverse primers of TRPV1 amplified a single band of 65 bp product, and TRPV1b primer sets that are common to both TRPV1 and TRPV1b amplified both 256 and 76 bp products using the reaction protocol for qRTPCR, further validating the specificities of the primers and also demonstrating the existence of TRPV1b splice variant in human DRG (Fig. 1b). TRPV1 mRNA was observed in a broad spectrum of tissues, with the highest abundance in neuronal tissues such as DRG and fetal brain (Fig. 1c). Approximately 39 000 copies of TRPV1 mRNA were expressed per 50 nanograms of total human DRG RNA (Fig. 1c); this corresponded to seven to eight copies of

Chemical cross-linking TRPV1 and TRPV1b proteins recombinantly expressed in HEK293 cells were cross-linked using methods described by Kedei et al. (2001). Briefly, 300 lg total cell lysate were incubated with freshly prepared cross-linking reagent, BS3, at room temperature for 30 min, with final concentrations ranging from 30 to 1000 lM. Then, 50 mM Tris pH 7.5 were added for another 15 min to quench the cross-linking reaction. The samples were immunoprecipitated using the TRPV1 ABRK-4 antibody, separated on 3–8% Nu-PAGE Tris-acetate gels (Invitrogen) and analyzed by western blot analysis using the TRPV1 P-19 antibody. Pulse-chase analysis HEK293 cells transfected with human TRPV1 and TRPV1b plasmids were metabolically labeled with EasyTag EXPRESS [35S] Protein Labeling Mix (150 lCi/mL) at 37C for 30 min. At the end of labeling, the radioactive medium was removed and cells were rinsed twice with normal growth medium to wash away the residual unincorporated radioactivity. Cells were either collected immediately for lysis, or were incubated in complete growth medium for varying lengths of time prior to lysis. Levels of radiolabeled TRPV1 protein at each time point were determined by immunoprecipitation followed by autoradiography. To immunoprecipitate the radiolabeled TRPV1 protein, cells were extracted with PBS buffer, containing 0.5% DOC, 0.5% NP-40, 5 mM methionine, 5 mM cysteine, 1 mg/mL bovine serum albumin (BSA) and 1· complete protease inhibitor cocktail, on ice for 10 min. The lysate was centrifuged at 10 000 g for 10 min at 4C. The supernatant fluid was diluted to 1 mL with lysis buffer, and was pre-cleared by incubation with 40 lL protein A agarose beads for 1 h followed by centrifugation. TRPV1 ABRK-4 antibody (5 lL)

Results



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Fig. 1 Expression of TRPV1 and TRPV1b mRNA in different human tissues. (a) Diagram showing the exon organization of TRPV1 and TRPV1b mRNA transcripts from exon 6 to exon 8. The location of forward and reverse primers used for amplification, as well as TRPV1- vs. TRPV1b-specific TaqMan probes used in quantitative RT-PCR, are illustrated by the gray boxes. (b) Conventional RT-PCR reactions of human DRG total RNA. TRPV1 (lane 2) and TRPV1b (lane 3) forward and reverse amplification primers were employed in RT-PCR reactions using reverse-transcribed human DRG cDNA as

template. A 65 bp product specific for TRPV1 (lane 2), and 76 bp and 256 bp products specific for TRPV1b and TRPV1, respectively (lane 3), were obtained. RT-PCR products were fractionated on 2% agarose gel and a 100 bp DNA ladder was loaded in lane 1. (c, d) Determination of TRPV1 and TRPV1b expression levels in different human tissues by qRT-PCR. The abundance of TRPV1 and TRPV1b transcripts is presented as number of mRNA copies in 50 nanograms of total RNA.



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TRPV1 mRNA per cell. TRPV1b mRNA levels were averaged at 2.7 ± 0.25% of TRPV1 in most of the tissues examined. The TRPV1b splice variant expression levels were highest in fetal brain, adult DRG and adult cerebellum, corresponding to 6–7% of TRPV1. Levels were low in thyroid, heart and thalamus, only 0.35 ± 0.06% of TRPV1, and very little or no expression was detected in salivary gland, thyroid gland and pancreas (Fig. 1d). TRPV1 and TRPV1b protein expression in transfected HEK293 cells and in rat DRG To study TRPV1b function, either by itself or in combination with TRPV1, cDNAs were subcloned into the mammalian expression vector, pCIneo, and were transiently transfected into HEK293 cells. Expression of both TRPV1 and TRPV1b was detected by immunoprecipitation using the TRPV1 N-terminal antibody, ABRK-1, followed by western blot analysis using the TRPV1 N-terminal antibody P-19

