Cloning and functional expression of a human orthologue of... : PAIN (original) (raw)
1. Introduction
Pain may be induced following exposure to several different classes of stimuli, including heat, mechanical or chemical stimuli, which are detected by nociceptor neurones (Sherrington, 1906). A group of chemical irritants, exemplified by capsaicin, an irritant vanilloid that is derived from chili peppers, are well known for their activity at nociceptor terminals. Although the molecular nature of the receptors responsible for transducing these painful stimuli has remained elusive, the tissue and in-vitro responses to the vanilloids have been studied for many years. Hopefully, an understanding of the mechanisms by which noxious stimuli are perceived will yield a route by which they can be manipulated for analgesic purposes (Bevan and Szolcsányi, 1990; Szallasi, 1994). Significant progress was made with the recent expression cloning of a functional cDNA for a rat vanilloid receptor (rVR1) (Caterina et al., 1997). The rVR1 was found to be responsive to capsaicin or heat and modulated by pH (Caterina et al., 1997; Tominaga et al., 1998). Variation between tissue responses to capsaicin and resiniferatoxin has suggested the presence of a family of vanilloid receptor genes, possibly for the transduction of different signals in response to a given stimulus, or in order to detect different ligands (Szallasi, 1994). The nature, however, of the endogenous ligands and direct mechanism of activation of VR1 in-vivo remain as outstanding questions. Furthermore, different species are more or less responsive to vanilloids (Holzer, 1991), highlighting an opportunity to separate the molecular pharmacology of vanilloid response from responses to endogenous or physiological stimuli (Szallasi, 1994).
With a view towards understanding the molecular basis of VR1 pharmacology and the long-term aim of producing therapeutics targeting human VR1 (hVR1), we have cloned hVR1 and report here its cDNA sequence, tissue expression profile and functional characterization. Together, these data suggest that this human vanilloid receptor is the human orthologue of the rat VR1 and will provide useful tools for the study of human pain.
2. Materials and methods
2.1. Isolation of hVR1 cDNAs
The public nucleotide databases were searched for human sequences similar to Rattus norvegicus VR1 using iterative BLAST searches. cDNA clones were obtained from the IMAGE consortium (Genome Systems Inc., St. Louis, MO). 5′ RACE (rapid amplification of cDNA ends; Frohman, 1993) was conducted using 2.5 μl Marathon-Ready cDNAs (Clontech, Basingstoke, UK) as templates and a gene-specific primer in combination with the AP1 primer (Clontech), which is complimentary to an adaptor ligated to the ends of all Marathon-Ready cDNAs. Secondary reactions were started with 1/25 of the product of the first, with a second gene-specific primer and AP2 (Clontech) as nested primers. Each RACE–polymerase chain reaction (PCR) mixture contained 2.5 μl of cDNA, 0.2 mM dNTP, 12 pmol primers, 1.5 mM MgCl2 and 20 units/ml of Ampli-Taq Gold polymerase (Perkin–Elmer Biosystems, Warrington, UK). The thermal cycle for the primary reaction began at 94°C for 10 min, followed by 5 cycles of 94°C for 30 s and 72°C for 3 min, then 25 cycles of 94°C for 30 s and 70°C for 3 min, and ended with 7 min at 70°C. The same cycle was used for the second reaction, except that the number of cycles at 94 and 70°C was increased to 30. The complete hVR1 open reading frame was amplified by reverse transcription (RT)–PCR: oligo dT-primed first-strand cDNAs were made from human brain and placental poly-A mRNAs (Clontech), using Superscript II reverse transcriptase (Life Technologies, Paisley, UK) according to the manufacturer's instructions. Subsequent high-fidelity PCR amplification from 1/500 of the RT reaction products was performed in 0.2 mM dNTP, 1.5 mM MgCl2, 40 units/ml of PfuTurbo DNA polymerase (Stratagene, Cambridge, UK), and 12 pmol PCR primers designed to amplify the complete hVR1 open reading frame (forward 5′-ACCATGAAGAAATGGAGCAGCACAG, and reverse 5′-AAGGCCCAGTGTTGACAGTG). The PCR thermal cycle began at 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 3 min, and ended with a further 5 min at 72°C.
PCR products were purified from agarose gels using Qiaquick Kit (Qiagen, Hilden, Germany) and subcloned using topoisomerase-activated pCR2.1-TOPO vector (Invitrogen, Leek, Netherlands). The RT–PCR amplified hVR1 open reading frame was cloned into the topoisomerase-activated pcDNA3.1-TOPO vector (Invitrogen). Plasmid DNA was prepared using Qiagen Minipreps (Qiagen). Clones were sequenced on both strands from standard vector primers and, where necessary, internal gene-specific primers, using an ABI automated sequencer. Sequences were assembled with a software package, Seqman (DNASTAR, Madison, WI), and the alignments optimized manually to give an overall consensus.
2.2. Chromosomal localization and genotyping
Chromosomal localization was identified by screening of a radiation hybrid panel as described (Li et al., 1998), using the following primers (Genosys, Cambridgeshire, UK) 5′-GAAATGGAGCAGCACAGACTTG and 5′-CCGAGTCACCCTTCCCAAAG. In the case of a discrepancy of any sample between two PCR runs, the sample was scored as ambiguous. The resulting string of 93 scores was submitted to the radiation hybrid mapping server at MIT (http://www-genome.wi.mit.edu/; Hudson et al., 1995). Cytogenic locations were inferred from these results using the markers shown to neighbour the genes via the Genome Directory.
