Molecular determinants of vanilloid sensitivity in TRPV1

and the from , (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous (anandamide, N-arachidonyl dopamine, N-oleoyldopamine and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable 3 H-RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (M547 and T550) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, 3 H-RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.


Introduction
The receptor for capsaicin (a small vanilloid molecule extracted from 'hot' chili peppers), designated Vanilloid Receptor 1 (aka VR1 and TRPV1: 1) has been cloned and shown to be a non-selective cation channel with high permeability to calcium. TRPV1 belongs to a super family of ion channels known as TRP's (transient receptor potential channels) several of which appear to be sensors of temperature (2,3). TRPV1 can be activated by exogenous agonists (capsaicin and RTX) and by physical stimuli such as heat (>42 0 C) and protons (pH 5). Possible endogenous ligands released during tissue injury have also been suggested including anandamide (arachdonyl ethanolamine or AEA) and products of lipoxygenases such as 12-HPETE, Narachidonyl dopamine (NADA) and N-oleoyldopamine ([OLDA] 4-7). Ji et al. (8) reported that TRPV1 is detectable at increased levels after inflammatory injury in rodents and speculated that the increased level of TRPV1 protein combined with the confluence of stimuli present in inflammatory injury states leads to a reduced threshold of activation of nociceptors that express TRPV1, i.e., hyperalgesia. Indeed the converse is true that TRPV1 deficient mice display reduced thermal hypersensitivity following inflammatory tissue injury (9).
Structure-function studies of this channel are in their infancy but fundamental observations have been reported. Publications of species differences, based upon differential binding of the radiolabeled TRPV1 agonist 3 H-RTX to dorsal root ganglia membranes, were recorded even before TRPV1 was cloned (10). Of note, rabbits were found to be resistant to the acute toxicity of capsaicin (11), and were found not to have 3 H-RTX binding sites (10). These observations have provided the basis for an approach to identify key regions involved in TRPV1 binding and activation by RTX and capsaicin by cloning TRPV1 from capsaicin-sensitive and insensitive species (rat, 1; human, 12; rabbit, 13,14; chicken, 15; guinea pig, 16). Rat and human TRPV1 have been pharmacologically characterized proving that capsaicin and RTX are indeed agonists of TRPV1 (capsaicin EC 50 :0.05-0.2 µM and RTX EC 50 :0.3-11 nM) transiently expressed in HEK293 cells (12,(17)(18)(19). Interestingly, these studies have indicated species differences in antagonism, such as the report that capsazepine blocks human but not rat TRPV1 response to low pH (18). Electrophysiological studies using membrane impermeable analogs of capsaicin (20), and mutational analysis of extracellular loops (21,22) have identified domains that contribute to capsaicin and proton activation respectively. These studies have demonstrated that capsaicin appears to function from the intracellular side and protons act on an extracellular site to activate TRPV1. We have previously reported the cloning of rabbit TRPV1 and that it is capsaicininsensitive but activated by heat (45°C) and protons (pH 5) in transiently expressed HEK293 cells (13). Jordt and Julius (15) have more recently shown that heterologously expressed chicken TRPV1 (gTRPV1) is similarly insensitive to activation by capsaicin but sensitive to heat (>42°C) and proton (pH 4.5) stimuli. Furthermore, Jordt and Julius (15) showed that the TM3/4 region of TRPV1 appeared to be responsible for capsaicin sensitivity. Experiments by other investigators have identified additional residues on the N-and C-terminal domains of TRPV1 that also appear to modify capsaicin sensitivity as well as 3 H-RTX binding (23,24).
We describe in the present study amino acids in TRPV1 critical for vanilloid sensitivity by utilizing rabbit TRPV1 (Oryctolagus cuniculus, oTRPV1) and selected mutations of the TM3/4 region. We determine, utilizing radioactive calcium ( 45 Ca 2+ ) uptake assays and whole cell patch clamp techniques, the sensitivity of oTRPV1 and mutants to the published activators of TRPV1.
Binding of 3 H-RTX was used to probe residues important for the high affinity binding of this ligand to TRPV1. Further, we examine the functional sensitivity of oTRPV1 and mutants to capsaicin site antagonists and show that gain of capsaicin sensitivity also confers competitive antagonist action at TRPV1. Lastly, we present a model of capsaicin and RTX bound to the TM3/4 region of rat TRPV1.