Fig. 2 Characterization of TRPV1 and TRPV1b proteins expressed in transfected HEK293 cells and in DRG. (a) TRPV1 and TRPV1b in transiently-transfected HEK293 cells (lanes 2 and 3) and in rat DRG protein lysates (lanes 4–9) were immunoprecipitated with TRPV1 N-terminal antibody ABRK-1 (lanes 4–7), TRPV1 C-terminal antibody ABRK-4 (lane 8) and antigen-depleted ABRK-1 anti-serum (lane 9), and western blotted with TRPV1 P-19 antibody. No signal was detected in mock-transfected HEK293 cells (lane 1) or antigendepleted ABRK-1 immunoprecipitated sample (lane 9). The expression of a-tubulin in 20 lg total protein lysate showed the integrity of the protein used in this study. The identities of different immune reactive bands are indicated on the right-hand side. The uncharacterized middle band is labeled ‘?’. (b) ABRK-1 immunoprecipitates from HEK293 cells (lanes 1 and 2), HEK293 cells transiently transfected with TRPV1 (lanes 3 and 4) or DRG lysates (lanes 5–7) were incubated in the absence or presence of PNGase F at 37C for 1 h. Lanes 5 and 7 were the same materials that were either incubated at 37C (lane 5) or loaded directly onto the gel (lane 7). Proteins were fractionated on 4–12% Bis-Tris NuPAGE gel and western blotted with TRPV1 P-19 antibody. (c) Of 250 lg total rat DRG lysates incubated in the absence or presence of CIP at 30C for 30 min, 25 lg were loaded onto 4–12% Bis-Tris gel and stained with Pro-Q Diamond for phosphoprotein (lanes 1–3); the rest of the lysates were immunoprecipitated with 5 lL ABRK-1 antibody, followed by western blot analysis using P-19 antibody (lanes 4–6).

(Fig. 2a). TRPV1 immunoreactive bands were detected with apparent molecular weights of 95–97 kDa, and a highly glycosylated form of TRPV1 at 120 kDa, consistent with the previous report by Jahnel et al. (2001). Western blot analysis of TRPV1b showed only one band at 88–90 kDa (Fig. 2a). Rat DRG tissue lysates yielded two major bands with apparent molecular weights of 93 and 97 kDa, but no highly glycosylated 120 kDa band. In addition, a band co-migrating with the recombinantly-expressed TRPV1b was detected in the DRG tissue extracts prepared from four different animals (Fig. 2a, lanes 4–7). This lower molecular weight band was not a non-specific protein, because no signal was detected in HEK293 cells without TRPV1 or when immunoprecipitating with antigen depleted ABRK-1 antiserum (Fig. 2a, lane 1 and lane 9), neither was it a degradation product of TRPV1 because the same band pattern was also detected following immunoprecipitation with the C-terminal antibody, ABRK-4 (Fig. 2a, lane 8). Moreover, examination of the abundant proteins a-tubulin (Fig. 2a) and GRP78 (data not shown) confirmed the integrity of the protein lysates. As the P-19 antibody recognized no other bands besides TRPV1 and TRPV1b in transfected HEK293 cells after immunoprecipitation with ABRK-1 or ABRK-4 antibodies, and rat TRPV1b homologue VR1L2 has been reported in the rat GenBank database (Accession number AB041029), we concluded that this band was endogenous VR1L2 (TRPV1b rat homologue) expressed in rat DRG. To characterize further whether the two major bands in DRG lysate represented the glycosylated versus unglycosyl-



ated full-length TRPV1, N-linked glycosidase PNGase F was used to treat the immunoprecipitates (Fig. 2b). In the positive control, full-length TRPV1 recombinantly expressed in HEK293 cells was deglycosylated upon enzyme treatment, because the 120 kDa highly glycosylated band and the 97 kDa partially glycosylated band both disappeared, and the bottom unglycosylated 95 kDa band became more intense. However, in DRG, the two major bands and the TRPV1b band all shifted down to lower molecular weight forms, but none of these bands merged together upon PNGase F treatment. As TRPV1 was reported to be N-linked glycosylated on Asn604 (Jahnel et al. 2001; Rosenbaum et al. 2002), we concluded that the two major bands in DRG were not different glycosylation forms of TRPV1. To exclude the possibility that the multiple bands were due to phosphorylation of TRPV1, CIP was used to treat rat DRG lysate. After incubating 250 lg lysate with 1 lL CIP enzyme at 30C for 30 min, 25 lg of the lysates were directly loaded onto SDS– PAGE for phosphoprotein detection using Pro-Q Diamond staining; the rest of the lysates were immunoprecipitated with ABRK-1 followed by western blot analysis with P-19 antibody (Fig. 2c). CIP treatment significantly reduced the overall phosphoprotein signals. Under the same conditions, no alteration of mobility was detected for ABRK-1 immune reactive signals, suggesting that the presence of the multiple bands was not due to phosphorylation of TRPV1. Taken together, these data suggest that in rat DRG, in addition to full-length TRPV1, there exists a small amount of TRPV1b and another unknown form of TRPV1 (either splice variant or some unknown post-translational modification) with an abundance similar to full-length TRPV1. Characterization of TRPV1b channel functions by electrophysiological assays TRPV1 and TRPV1b channel functions were investigated in transiently-transfected HEK293 cells using electrophysiological techniques. HEK293 cells transfected with fulllength TRPV1 were activated by 1 lM capsaicin and low pH (5.0). In contrast, no significant activation by these stimuli was observed in the TRPV1b-transfected HEK293 cells (Figs 3a–d), although equivalent amounts of TRPV1b and TRPV1 proteins were expressed [TRPV1 capsaicin response: 3066 ± 1434 pA (n ¼ 8), pH 5.0 response: 5506 ± 1335 pA (n ¼ 7); TRPV1b capsaicin response: 26 ± 4.2 pA (n ¼ 10), pH 5.0 response: 991 ± 291 pA (n ¼ 10); vector-transfected cells capsaicin response: 36 ± 14.6 pA (n ¼ 4), pH 5.0 response: 286 ± 79 pA (n ¼ 4)]. The pH responses in TRPV1b-expressing cells were very weak compared with TRPV1-expressing cells. The differences between TRPV1b and vector-transfected cells were not considered significant when analyzed using unpaired Student’s t-test (Fig. 3b). In order to characterize the identity of the currents in response to pH 5.0 observed in TRPV1b-transfected cells further, ASIC blocker,