The population frequency of polymorphisms was determined by screening a collection of 123 U.S. citizens with average ethnic background, using PCR amplification and the allele-specific primers (Genosys) 5′-GTGGAAAACCCGAACAAGAAGTCGA CorT and 5′-AACCGCCCTGCACGTTCTCAAGAC. Each DNA was subjected to two allele-specific PCR amplifications, using common forward primer and either allele-specific primer. Products were electrophoresed on 3% agarose gels and scored for the presence or absence of both alleles.
2.3. Analysis of expression of VR1 mRNA in CNS and peripheral tissues
Tissue expression of human and rat VR1 was studied using TaqMan quantitative RT–PCR (Gibson et al., 1996). Human poly(A)+ RNA samples obtained from Clontech were typically pooled samples from 8 to 69 non-diseased individuals, both male and female (age range 10–78 years). For analysis of mRNA expression in human dorsal root ganglion (DRG), total RNA from DRG and whole brain (Clontech) was used, due to limited material. Rat tissues were dissected from 3 to 20 Sprague–Dawley rats, depending on tissue size. Total RNA was extracted from rat tissues using Trizol (Life Technologies). cDNA was produced from the RNA by oligo-dT priming and reverse transcription using SuperScriptII RT (Life Technologies) according to the manufacturer's instructions. TaqMan reactions were conducted using the following flanking primers: 5′-GCTCAGCCCGAGGAAGTTT and 5′-ACTCTTGAAGACCTCAGCCTCC, and a TaqMan fluorogenic probe 5′-TGCGACAGTTTTCAGGGTCTCTAAAGCC for hVR1 (Perkin–Elmer Biosystems). The human glyceraldehyde phosphate dehydrogenase (GAPDH) probe consisted of 5′-GGAAGCTCACTGGCATGGC and 5′-TAGACGGCAGGTCAGGTCCA primers with a 5′-CCCCACTGCCAACGTGTCAGTG fluorogenic probe. The rVR1 probe consisted of 5′-TTTCAGGGTGGACGAGGTAAA and 5′-AGTTGCCTGGGTCCTCGTT flanking primers with a 5′-ATACCCACATTGGTGTTCCAGGTAGTCCA probe. Rat GAPDH probes were 5′-GAACATCATCCCTGCATCCA and 5′-CCAGTGAGCTTCCCGTTCA flanking primers with a 5′-CTTGCCCACAGCCTTGGCAGC probe. Ta-qMan reactions were performed using the cDNA prepared from 50 ng total RNA or 10 ng polyA+ RNA. TaqMan PCR mastermix (Perkin-Elmer Biosystems) was added to each cDNA sample: 2.5 μl TaqMan buffer, 6 μl MgCl2 (25 mM), 2 μl dATP/dUTP/dCTP/dGTP (10 mM), 0.25 μl uracil-_N_-glycosylase, 0.5–1 μl forward primer (10 μM), 0.5–1 μl reverse primer (10 μM), 0.5 μl TaqMan probe (5 μM), 0.125 μl TaqGOLD (Perkin Elmer Biosystems), PCR-grade water to 25 μl (including sample). The TaqMan PCR was conducted using an ABI Prism 7700 with cycling parameters of: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were analysed using the Power Macintosh software accompanying the ABI Prism 7700. The fluorescent signal was compared to appropriate dilutions of genomic DNA standard and the mRNA-derived values for each sample calculated by the subtraction of the low levels of genomic DNA contaminating the RNA samples. Genomic DNA contamination was assessed by amplification of the same quantity of the RNA samples with omission of reverse transcriptase from the cDNA synthesis step. Both rat and human data correspond to means of triplicate RT reactions using pools of each tissue RNA. Results were expressed as %VR1/GAPDH. A fragment of hVR1 was amplified from a human DRG library by PCR using the primers 5′-CTCCTACAACAGCCTGTAC and 5′-AAGGCCCAGTGTTGACAGTG and visualized after electrophoresis on a 1.5% agarose gel.
2.4. Expression in Xenopus oocytes
Surgery was carried out in accordance with the United Kingdom Animals (Scientific procedures) Act 1986 and conformed to SmithKline Beecham ethical standards. Oocytes were removed, dissociated and washed as previously described (Meadows et al., 1998). Stage V and VI oocytes were removed to modified Barth's solution (MBS) containing (mM): NaCl 88, KCl 1, NaHCO3 2.4, HEPES 15, MgSO4 0.82, Ca(NO3)2 0.33, CaCl2 0.41; buffered to pH 7.4 with NaOH and containing gentamicin (0.1 mg/ml). Human VR1.pcDNA3.1 plasmid was injected directly into the nuclei (0.5–1.5 ng/oocyte). Control oocytes were injected with hVRL1.pcDNA3.1 plasmid, which encodes a pH and capsaicin unresponsive gene. Oocytes were incubated in MBS plus gentamycin at 22°C prior to use in electrophysiological recordings. All experiments were performed at room temperature (20–24°C). Oocytes were placed in a recording chamber 1–3 days after injection and continuously perfused (14 ml/min) with a solution containing (mM): NaCl 88, KCl 1, NaHCO3 2.4, HEPES 15, MgCl2 1, BaCl2 0.1 (adjusted to the appropriate pH with 1 M NaOH or 5 M HCl). Solution was applied using large bore tubing (internal diameter 1.5 mm) which facilitated rapid solution exchange (half-time 350–1000 ms). Recordings were obtained using the two-electrode voltage-clamp technique with a Warner oocyte clamp (OC725B), holding potential −60 mV. Electrodes used were low resistance (0.5–3 MΩ) when filled with 3 M KCl. Currents were evoked in response to capsaicin (Sigma-Aldrich, Poole, UK) or pH shift, with a 30 s preincubation with capsazepine (Sigma–Aldrich) where applicable, applied through the perfusion system until the maximum current amplitude was reached. Current responses, digitized at 2 kHz and filtered at 1 kHz, were stored for later analysis using CHART (CED) software. The concentration–response curves were fitted to the logistic equation using Origin software.