Molecular biology
A cDNA library was made in pSPORT vector from poly A + containing RNA extracted from dorsal root ganglia dissected from New Zealand white rabbits. The library was screened at high stringency (2X SSC, 65 0 C) with a rTRPV1 probe (bases 1063-2185, the sequence with Accession number AF029310). Several clones were isolated and the longest full-length clone, designated

Functional assays
The activation of TRPV1 is followed as a function of cellular uptake of radioactive calcium 100 µl of α 1 -acid glycoprotein (2mg/ml; Sigma) was added into the binding mix and incubated for an additional 10 min to reduce nonspecific binding. The bound and free ligands were separated by centrifugation in a Beckman 12 microfuge. The tip of the microfuge tubes containing the cell pellet was cut off and the bound radioactivity determined by scintillation counting (Wallac). Data was analyzed using GraphPad Prism.

Electrophysiology
HEK293 cells transiently expressing the TRPV1 channels were maintained at 37ºC in a 5% CO 2 atmosphere. Whole-cell membrane currents were recorded using the whole-cell patchclamp technique (26). The external calcium-free recording solution contained 140mM NaCl, 5mM potential at -60 mV. A "sewer-pipe" perfusion system (Rapid Solution Changer model RSC-200, Bio-Logic Science Instrument SA, France) was used to apply solutions directly to the cell under study. Capsaicin and Ruthenium Red were dissolved directly into external recording solution.
Recording solutions were adjusted to the desired pH by adding HCl. Data was analyzed using pClamp v8.0 and Prism v3.02 (GraphPad Software Inc., USA) software.

Molecular Modeling
Molecular modeling was carried out using Insight II (2000) software (Accelrys Inc., USA).
Transmembrane helices and connecting segments were modeled using the Biopolymer module of Insight II (2000). RTX and capsaicin structures were generated and minimized using Insight II tools.

Results
All studies of mutant TRPV1 function were conducted using transient transfections in HEK293 cells. Transient transfections of HEK 293 cells followed by immunohistochemical staining for TRPV1 protein indicated that all TRPV1 cDNA's studied in this report appeared to be expressed. However, this technique did not allow for quantitative analysis of the number of functional channels expressed on the cell surface and as such all of the data presented here are discussed as relative activity. Interpretation of 45 Ca 2+ uptake assays utilizing different TRPV1 mutants assumed that all have the same permeability to calcium ions. oTRPV1 gain of function mutants were also characterized by stable expression in CHO cells.

oTRPV1 is less sensitive to capsaicin activation than rat TRPV1
To determine if oTRPV1 is indeed less sensitive to vanilloids than other species, oTRPV1 was cloned from a bacterial colony screen of a rabbit DRG cDNA library utilizing hybridization with a radiolabeled 32 P-rTRPV1 probe. A cDNA clone (2.4kb) was identified; whose predicted protein sequence had high homology to rTRPV1 (86% identity and 91% similarity) and hTRPV1 (87% identity and 92% similarity; Table 1; Figure 1A). In situ hybridization of rabbit dorsal root ganglia (DRG) sections with a probe generated from the oTRPV1 cDNA revealed strong labeling by guest on July 8, 2020 http://www.jbc.org/ Downloaded from of cells in the DRG ( Figure 1B) with expression restricted to the small and medium diameter cell bodies consistent with that seen in other species. Studies showed that conditions capable of robustly activating rTRPV1 had a mixed effect on oTRPV1. Whereas oTRPV1 was activated by heat (45°C) or pH 5 similar to rTRPV1, it was not activated by capsaicin at supra-maximal activation concentrations for rTRPV1 ( Figure 1C). Further, oTRPV1 transfected HEK293 cells did not show any specific 3 H-RTX binding, whereas rTRPV1 transfected cells showed specific binding with K D value of 0.089 + 0.01 nM.
Low pH elicited a much smaller current of 1.02 + 0.93 pA/pF (n=6 cells) in mock-transfected HEK293 cells, which indicated that the proton-activated current in (rat and rabbit) TRPV1transfected cells was primarily mediated by TRPV1. Large currents were also observed in response to 1 and 10µM capsaicin in rTRPV1-transfected cells. In contrast, 1 µM capsaicin failed to generate any current in oTRPV1-transfected cells, although 10 µM evoked a small current ( Figure 1D). These experiments confirmed that oTRPV1 was functionally expressed in HEK293 cells and that oTRPV1 was much less sensitive to activation by capsaicin than rTRPV1.
However, the small current elicited with 10 µM capsaicin suggested a rudimentary capsaicin-site in oTRPV1. The ability of ruthenium red to block oTRPV1 was also tested in patch-clamp studies. Ruthenium red (10µM) application 10s after pH 5 activation blocked 83.4 + 8.2% of the oTRPV1 current (n=5 cells; Figure 1E). These data verified that proton activation of oTRPV1 is sensitive to pore blockade similar to rTRPV1. In summary, sequence similarities to TRPV1s from other species, the activation profile of oTRPV1 by proton and heat, blockade of the proton and heat responses by ruthenium red, and the expression pattern of oTRPV1 mRNA in rabbit dorsal root ganglia confirm that oTRPV1 is the rabbit orthologue of TRPV1. The limited sensitivity of oTRPV1 to capsaicin and RTX and lack of detectable [