A-317567, was used in electrophysiological analysis (Dube et al. 2005). TRPV1b currents can be blocked by 30 lM ASIC channel blocker from 631.6 ± 161.4 pA (n ¼ 21) to 26 ± 7.5 pA (n ¼ 5), values that are not different from the background signals without acid stimulation (27 ± 4.5 pA n ¼ 10), while TRPV1 currents are marginally affected 96.1 ± 2.6% (n ¼ 3) of the current remained (data not shown) (Fig. 3c). These results demonstrated that pH-sensitive currents in TRPV1b-transfected cells were due to activation of ASIC channels endogenously expressed in HEK293 cells. Stimulation of TRPV1b-transfected HEK293 cells with heat (50C) evoked a response of 93 ± 25 pA (n ¼ 10), which is much weaker than that of TRPV1-expressing cells (633 ± 170 pA n ¼ 8) and no greater than that in vector-transfected HEK cells (84 ± 32 pA n ¼ 4) (Fig. 3d). TRPV1 was recently reported to be activated by changes in membrane voltage. The voltage-dependent activation curve was shifted to more physiologically relevant potentials following ligand binding or an increase in temperature (Voets et al. 2004). To verify that TRPV1b was inactive, we tested the responses to different stimuli over a range of membrane potentials using both ramp protocols, and by holding the membrane at the more depolarized potential of ) 25 mV. Voltage ramps from ) 80 to + 80 mV evoked a small, endogenous, outwardly-rectifying, voltage-dependent current that was activated in all HEK cells tested. However, there were no TRPV1b-specific currents in response to acidic pH (pH 5.0), 1 lM capsaicin or 50C heat stimulation. The small outward currents were not any different from those observed when ramps were run at room temperature (22–24C) or in vector-transfected cells following heat stimulation (Fig. 3e). In contrast, cells transfected with full-length TRPV1 responded to all stimuli, with currents showing a characteristic outward rectification (Fig. 3e). In addition, there was no difference in heat-activated currents between cells transfected with TRPV1b (53.4 ± 8.2 pA, n ¼ 6) and vector alone (32.6 ± 10.5 pA, n ¼ 3) when the membrane potential was held at )25 mV (data not shown). Detection of TRPV1b cell-surface expression by biotinylation Compared with full-length TRPV1, TRPV1b lacks 60 amino acids that encode half of the third ankyrin domain and 44 amino acids thereafter. The deleted region is neither the proposed capsaicin binding site (Jordt and Julius 2002), nor the pore loop that is presumably located between the fifth and sixth transmembrane domains of TRPV1 (Garcia-Martinez et al. 2000; Ferrer-Montiel et al. 2004). Based on the studies described above, it was not clear whether TRPV1b was incapable of responding to the stimuli, or whether the function was not detected due to lack of cell-surface expression. In order to determine the distribution of TRPV1/1b proteins between the plasma membrane and the



Fig. 3 Functional studies of TRPV1b in transfected HEK293 cells. Representative traces of HEK293 cells transfected with TRPV1, TRPV1b and mammalian expression vector, pCIneo, in response to 1 lM capsaicin (a), protons at pH 5.0 (b), 30 lM ASIC blocker, A317567, together with protons at pH 5.0 (c) and thermal stimulus at 50C (d), measured with electrophysiological assays. Scale bars represent 500 pA/ 1 s, 1 nA/1 s, 200 pA/500 ms and 75 pA/ 1 s in a, b, c and d, respectively. Peak currents under different conditions were analyzed as mean ± SE (n ¼ 4–10) and plotted in bar graphs. (e) TRPV1b was not activated over a range of voltage ramps in response to TRPV1 stimuli. Representative current traces of voltage ramps evoked in HEK293 cells transfected with TRPV1, TRPV1b or vector at room temperature (22–24C), and in response to 50C thermal stimulation. Currents were measured during 1600 ms voltage ramps from ) 80 mV to + 80 mV.