2.5. Functional expression in mammalian cells
HEK293 cells were transfected with hVR1.pcDNA3.1 using Lipofectamine Plus (Life Technologies), according to the manufacturer's protocol. A stably transfected clone was also isolated by selection in 400 μg/ml G418.
Stable cells, or cells 24 h after transient transfection, were plated onto 19-mm coverslips coated with poly-L-lysine 30 000 cells/cm2 in modified Eagle's medium supplemented with non-essential amino acids, 10% foetal calf serum and glutamine (Life Technologies).
Whole-cell patch-clamp experiments were performed with standard methods as described elsewhere (Smart et al., 2000). The extracellular solution consisted of (mM): NaCl, 130; KCl, 5; BaCl2, 2; MgCl2, 1; glucose, 30; HEPES–NaOH, 25; pH 7.3. Electrodes were filled with (mM): CsCl, 140; MgCl2, 4; EGTA, 10; HEPES–CsOH, 10; pH 7.3. Capsaicin-containing solutions were prepared from a 10 mM stock in dimethylsulphoxide. Drugs were applied with an automated device for fast switching of solutions (Warner Instruments SF-77B; time for solution exchange ∼30 ms). To achieve rapid temperature jumps, the solution supplied to one of the bank of glass tubes in the solution exchanger was first passed through a heating device (Warner Instruments in-line heater SH-27A), allowing the temperature of the solution flowing from that barrel to be elevated in a controlled manner. The system was calibrated using two miniature thermocouples (∼100 μm diameter, Omega Instrument) placed at the input to the glass tube, through which the heated solution flowed, and adjacent to its output in the location usually occupied by the recorded cell. During experiments the latter thermocouple was removed. Currents were recorded at a holding potential of −70 mV using an Axopatch 200B amplifier. Data acquisition and analysis were performed using the pClamp7 software suite and Origin software.
For calcium imaging experiments the medium was replaced with Neuron tyrode (mM): NaCl 145, KCl 2.5, CaCl2 1.5, MgCl2 1.2, Hepes 10, glucose 10; pH 7.4, containing 5 μl/ml Fluo 4-AM and dye-loading supplement 2 μl/ml of Pluronic F-127 (20% w/v in dimethylsulphoxide) (Molecular Probes, Leiden), and incubated at 37°C for 20 min. The cells were washed three times with Neuron tyrode to remove excess dye. Test solutions were perfused onto the cells, via a six-channel solution changer (VC-6, Warner Instrument Corp.) at a rate of 4 ml/min and thermostatically maintained at 37°C (TC-324B temperature controller, Warner Intrument Corp.). Cells were imaged using a Zeiss Axiovert 135 microscope and FLUAR 40×/1.30 oil immersion objective and CCD camera (Photonic Sciences) at excitation wavelength 490 nm and emission 530 nm. Images were collected and analysed using NIH Image software. A fluorescence image was averaged over four frames (at 25 frames/s, total exposure time=0.15 s) and each image taken every 5 s. Intensifier and video gain settings were adjusted so that naïve Fluo-4 AM-loaded cells were visualized at the lower end of the pseudocolour scale providing the greatest dynamic range for possible increases in intracellular calcium signalling.
3. Results
3.1. Cloning of cDNA for human vanilloid receptor-1
A BLAST comparison of AF029310 (GenBank accession for the rVR1 cDNA) against public databases identified the EST NCBI:99815 (Merck/Washington University EST project; GenBank accession no. T48002), by its homology at the nucleic acid level to the 3′ end of the coding region of rVR1. The EST was derived from a human foetal spleen cDNA library clone (IMAGE accession no. 71579), the insert from which was completely sequenced and found to contain a further 1.2 kb of sequence extending 3′. A database search with this new sequence revealed a cluster of ESTs (UniGene accession number Hs.111965) derived from brain, kidney, promyelocyte, foetal liver/spleen, stomach and ovary, that matched the 3′ end of the clone 71579 insert. Clone 71579 terminated at its 3′ end in a polyA stretch that was preceded by a consensus polyadenylation sequence (5′-AATAAA-3′) 17 bases upstream, confirming that the 3′ end of the transcript had been reached. Comparison of the sequence of clone 71579 with rVR1 indicated that approximately 2.4 kb of coding sequence was missing. To complete the coding region, primers derived from the available sequence were used for RACE (Frohman, 1993). Three sequential 5′ RACE experiments yielded a total of 11 independent products from leukocyte, skeletal muscle, thymus, and promyelocytic leukaemia Marathon-Ready human cDNAs, that in composite extended the hVR1 sequence by 3341 nucleotides 5′ to IMAGE clone 71579. Comparison with the rVR1 sequence, and the presence of termination codons in all three reading frames in the 220 nucleotides immediately 5′ to the putative initiation codon, which complied with Kozak's rules (Kozak, 1991), indicated that the complete coding region had been found (Fig. 1A). The 4803 bp hVR1 cDNA sequence includes a 2517 bp open reading frame. The open reading frame was then amplified from cDNA prepared from brain and placental poly(A)+ RNA and an identical insert sequence determined from both brain and placental clones. Accession number AJ277028.