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intracellular compartment, cell-surface biotinylation followed by western blot analysis was performed. In HEK293 cells transfected with full-length TRPV1, the exclusive detection of Na+/K+ ATPase on the cell surface and cytochrome c in the intracellular fraction suggested that this method successfully separated plasma membrane proteins from intracellular proteins (Fig. 4a). Both the glycosylated and unglycosylated forms of TRPV1 were found in both the plasma membrane and intracellular fractions. Comparing the TRPV1 immune reactive signal intensities, cell-surface TRPV1 accounts for 6.0 ± 2.5% (n ¼ 3) of the total TRPV1 in the cells (Fig. 4a). This is also consistent with previous reports that determined the subcellular localization of TRPV1 using immunofluorescence studies (Olah et al. 2001; Liu et al. 2003). In HEK293 cells transfected with either 25 lg TRPV1b or 10 lg TRPV1 alone, when comparable amounts of total cellular TRPV1 and TRPV1b proteins were expressed, TRPV1b was detected on the cell surface at levels similar to TRPV1 (Fig. 4b, lanes 1 and 2). However, in cells double-transfected with 10 lg TRPV1 and 25 lg TRPV1b, TRPV1 cell-surface expression was reduced to 23% of that in cells transfected with TRPV1 alone (Fig. 4b, lane 3). This suggests that the presence of TRPV1b might interfere with the trafficking of TRPV1 to the plasma membrane.

TRPV1b forms homotetramer in transfected HEK293 cells TRP channel N-terminal ankyrin repeats have been shown to play an important role in tetrameric complex formation. Deletion of the third ankyrin repeat in TRPV6 resulted in a non-functional channel that did not form tetramers (Erler et al. 2004). In order to determine whether TRPV1b, with an incomplete third ankyrin repeat, was able to form a tetrameric complex, BS3 cross-linking reactions were performed on TRPV1- and TRPV1b-transfected HEK293 cell lysates, followed by immunoprecipitation and western blot analysis. In a small reaction volume (300 lg protein in 100 lL buffer), multiple forms of TRPV1 and TRPV1b complexes were observed upon cross-linking. With BS3 concentrations from 0 to 100 lM, TRPV1 and TRPV1b existed primarily as monomers and dimers, while at BS3 concentrations of 300 lM or higher, TRPV1 and TRPV1b trimers and tetramers became more evident (Fig. 5). To exclude the possibility that TRPV1b multimeric complex formation detected by cross-linking reactions were artifacts due to protein overexpression in HEK293 cells, we diluted the cell lysate 10-fold (300 lg protein in 1000 lL buffer) before the cross-linking reaction was performed. To our surprise, only tetramer and higher molecular weight protein



complexes, but no TRPV1b monomers or dimers, were observed in the presence of 300 lM and 1000 lM BS3 reagent. This suggests that TRPV1b predominantly exists as tetramer, even in the exogenously overexpressed cell system. As a negative control, HEK293 cells overexpressing P2X7 were cross-linked under the same conditions, and no tetrameric complex formation was observed (data not shown). Therefore, TRPV1b formed homomeric complexes similarly to full-length TRPV1. These results demonstrated that the 60 amino acids encoded by exon 7 were not required for protein trafficking to the plasma membrane or complex formation but rather, appeared to play a crucial role in maintaining normal TRPV1 channel function.

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Fig. 4 Cell-surface expression of TRPV1 and TRPV1b proteins in transiently-transfected HEK293 cells. (a) TRPV1 transiently transfected in HEK293 cells was surface biotinylated and separated from the intracellular protein by streptavidin beads. The distribution of cellsurface TRPV1, captured by streptavidin beads, and intracellular TRPV1 protein, immunoprecipitated with ABRK-1 antibody, was analyzed by western blot analysis using TRPV1 P-19 antibody (top panel). Detection of the plasma membrane protein Na+/K+ ATPase in the biotinylated fraction and the mitochondrial protein, cytochrome c, in the intracellular fraction by western blot analysis suggests successful separation of cell-surface proteins using this method (middle and bottom panels). (b) HEK293 cells transiently transfected with 10 lg TRPV1 and/or 25 lg TRPV1b plasmids were surface-labeled with biotin. Cell-surface TRPV1 and TRPV1b were detected by western blot using TRPV1 P-19 antibody after streptavidin bead pull-down (upper panel). Total TRPV1 and TRPV1b expressed in transfected HEK293 cells was immunoprecipitated with ABRK-4 antibody and immunoblotted with the P-19 antibody (lower panel).

TRPV1b associates with TRPV1 and inhibits its channel activity To determine whether TRPV1b was able to form heteromeric complexes with TRPV1, a co-immunoprecipitation experiment was performed on HEK293 cells transiently transfected with both HA-tagged TRPV1 (HA-TRPV1) and -untagged TRPV1b. Cell lysates were incubated with HA antibody and then analyzed for the presence of HA-TRPV1 and TRPV1b in the immune complex by western blotting with the TRPV1 P-19 antibody. As shown in Fig. 6(a), the HA antibody not only immunoprecipitated HA-TRPV1, but also co-precipitated the untagged TRPV1b that had been co-transfected with HA-TRPV1 (top panel), suggesting that TRPV1b formed complexes with HA-TRPV1. The bottom panel shows that similar amounts of HA-TRPV1 and TRPV1b proteins were present in the total cell lysates. As TRPV1b co-assembled with full-length TRPV1, it was possible that TRPV1b could affect TRPV1 activity. To test this hypothesis, HEK293 cells were transfected with different ratios of TRPV1b and TRPV1 cDNAs. The expression of

Fig. 5 TRPV1b tetrameric complex formation in transfected HEK293 cells. Lysates of HEK293 cells transiently transfected with TRPV1 or TRPV1b prepared at the concentration of 300 lg/100 lL or 300 lg/ 1000 lL were incubated with different concentrations of cross-linking reagent BS3 at room temperature (22–24C) for 30 min, followed by immunoprecipitation with the TRPV1 ABRK-4 antibody. Immunoprecipitated proteins were fractionated on 3–8% NuPAGE Tris-acetate gel and western blotted with TRPV1 P-19 antibody. The predicted monomer, dimer, trimer and tetramer are indicated on the right-hand side of the blots.



TRPV1 and TRPV1b under different conditions was determined by immunoprecipitation and western blot analysis. Interestingly, the level of TRPV1 protein expression

appeared to increase as more TRPV1b was expressed, despite the fact that the amount of transfected TRPV1 plasmid remained constant. When we plotted the protein

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Fig. 6 TRPV1b assembled with TRPV1 and inhibited TRPV1 activity in a dose-dependent manner. (a) Co-immunoprecipitation of TRPV1b with HA-TRPV1. HEK293 cells transiently transfected with HA-tagged TRPV1, untagged TRPV1b or both were immunoprecipitated with the HA antibody, 3F10, followed by western blot analysis with the TRPV1 P-19 antibody (upper panel). Total TRPV1 and TRPV1b protein expression was determined by immunoprecipitation with TRPV1 ABRK-4 antibody, followed by western blot analysis with the P-19 antibody (lower panel). (b) TRPV1 and TRPV1b protein co-expression in transiently-transfected HEK293 cells. TRPV1 and TRPV1b protein expression in HEK293 cells transfected with 0.5 lg TRPV1 plasmid alone or together with 2.5 lg (5·), 5 lg (10·), 7.5 lg (15·) and 10 lg (20·) TRPV1b plasmid was determined by immunoprecipitation from 200 lg total cell lysate, followed by western blot analysis. pCIneo vector DNA was added as needed to equalize the total amount of DNA (16 lg) under all conditions (upper panel). a-tubulin levels from 20 lg total proteins were measured to indicate that equal amounts of proteins were used for TRPV1/TRPV1b immunoprecipitation, shown

above (lower panel). (c–e) TRPV1b inhibited TRPV1 activities in a dose-dependent manner. HEK293 cells co-transfected with 0.5 lg TRPV1 plasmid and varying amounts of TRPV1b plasmid were loaded with fluo-4/AM. Fluorescence changes in response to 50 nM capsaicin (c), protons at pH 5.0 (d) and 3 lM NADA (e) were measured using a fluorometric imaging plate reader (FLIPR). Data were analyzed based on peak fluorescence and were plotted as a percentage of the peak signal from cells expressing TRPV1 alone (mean ± SE, n ¼ 8). (f) Representative traces of HEK293 cells transfected with 0.5 lg TRPV1 alone or together with 10 lg TRPV1b in response to a 50C thermal stimulus using electrophysiological assays (upper panel). Data were analyzed and are presented as a percentage of the peak signal from cells expressing TRPV1 alone (mean ± SE, n ¼ 11). Dotted line shows the current observed in untransfected cells. The reduction of current amplitude in TRPV1b and TRPV1 co-transfected cells was considered significant (*p < 0.002) using unpaired Student’s t-test (lower panel).