Primary structure of human and rat vanilloid receptor-1 (VR1). (A) Nucleotide and amino acid sequences of the human VR1 cDNA. (B) Amino acid sequence comparison for hVR1 and rVR1. Annotations show conserved predicted domains and consensus sites: transmembrane domains, bold underline; pore loop, dotted underline; ankyrin repeats, boxed; protein kinase A phosphorylation sites, filled diamond; _N_-glycosylation site, filled square; Val/Ile polymorphism, I.
The hVR1 cDNA was found to possess significant homology to the rVR1, with 86% identity and 92% similarity at the amino acid level (Fig. 1B). This level of homology strongly suggests that hVR1 is the orthologue of rVR1 and not of the recently identified VRL-1 (Caterina et al., 1999) to which the homology is much lower, at 51% identity. The region of lowest homology is in sequence N-terminal of the ankyrin repeats where amino acid identity falls to ∼70 and ∼40% over the N-terminal 200 and 100 residues, respectively. Homology is also reduced at the C-terminus. The protein structures of rVR1 and hVR1 were analysed and compared using GCG, TMPRED, PROSITE (Bairoch et al., 1997) software, and using Hidden Markov Models (HMM) with HMMER-2 software (Washington University, St. Louis, 1998) on databases of protein families such as SMART (Schultz et al., 1998) and Pfam (Sonnhammer et al., 1998). These analyses show that rVR1 and hVR1 share predicted transmembrane regions, ankyrin repeat domains and predicted phosphorylation sites (Fig. 1B). Only those ankyrin repeat-like regions that were clear in the HMMER-2 prediction are annotated but visual inspection of the protein sequences suggests that there may be a further region starting at position 283 that could also contribute to this overall ankyrin repeat domain. Protein kinase A phosphorylation sites at Ser502 and Thr371 are conserved between the rat and human VR1. The site at rat Thr145 is altered, however, by the change from Arg to His143, and a new predicted site is introduced in hVR1 at Ser005 (Fig. 1B).
3.2. Chromosomal localization
The chromosomal localization of hVR1 was determined by screening of a radiation hybrid panel. The nearest-neighbour markers to hVR1 in the radiation hybrid mapping MIT database were found to be WI-9674 at −22.06 cR (lod>3.0) and WI-5436 at −39.3 cR. The failure to identify a marker on the opposing flank of hVR1 is a reflection of the limited number of markers in this region. The inferred cytogenic location is 17p13. Referral to the Online Mendelian Inheritance in Man (OMIM) database (http://www3.ncbi.nlm.nih.gov/htbin-post/Omim) revealed no obvious disease association with hVR1.
3.3. Identification of a Val/Ile polymorphism
During sequencing of RACE fragments for hVR1, a possible polymorphism at nucleotide 1753 was observed, with a single base substitution of guanine to adenine resulting in a valine to isoleucine amino acid substitution at position 585. A genotyping screen of 123 randomly selected individuals, of mixed race, showed that both alleles are common, with the largest proportion of the population being heterozygous (51%), Val/Val (15%), Ile/Ile (34%). Both polymorphs give rise to functional channels, that are responsive to capsaicin and protons (data not shown).
3.4. VR1 mRNA expression in brain and peripheral tissues
Tissue expression of hVR1 and rVR1 was analysed using TaqMan quantitative PCR (Gibson et al., 1996; Lie and Petropoulos, 1998) across a wide range of peripheral tissues and in brain. The data are presented normalized to the house-keeping gene GAPDH in order to correct for RNA quantity and quality. Similar data are obtained when normalized to RNA mass or other house-keeping genes such as _β_-actin. A uniformly low expression of hVR1 was found throughout tissues tested (Fig. 2A). Only very weak signals could be detected by Northern analysis (data not shown); however, the TaqMan measurements were above background. Total RNA from human DRG was analysed alongside total RNA from human whole brain in order to allow cross-reference between parts A and B in Fig. 2. Human VR1 was found to be most highly expressed in DRG. Human VR1 could also be detected in a human DRG cDNA library, using PCR and primers to the 3′ end of the hVR1 coding region that amplify a 676 bp fragment (Fig. 2D). Rat VR1 transcript expression (Fig. 2C) was also found to be most highly expressed in DRG, as previously described by Caterina et al. (1997).
Tissue expression profiles for rat and human vanilloid receptor-1. cDNA prepared from poly(A)+ mRNA (A) or total RNA extracted from human (B) tissues, or rat (C) tissues, was used in TaqMan analysis as described in Section 2. Data is normalized as % vanilloid receptor-1/glyceraldehyde phosphate dehydrogenase and shown as mean±SEM for triplicate reverse transcription reactions from each RNA pool. (D) Specific hVR1 primers were used to amplify, by 35 cycles of PCR, a 676 bp fragment of hVR1 from human DRG library (lane 1), cloned hVR1 cDNA (lane 2) or no DNA control (lane 3), molecular weight markers (M). Products were visualized after electrophoresis in a 1.5% agarose gel.
3.5. Functional response of human VR1 to capsaicin, pH and temperature
In order to determine whether or not the hVR1 protein is a functional orthologue of rVR1, the full-length hVR1 open reading frame was amplified from a placental cDNA library and subcloned into pcDNA3.1. Xenopus oocytes were injected with the hVR1.pcDNA3.1, or control plasmid and membrane currents were measured by two-electrode voltage clamp following application of capsaicin or pH shifts. Increasing concentrations of capsaicin evoked inward currents of increasing amplitude (Fig. 3A) that were not observed in oocytes injected with a control plasmid (_n_=68, four different batches of oocytes, data not shown). The EC50 of the capsaicin activated response was 640 nM and responses to 1 μM capsaicin (1.18±1.72 μA, _n_=10) were completely blocked by 10 μM capsazepine (0±0 μA, _n_=4) (Fig. 3B). A reduced capsaicin response was obtained after extended washout of capsazepine, indicating that the blockade was partially reversible (Fig. 3B). Protons alone evoked a response at pH 5.0 but not at pH 6.5 or 7.4 (Figs. 3C and 4A). As well as acting as an agonist, protons have also been shown to potentiate the activity of rVR1 (Tominaga et al., 1998). A pH of 6.5 has a similar effect on hVR1 (Fig. 4A). Acid and alkali shifts have opposing effects on the efficacy and potency of capsaicin for hVR1 (Fig. 4B).
Current responses to capsaicin or pH shifts in oocytes expressing human vanilloid receptor-1 (VR1). Oocytes were injected with hVR1.pcDNA3.1 plasmid and incubated for 2–3 days. Currents were recorded using the two-electrode voltage clamp technique from a holding potential of −60 mV. (A) Representative current profiles recorded upon exposure to 30 nM–1 μM capsaicin. Horizontal bars indicate the period of capsaicin application. Data were obtained from a single oocyte. (B) Sample trace from a single oocyte of the effect of 10 μM capsazepine on capsaicin-induced currents. (C) Pooled normalized concentration response curves to capsaicin (▪) or pH (○). Data obtained from each cell were normalized to the cell's response to 10 μM capsaicin or pH 5, respectively. Each data point represents mean±SEM (_n_=5 or more).
Potentiation of current responses to capsaicin by pH, in oocytes expressing human VR1. (A) Sample trace of the current evoked by a shift from pH 7.4 to 5.0, pH 6.5 or in response to capsaicin at pH 7.4 or 6.5. (B) Concentration–response data to capsaicin at pH 6.5 (▪), 7.4 (▵) or 8.0 (•). Response amplitude was normalized to the response to 1 μM capsaicin at pH 7.4 and mean±SEM plotted (_n_=6 or more).
In addition to the basic electrophysiological characterization work in Xenopus oocytes presented above, we used patch-clamp techniques to characterize the properties of macroscopic hVR1-mediated currents in hVR1 transfected HEK293 cells (hVR1.HEK). In untransfected HEK293 cells, voltage-clamped at −70 mV, capsaicin at concentrations as high as 30 μM failed to produce any significant currents (Fig. 5A, upper panel). In contrast, in the majority of hVR1.HEK cells 1 μM capsaicin activated a significant inward current (Fig. 5B, upper panel). In agreement with data from Xenopus oocytes, extracellular acidification activated a slowly gating inward current in hVR1.HEK cells (Fig. 5B, lower panel), but not in untransfected HEK cells (Fig. 5A, lower panel). However, a fast proton gated current, probably reflecting the expression of an endogenous ASIC, was observed in both hVR1- and wildtype HEK293 cells (Gunthorpe et al., unpublished observations). Both the acid- and capsaicin-gated currents in hVR1.HEK cells were outwardly rectifying with a reversal potential close to 0 mV (Fig. 5C).
Whole-cell current responses to capsaicin or protons in wild-type and hVR1 transfected HEK293 cells. (A) Example trace of whole-cell current in wild-type HEK293 cells exposed to 30 μM capsaicin (upper panel) (_n_=10) or pH 5.3 (lower panel) (_n_=10). (B) Example trace of current evoked in HEK293, transfected with hVR1, exposed to 1 μM capsaicin (upper panel) or pH 5.3 (lower panel). (C) Current–voltage properties of hVR1 gated by protons or capsaicin, determined using a ramp protocol (−70 to +70 mV at 0.14 mV/ms) applied during the plateau phase of the response (_n_=3).
Human VR1.HEK cells were also exposed to capsaicin, with or without the antagonist capsazepine, and imaged for cytoplasmic calcium changes using Fluo-4. Capsaicin (1 μM) evoked robust increases in cytoplasmic Ca2+ concentrations, which were reversibly blocked by pretreatment with capsazepine (10 μM) (data not shown), as was shown also in oocyte recordings (Fig. 3B). The increase in cytosolic calcium concentration is consistent with recordings of elevated calcium accumulation in DRG neurones in response to capsaicin (Wood et al., 1988).