levels of TRPV1 against the levels of co-transfected TRPV1b, a positive correlation was observed with a slope of 0.42 ± 0.15 (n ¼ 11, r2 ¼ 0.46, p ¼ 0.022). A 20-fold excess of TRPV1b plasmid (10 lg) yielded fourfold higher TRPV1b protein expression over TRPV1, determined by densitometry using the NIH IMAGE (1.62) program (Fig. 6b). In HEK293 cells transfected with 0.5 lg TRPV1 plasmid together with varying amounts of the TRPV1b plasmid, TRPV1b inhibited TRPV1 activity dose-dependently in response to capsaicin, protons, the endogenous vanilloid, N-arachidonoyl-dopamine (NADA), and heat. Co-transfection of 0.5 lg TRPV1 plasmid with 2.5 lg and 10 lg TRPV1b plasmid inhibited 50 nM capsaicin-induced calcium influx by 54.2 ± 7.8% and 96.0 ± 2.3% (n ¼ 8), respectively, in the FLIPR assay (Fig. 6c). In addition, co-transfection with 10 lg TRPV1b (20·) inhibited 85.9 ± 3.0% (n ¼ 8) of TRPV1 activity at pH 5.0, and 92.1 ± 2.5% (n ¼ 8) of TRPV1 activity induced by 3 lM NADA (Figs 6d and e). In electrophysiological studies, co-transfection of 10 lg TRPV1b (20·) inhibited 50.1 ± 7.0% (n ¼ 11) of TRPV1 thermal response at 50C (Fig. 6f). These data are consistent with TRPV1b acting as a negative modulator of TRPV1 in response to capsaicin, protons, noxious heat and endogenous vanilloids. Determination of TRPV1 and TRPV1b protein stabilities by pulse-chase analysis It was also noticed that in transfection experiments, equal amounts of TRPV1 and TRPV1b plasmids do not express equal amounts of proteins. Total cellular TRPV1b protein was 38.4 ± 5.4% (n ¼ 5) and cell-surface TRPV1b was 17.3 ± 2.7% (n ¼ 5) of TRPV1 when equal amounts of plasmids were used to transfect HEK293 cells separately. When co-transfected into HEK293 cells, total cellular TRPV1b was 29.4 ± 11.9% (n ¼ 5) and cell-surface TRPV1b was 35.3 ± 5.2% (n ¼ 5) of that of TRPV1 (Fig. 7a). More TRPV1b plasmid was required to achieve expression levels equivalent to TRPV1, suggesting that the TRPV1b protein might not be as stable as TRPV1. To compare the stabilities of TRPV1b and TRPV1, pulse-chase analysis was performed. TRPV1 (10 lg) and TRPV1b (25 lg) plasmid DNA were used to transfect HEK293 cells separately. Then, 30 h after transfection, cells were metabolically labeled with [35S]-methionine for 30 min and chased with complete growth medium for different lengths of time. Both TRPV1 and TRPV1b were synthesized at similar levels during metabolic labeling (Fig. 7b). However, TRPV1b was less stable than TRPV1. TRPV1 was detectable even 30 h after initial synthesis, whereas no TRPV1b was detectable after 12 h. Immediately after metabolic labeling (0 h), TRPV1 and TRPV1b primarily existed as 95 kDa and 88 kDa unglycosylated proteins. After 6 h of chase, a partially (97 kDa) and a highly glycosylated form (120 kDa) of TRPV1 were detected, suggesting the matur-

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Fig. 7 TRPV1 and TRPV1b protein stabilities determined by pulsechase analysis. (a) Relative abundance of total and cell-surface levels of TRPV1 and TRPV1b proteins in single and double-transfected HEK293 cells with equal amounts of plasmids were determined using IMAGEQUANT 5.2. Protein levels were analyzed as mean ± SE (n ¼ 5) and plotted in bar graphs. The differences between total cellular levels of TRPV1 and TRPV1b, and cell-surface levels of TRPV1 and TRPV1b, in singly-transfected experiments, and cell-surface levels of TRPV1 in single and double-transfected cells, were considered significant when analyzed using paired Student’s t-test (**p < 0.0003; ***p < 0.0001). (b) HEK293 cells transiently transfected with 10 lg TRPV1 plasmid or 25 lg TRPV1b plasmid were metabolically labeled with [35S]-methionine for 30 min and chased with complete growth medium for 6, 12, 22 and 30 h. The amount of labeled TRPV1 and TRPV1b proteins at the end of different time points were determined by immunoprecipitation with ABRK-4 antibody followed by autoradiography.

ation of TRPV1. In contrast, at the same time point, TRPV1b remained as the 90 kDa partially-glycosylated form. As complex glycosylation occurs in late secretory organelles such as the Golgi and Trans-Golgi network, the lack of a highly glycosylated form of TRPV1b suggests a different trafficking route after synthesis. Discussion

In this study, we characterized the human TRPV1 splice variant TRPV1b that is lacking exon 7. TRPV1 exon 7 is prone to frequent alternative splicing, possibly due to the presence of abnormal splice sites at the intron/exon junction (Xue et al. 2001). Alternative splicing around exon 7 that results in either complete or partial deletion of the exon have been reported in rat (VR1L2 and VR.5¢sv) (Schumacher et al. 2000; Xue et al. 2001), in human (TRPV1b) (Lu et al. 2005) and in mouse (TRPV1b) (Wang et al. 2004). While mouse TRPV1b was characterized as a non-functional