Rat VR1 is activated by increases in heat above a threshold of approximately 42°C (Caterina et al., 1997) which is in line with the observation of a current of very similar properties and temperature dependence in rodent DRG neurones (Cesare and McNaughton, 1996). Temperature jumps from room temperature (24–25.5°C) to temperatures up to 52°C were used to examine if hVR1 expressed in HEK293 cells could be activated in a similar way. Elevations in extracellular temperature produced small inward currents in untransfected HEK293 cells clamped at −70 mV (Fig. 6A, upper panel). These heat-activated currents in wild-type cells had rapid and near symmetrical activation and deactivation kinetics and were not associated with any appreciable increase in current noise (Fig. 6A). With temperature shifts to between 25 and 48°C the magnitude of these inward currents appeared linearly related to the amplitude of the temperature jump. Between 48 and 52°C there appeared to be a somewhat greater current activation and consequently the relationship between temperature and current became steeper (Fig. 6B). Similar currents were also observed in hVR1.HEK cells that failed to respond to capsaicin. As previously suggested by others (Cesare and McNaughton, 1996), these currents probably arise predominantly from basic physicochemical changes in the cell, including a change in the resistance of the seal between the electrode and cell membrane. In line with this there is a strong positive correlation between holding current at room temperature and the current produced by a temperature jump (data not shown).
Whole-cell current responses of wild-type and hVR1 transfected HEK293 cells in response to temperature. (A) Example traces of whole-cell currents in response to temperature shifts from room temperature to the indicated temperature in wild-type (upper panel) or hVR1-transfected (lower panel) HEK293 cells. (B) Temperature dependency of current response from two independent batches of wild-type HEK293 cells. (C) Temperature-response curves for the example shown in A (○) and for pooled data from hVR1.HEK293 cells (•) (mean±SEM, _n_=10). (D) The mean current–voltage relationship of the heat-activated current in seven capsaicin-responsive hVR1.HEK293 cells. The relationship in each cell was determined using a voltage ramp applied before and during a heat challenge to 50°C. These were then normalized to the amplitude of the heat-gated current at −70 mV, before averaging across cells.
In capsaicin-responsive hVR1.HEK cells, an additional distinctive component of temperature-induced current response was observed. This current became apparent with jumps to temperatures at ∼44°C and greatly increased in amplitude as the temperature was further increased (Fig. 6A,C). Statistical comparison of the amplitude of the responses to a 50°C challenge in capsaicin responsive hVR1.HEK cells (385±109 pA, _n_=13) and wild-type HEK293 cells (39.8±2.2 pA, _n_=12) revealed a highly significant difference (P<0.008, unpaired Student's _t_-test). Unlike the small currents produced by heat in untransfected HEK293 cells, the response to heat in capsaicin-sensitive hVR1.HEK cells was associated with a considerable increase in current noise, indicative of the activation of a channel of relatively large single channel conductance. Activation of the current was quite slow, but deactivation upon cooling was more rapid giving the hVR1-dependent heat-induced current a clearly asymmetric kinetic profile (Fig. 6A). Paralleling the situation observed when hVR1 is activated by capsaicin or acid pH (Fig. 5), the heat-gated current exhibited a reversal potential close to 0 mV and substantial outward rectification (Fig. 6D).
These responses to capsaicin, protons and temperature are very similar to those reported for rVR1 (Caterina et al., 1997; Tominaga et al., 1998) and suggest that the cloned cDNA is the human orthologue of the rat VR1.
4. Discussion
We have cloned a human cDNA, homologous to the reported rat vanilloid receptor, that is responsive to both capsaicin, pH and heat. The hVR1 cDNA has a high degree of homology with the rVR1 (Caterina et al., 1997), 92% similarity at the amino acid level, with divergence located primarily in the very N- and C-terminal coding regions. Because of the close homology and parallel functional characteristics with the rVR1, we have termed the human cDNA, human vanilloid receptor-1.
Cloning of a number of orthologues from different species would be expected to greatly facilitate an understanding of the molecular pharmacology of the vanilloid receptor-1, and to provide insights into which domains and residue changes between species are responsible for the significant inter-species variation in responses to capsaicin or resiniferatoxin (reviewed by Szallasi, 1994). Comparison between the human and rat VR1 cDNAs, which both code for capsaicin responsive channels, shows very high conservation across the transmembrane domains, ankyrin repeats, putative pore loop and preservation of particular predicted phosphorylation sites. The conserved phosphorylation sites may be involved in modulatory mechanisms such as desensitization (Docherty et al., 1996; Lopshire and Nicol, 1998). Capsaicin has been shown to mediate its effect on the intracellular side of the receptor (Jung et al., 1999); however, the high homology between rat and human primary structures reduces the opportunity to locate a common minimal binding site for capsaicin by simple comparison of these native sequences. It will be interesting to compare further species orthologues and other members of this channel family for their responses to capsaicin.
During the cloning of hVR1 we identified a valine/isoleucine polymorphism at position 585 and it is possible that this polymorphism may play a role in familial inheritance of changes in susceptibility to pain. Both alleles, however, were found to be common in a random sample of the human population and the substitution represents a conservative change when compared to the rat, which has a leucine in the equivalent position. Furthermore, we have been unable to detect a functional difference between the polymorphs, suggesting that the substitution does not critically alter receptor structure/function.