dominant-negative regulator of full-length TRPV1, and human TRPV1b was found not to respond to capsaicin and protons expressed in Xenopus oocytes, the functions of the other splice variants, especially their effects on TRPV1 function, are not clear. The mRNA expression profiles of TRPV1 and TRPV1b in 35 different human tissues were examined by real-time quantitative RT-PCR. Interestingly, the TRPV1 mRNA level was 12 773 copies/50 ng total RNA in heart, much higher than that in the left ventricle and left atrium (3006 and 6834 copies/50 ng total RNA, respectively). This discrepancy of TRPV1 abundance between regional and whole heart tissue may suggest that TRPV1 expression varies significantly in different cell types of the heart. It has been reported that TRPV1 mRNA was detected in aortic smooth muscle, and TRPV1 function was detected in nerve fibers innervating epicardial surface blood vessels (Strecker et al. 2005; Yang et al. 2006). One possible explanation is that the left ventricle and atrium are composed primarily of cardiac muscles that express very low TRPV1, but the whole heart includes not only cardiac muscle but also nerve terminals innervating the heart, which may contribute to the higher TRPV1 level detected. The relative abundance of TRPV1b mRNA to TRPV1 mRNA is constant in most of the tissues examined at about 2.7%. Highest TRPV1b levels (6–7% of TRPV1) were detected in fetal brain, cerebellum and DRG, suggesting that these tissues are most likely subject to regulation by TRPV1b. It was previously reported by Lu et al. (2005) that TRPV1 mRNA co-existed in about 86% of the TRPV1b-expressing trigeminal ganglion neurons examined using single cell RT-PCR. This result raises the possibility that mutual regulation between TRPV1 and TRPV1b might take place in the same neuronal cell in vivo. To date, apart from mRNA, no TRPV1 splice variant protein has been detected in native tissues such as DRG, making it difficult to speculate about the physiological roles of these splice variants in regulating TRPV1 and in inflammatory pain sensation in vivo (Caterina et al. 2000; Davis et al. 2000). Studies of TRPV1 protein endogenously expressed in rat DRG have been reported by several groups using immunoprecipitation followed by western blot analysis. The major signal detected was a single band with no glycosylated form of TRPV1 (Kedei et al. 2001; Rutter et al. 2005). However, the identities of TRPV1 and the splice variants expressed in DRG remained unclear, probably due to low abundance of the protein or poor resolution on gel fractionation. Previously, when we fractionated the immunoprecipitated TRPV1 protein on 4–12% Bis-Tris gel under conventional conditions, i.e. 125 V constant voltage for 1.5 h (when the dye front was at the bottom of the gel), we observed only a single band on the western blot, exactly as described in the papers by Kedei et al. (2001) and Rutter et al. (2005). However, when the gel was fractionated extensively such that the 30 kDa marker was at the bottom

of the gel, TRPV1 immunoreactive proteins separated into multiple bands of varying apparent molecular weight. When the same fractionation conditions were followed, the same band pattern was repeated every time. This is the first report showing that in rat DRG, the previously reported single TRPV1 band is actually composed of two equally intense bands. Besides the major doublet bands, a small molecular weight band co-migrates with recombinant TRPV1b in DRG. In addition, by treating TRPV1 immunoprecipitates with Nlinked glycosidase PNGase F, we demonstrated that the doublet and TRPV1b (or VR1L2) are three glycosylated but unique TRPV1 variants. Calf intestine alkaline phosphatase, which is a non-specific protein phosphatase, does not change mobility of the three TRPV1 immune reactive bands, suggesting that the multiple bands do not represent the different phosphorylation status of TRPV1. Based on the apparent molecular weights, we estimated that the higher band of the doublet is about 10–20 amino acids larger than the lower band. Although we were not able to amplify products other than TRPV1 and TRPV1b using RT-PCR on human DRG total RNA (data not shown), we cannot exclude the possibility that we did not separate the isoform with a 30– 60 nucleotide deletion by agarose gel fractionation, or that one band in the doublet represents a novel post-translationally-modified form of TRPV1. Although the identity and function of this TRPV1 isoform is unknown, and we are not certain whether it is also observed in human DRG, the high abundance of this TRPV1 splice variant in rat DRG suggests that it may play an important role. TRPV1b is not activated by capsaicin, protons or heat when expressed in mammalian cells, due to the absence of exon 7. Our results are consistent with the conclusion of Lu et al. (2005) on capsaicin and acidic pH responses, but they differ with regard to the heat response. Lu and co-workers reported that TRPV1b expressed in Xenopus oocytes formed functional channels that responded to thermal stimuli with a threshold of 47C (Lu et al. 2005). However, in their study, the absolute current magnitudes of the TRPV1b temperatureactivated responses were not provided, nor were values given for temperature responses in mock-injected oocytes. These authors also showed that a small proportion of trigeminal ganglion neurons that express TRPV1b, but not TRPV1 mRNA, were unresponsive to capsaicin and acid but responsive to heat stimulus. Taken together, their results suggested that TRPV1b may be involved in the thermal response in these neurons. However, the possibility cannot be excluded that other thermal-sensitive receptors mediate this response. In addition, Lu et al. were not able to block heat responses in TRPV1- or TRPV1b-injected oocytes with 10 lM capsazepine, while we had previously shown that capsazepine was able to block the TRPV1-mediated heat response, with an IC50 of 62 nM, in transfected HEK293 cells (Neelands et al. 2003), suggesting that differences between the two expression and assay systems should be taken into



consideration as well. As we did not detect TRPV1bmediated heat responses in mammalian cells, TRPV1b functional role in vivo is arguable. The mechanism by which TRPV1 exon 7 alters TRPV1 channel property is not known. We demonstrated that this region is not required for cell-surface expression because comparable amounts of TRPV1 and TRPV1b proteins were observed on the plasma membrane when their protein expression levels were equivalent (Fig. 4b, lanes 1 and 2). The consequences of exon 7 deletion in TRPV1b are comparable to those of the mouse TRPV1b splice isoform, which contains a deletion corresponding only to the last 10 amino acids in exon 7 (Wang et al. 2004). Domains responsible for TRPV channel tetrameric complex formation have been reported to involve the C-terminal TRP-like domain (Chang et al. 2004; Garcia-Sanz et al. 2004) and the N-terminal ankyrin repeats (Chang et al. 2004; Erler et al. 2004). Although previous studies showed that a TRPV6 splice variant lacking the third ankyrin repeat failed to form homotetramers (Erler et al. 2004), TRPV1b missing part of the third ankyrin domain is able to from homotetramers, as demonstrated by cross-linking reactions, and to form heterocomplexes with TRPV1, as determined by co-immunoprecipiatation (Figs 5 and 6a). However, TRPV1b showed differences in post-translational modifications. Unlike TRPV1, which is both highly and partially glycosylated when expressed in HEK293 cells, TRPV1b is only partially glycosylated (Figs 2a and 7), suggesting a different trafficking route in the cells. It is also noted that when TRPV1b is co-expressed with TRPV1, the level of cell-surface TRPV1 is significantly lower than that in cells transfected with TRPV1 alone (Figs 4b and 7a). This supports the hypothesis that TRPV1b alters TRPV1 trafficking through protein–protein interactions. TRPV1b was very unstable compared with TRPV1, suggesting that it may be targeted for degradation. This is consistent with a previous report showing that a partial deletion in this region also resulted in channel instability in mouse TRPV1b (Wang et al. 2004). The apparent instability of TRPV1b implies that the inhibitory effect of TRPV1b on TRPV1 channel activity may be a transient and tightly regulated process. Compared with full-length TRPV1, overall TRPV1b abundance is low both at the mRNA level (7% of TRPV1 in DRG) and at the protein level (10–20% of TRPV1 in naı¨ve rat DRG, depending on whether one or two bands in the doublet are considered) (Figs 1 and 2a). In the light of these data, it is questionable whether TRPV1b is able to affect TRPV1 function to a significant extent. In our study, we found that when TRPV1 and TRPV1b plasmids were co-transfected into HEK293 cells, 23 ± 9.5% of TRPV1b protein was able to reduce TRPV1 levels on the cell surface from 100% to 41 ± 4.1% (n ¼ 5) (Fig. 7a). This suggests that by forming heteromultimers with TRPV1, TRPV1b function is amplified, depending on the stoichiometry of the

heterocomplex. In addition, although TRPV1b levels are low in naı¨ve animals, we cannot exclude the possibility that TRPV1b may be up-regulated under other physiological or pathological situations. In HEK293 cells co-transfected with different ratios of TRPV1 and TRPV1b plasmids, TRPV1b protein inhibited TRPV1 channel activity in a dose-dependent manner. Although 20-fold more TRPV1b plasmid (or fourfold more TRPV1b protein) was required for complete inhibition of TRPV1 activity (Fig. 6b), the apparent low efficacy of TRPV1b could be due to the limited amounts of TRPV1b that associated with TRPV1. This is because TRPV1b has the tendency to form both homotetramers and heterotetramers, and it is assumed that only the heterocomplexes with TRPV1 allow TRPV1b to exert the inhibitory effects. In addition, we also observed that more TRPV1 protein was expressed when TRPV1b plasmid levels increased in the co-transfection experiment. Up-regulation of TRPV1 becomes most prominent when 20-fold more TRPV1b plasmid is applied in transfection (Fig. 6b), which can significantly mask the effect of TRPV1b. Thus, the inhibition of TRPV1b on TRPV1 may be underestimated in the co-transfection experiments. In conclusion, this study describes the expression profile of TRPV1 and TRPV1b mRNA in different human tissues, and the detection of endogenous TRPV1 and TRPV1b proteins in rat DRG. Using biochemical and electrophysiological techniques, we have demonstrated that TRPV1b cannot be activated by known TRPV1 agonists such as capsaicin, protons and noxious heat. When co-expressed with TRPV1 in HEK293 cells, TRPV1b associates with TRPV1 and inhibits its channel activity. Thus, we hypothesize that TRPV1b can potentially function as a naturally-existing negative regulator of TRPV1. As constitutive TRPV1 activation leads to severe cytotoxicity and cell death (Olah et al. 2001; Karai et al. 2004), fine tuning of the channel activity is crucial for maintaining normal cell function and viability. Besides the regulation mediated by kinases, phosphatases, phospholipids and binding proteins that sensitize or desensitize TRPV1 (Bhave et al. 2002; Numazaki et al. 2002, 2003; Prescott and Julius 2003), the presence of the TRPV1 splice variant may provide another mechanism for down-regulating channel activity. Conditions leading to upregulation of TRPV1b levels, reducing TRPV1 cell-surface distribution or promoting TRPV1b and TRPV1 heterocomplex formation may all negatively affect TRPV1 channel activity. Compared with other regulatory mechanisms, regulation via TRPV1b might be a more specific and long-lasting approach, with minimum side effects towards cellular events that are tightly regulated by these multi-substrate enzymes. References Bhave G., Zhu W., Wang H., Brasier D. J., Oxford G. S. and Gereau R. W., 4th (2002) cAMP-dependent protein kinase regulates



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