The expression profile of hVR1 is similar to that of rVR1 and is elevated, relative to other tissues, in the DRG. An unexpected observation, however, was that hVR1 is expressed at low levels in a wide range of tissues. Recently, expression of rVR1 has been reported to be more widespread than previously thought (Caterina et al., 1997), with mRNA expression reported in the hypothalamus, striatum, cerebellum and cerebral cortex (Sasamura et al., 1998). Our own studies show mRNA expression for rVR1 in brain and peripheral tissues but at levels of expression very much lower than that found in DRG. Such small TaqMan signals, as seen in most tissues, may arise from two possible sources, either from trace mRNA in the afferent axons found arborizing through tissues, or from expression of VR1 in small populations of cells not previously thought to express VR1. The latter is consistent with the observation that hVR1 ESTs and RACE products were obtained from libraries from a number of different tissues including brain, placenta, spleen and also from clonal HL-60 and CCRF-CEM cell lines of non-neuronal origin. In addition, capsaicin responses have been observed in mast cells and in the C6 glioma cell line (Bíró et al., 1998a,b), suggesting that functions for vanilloid receptors in non-neuronal cell types may yet be discovered. Detailed in situ hybridization and immunohistochemical analysis is required in order to determine which of the cell populations in these tissues expresses VR1. The first of such studies (Tominaga et al., 1998) highlighted the presence of rVR1, detected by immunohistochemistry, in cell bodies of primary afferent neurones but also noted expression in axons arborizing through the bladder smooth muscle tissue. Such studies will guide subsequent work upon isolated cell types.
Aside from the conserved primary structure between the rat and human VR1 cDNAs, the very similar responses to capsaicin, pH and temperature strongly suggest that hVR1 is the orthologue of rVR1. Both show a very similar response profile to capsaicin; hVR1 expressed in oocytes has an EC50 of approximately 640 nM for capsaicin, which is close to that of 719 nM reported for rVR1 (Caterina et al., 1997), and consistent with measurements in primary rat DRG neurones (Wood et al., 1988) and responses in human DRG neurones (Baumann et al., 1996). Furthermore, the capsaicin response of native, recombinant rVR1 and hVR1 receptors can be completely blocked by the antagonist capsazepine (Bevan et al., 1992). Human VR1 also responds to low pH in a similar fashion to rVR1, showing an ability to respond to low pH (pH 5.0) alone or an enhanced response to capsaicin at proton concentrations (pH 6.5) that are otherwise ineffective alone. Lastly, the temperature response of hVR1, particularly the threshold at 42–44°C, is similar to that seen for rVR1 expressed in HEK293 cells (Caterina et al., 1997) and in primary DRG neurones (Cesare and McNaughton, 1996). Humans and rats have a similar threshold for noxious heat and it is interesting to note the similar threshold of VR1 between the species.
Prior to the cloning of rVR1, various pieces of pharmacological data suggested the presence of more than one VR, for example the differing responses to different vanilloids (Liu et al., 1998), or the R and C receptor subtypes identified on the basis of a high affinity for RTX or capsaicin (reviewed by Szallasi and Blumberg, 1999). By using recombinant rVR1 it has now been shown that rVR1 can account for both the putative R and C activities (Szallasi et al., 1999). Some of the remaining pharmacological diversity might be explained by the formation of heteromeric channels composed of VR1 and other channel subunits, by analogy to other ligand-gated channels that form heteromeric complexes such as P2X (Lewis et al., 1995). These would be likely to have differing sensitivity to ligands or altered gating properties but heterologous subunits are yet to be identified. A thorough pharmacological characterization of the responses of hVR1 to the many vanilloids will contribute to that characterization and is the subject of ongoing work. Vanilloid receptors represent an exciting new avenue in the understanding of sensory mechanisms, and the reporting of hVR1 provides an opportunity for the development of VR1 antagonists and other tools to further human pain research.
Acknowledgements
The authors gratefully acknowledge the support of Dr R. Russell and M. Sims for technical advice and core services, and Dr R. Ravid, Netherlands Brain Bank, for supply of human adult brain tissue.
References
Bairoch A, Bucher P, Hofmann K. The PROSITE database, its status in 1997. Nucleic Acids Res. 1997;25:217-221.
Baumann TK, Burchiel KJ, Ingram SL, Martenson ME. Responses of adult human dorsal root ganglion neurons in culture to capsaicin and low pH. Pain. 1996;65:31-38.
Bevan S, Szolcsányi J. Sensory neuron-specific actions of capsaicin: mechanisms and applications. Trends Pharmacol Sci. 1990;11:330-333.
Bevan S, Hothi S, Hughes G, James IF, Rang HP, Shah K, Walpole CSJ, Yeats JC. Capsazepine: a competitive antagonist of the sensory neurone excitant capsaicin. Br J Pharmacol. 1992;107:544-552.
Bíró T, Brodie C, Modarres S, Lewin NE, Ács P, Blumberg PM. Specific vanilloid responses in C6 rat glioma cells. Mol Brain Res. 1998a;56:89-98.
Bíró T, Maurer M, Modarres S, Lewin NE, Brodie C, Ács G, Ács P, Paus R, Blumberg PM. Characterization of functional vanilloid receptors expressed by mast cells. Blood. 1998b;91:1332-1340.
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat activated ion channel in the pain pathway. Nature. 1997;389:816-824.
Caterina MJ, Rosen TA, Tominaga M, Brake AJ, Julius D. A capsaicin-receptor homologue with a high threshold for noxious heat. Nature. 1999;398:436-441.
Cesare P, McNaughton P. A novel heat-activated current in nociceptive neurons and its sensitization by bradykinin. Proc Natl Acad Sci USA. 1996;93:15435-15439.
Docherty RJ, Yeats JC, Bevan S, Boddeke HW. Inhibition of calcineurin inhibits the desensitization of capsaicin-evoked currents in cultured dorsal root ganglion neurones from adult rats. Pflügers Arch. 1996;431:828-837.
Frohman MA. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE. Methods Enzymol. 1993;218:340-356.
Gibson UE, Heid CA, Williams PM. A novel method for real time quantitative RT–PCR. Genome Res. 1996;6:995-1001.
Holzer P. Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol Rev. 1991;43:144-201.
Hudson T, Stein L, Gerety S, Ma J, Castle A, Silva J, Slonim DK, Baptista R, Kruglyak L, Xu S-H, Hu X, Colbert AME, Rosenberg C, Reeve-Daly MP, Rozen S, Hui L, Wu X, Vestergaard C, Wilson KM, Bae JS, Maitra S, Ganiatsas S, Evans CA, DeAngelis MM, Ingalls KA, Nahf RW, Horton LT Jr, Anderson MO, Collymore AJ, Ye W, Kouyoumjian V, Zemsteva IS, Tam J, Devine R, Courtney DF, Renaud MT, Nguyen H, O'Connor TJ, Fizames C, Faure S, Gyapay G, Dib C, Morissette J, Orlin JB, Birren BW, Goodman N, Weissenbach J, Hawkins TL, Fote S, Page DC, Lander ES. An STS-based map of the human genome. Science. 1995;270:1945-1954.
Jung J, Hwang SW, Kwak J, Lee SY, Kang CJ, Kim WB, Kim D, Oh U. Capsaicin binds to the intracellular domain of the capsaicin-activated ion channel. J Neurosci. 1999;19:529-538.
Kozak M. Structural features of eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem. 1991;266:7-19870.
Lewis C, Neidhart C, Holy C, North RA, Buell G, Surprenant A. Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature. 1995;377:432-435.
Li X, Bouzyk MM, Wang X. Assignment of the human oxidized low-density lipoprotein receptor gene (ORL1) to chromosome 12p13.1-p12.3 and identification of a polymorphic CA-repeat marker in the OLR1 gene. Cytogenet Cell Genet. 1998;82:34-36.
Lie YS, Petropoulos CJ. Advances in quantitative PCR technology: 5′ nuclease assays. Curr Opin Biotechnol. 1998;9:43-48.
Liu L, Szallasi A, Simon SA. A non-pungent resiniferatoxin analogue, phorbol 12-phenylacetate 13-acetate 20-homovanillate, reveals vanilloid receptor subtypes on rat trigeminal ganglion neurons. Neuroscience. 1998;84:569-581.
Lopshire JC, Nicol GD. The cAMP transduction cascade mediates the prostaglandin E2 enhancement of the capsaicin-elicited current in rat sensory neurones: whole-cell and single-channel studies. J Neurosci. 1998;18:6081-6092.
Meadows HJ, Kumar CS, Pritchett DB, Blackburn TP, Benham CD. SB-205384: a GABAA receptor modulator with novel mechanism of action that shows subunit selectivity. Br J Pharmacol. 1998;123:1253-1259.
Sasamura T, Sasaki M, Tohda C, Kuraishi Y. Existence of capsaicin-sensitive glutamatergic terminals in rat hypothalamus. NeuroReport. 1998;9:2045-2048.
Schultz J, Milpetz F, Bork P, Ponting CP. SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci USA. 1998;95:5857-5864.
Sherrington CS. The integrative action of the nervous system. New York: Scribner; 1906.
Smart D, Gunthorpe MJ, Jerman JC, Nasir S, Gray J, Muir AI, Chambers JK, Randall AD, Davis JB. The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br J Pharmacol. 2000;129:227-230.
Sonnhammer EL, Eddy SR, Birney E, Bateman A, Durbin R. Pfam: multiple sequence alignments and HMM-profiles of protein domains. Nucleic Acids Res. 1998;26:320-322.
Szallasi A. The vanilloid (capsaicin) receptor: receptor types and species differences. Gen Pharmacol. 1994;25:223-243.
Szallasi A, Blumberg PM. Vanilloid (capsaicin) receptors and mechanisms. Pharmacol Rev. 1999;51:159-211.
Szallasi A, Blumberg PM, Annicelli LL, Krause JE, Cortright DN. The cloned rat vanilloid receptor VR1 mediates both R-type binding and C-type calcium response in dorsal root ganglion neurons. Mol Pharmacol. 1999;56:581-587.
Tominaga M, Caterina MJ, Malmberg A, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI, Julius D. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron. 1998;21:531-543.
Wood JN, Winter J, James IF, Rang HP, Yeats J, Bevan S. Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture. J Neurosci. 1988;8:3208-3220.
Keywords:
Capsaicin; Protons; pH; Temperature; Nociception
© 2000 Lippincott Williams & Wilkins, Inc.