Residue 550 is an important determinant for vanilloid sensitivity in oTRPV1
A rat-rabbit chimera (r/o chimera) of TRPV1 was constructed by transfer of transmembrane domains 3 through 4 (amino acids S505 to T550) from rTRPV1 to oTRPV1, because Jordt and Julius (15) previously showed that the TM3/4 region of TRPV1 appears to be responsible for capsaicin sensitivity. Functional analysis of transiently transfected cells by 45 Ca 2+ uptake showed that the r/o chimera gained sensitivity to vanilloids (EC 50 for capsaicin: 0.051 + 0.029 µM and RTX: 11 + 5 nM) similar to rTRPV1 ( Figure 2A). Sensitivity of the r/o chimera to capsaicin was also characterized by electrophysiology. Currents evoked by pH 5 and 1 µM capsaicin were similar in the chimera and rTRPV1 ( Figure 1D, 2A, bottom panel). In addition, we also made a human-rabbit chimera (h/o chimera) transferring the S505-T550 from hTRPV1 to oTRPV1. Similar to r/o chimera, functional analysis showed that h/o chimera gained sensitivity to capsaicin (Figure 2A), further confirming that the TM3/4 region is responsible for vanilloid sensitivity.
Amino acid sequence alignment of the 505-550 region indicated that ten amino acids are different between rat and rabbit TRPV1 and six amino acids are different between human and rabbit TRPV1 ( Figure 2C). To determine which residues within this region were responsible for gain of functional sensitivity to vanilloids in oTRPV1, we mutated the residues that are different in rabbit from both rat and human TRPV1 (A505S, A520S, C534R, T540S and I550T). Remarkably, changing the single residue at 550 in rabbit to the corresponding residue found in rat and human TRPV1 (I550T) was sufficient to confer gain of function for activation by capsaicin ( Figure 2B).
Dose response curves in 45 Ca 2+ -uptake experiments indicated that the EC 50 of capsaicin at the oTRPV1 channel was 14.8 + 7.9 µM whereas it was 0.016 + 0.006  µM for rTRPV1, 0.051 + 0.029  µM for the r/o chimera (described above), and 0.052 + 0.034  µM for oTRPV1-I550T. In addition to the above studies using transiently transfected HEK293 cells, we have also verified oTRPV1-I550T sensitivity to vanilloids in stably expressing CHO cells. Vector transfected HEK293 or parental CHO cells did not show significant [PPAHV], NADA and OLDA) were inactive up to 40 µM at wild type oTRPV1, but functioned as potent agonists at oTRPV1-I550T (Table 2)

T550 is an important determinant for vanilloid sensitivity in rat and human TRPV1
To further verify that the T550 found in native rat and human TRPV1 contributes to vanilloid sensitivity of TRPV1, we conducted a loss of function study by substituting the natural threonine with the oTRPV1 isoleucine-550 residue. Dose response curves in 45 Ca 2+ -uptake experiments demonstrated that the EC 50 of capsaicin at the mutant rTRPV1-T550I is shifted to 0.608 + 0.032  µM from 0.057 + 0.014 µM at wild type rTRPV1 channel, about a ten fold loss in sensitivity ( Figure 3A). EC 50 of capsaicin at the mutant hTRPV1-T550I is shifted to 4.58 + 0.6 µM from 0.12 + 0.05 µM at wild type hTRPV1 channel, approximately a 40-fold loss in sensitivity by guest on July 8, 2020 http://www.jbc.org/ Downloaded from ( Figure 3A). Patch clamp recordings also confirmed loss of capsaicin sensitivity in rTRPV1-T550I and hTRPV1-T550I; 1 µM capsaicin failed to generate any current in TRPV1-T550Itransfected cells, although 10 µM did evoke currents ( Figure 3B). Protons (pH 5) still evoked large currents in TRPV1-T550I-transfected cells indicating that only capsaicin sensitivity was reduced. Gain of vanilloid sensitivity with I550T mutation in oTRPV1 and loss with a reverse mutation in rat and human TRPV1-T550I strongly suggests that T550 is one of the critical molecular determinants for TRPV1 activation by vanilloids.
Based on their mutational data, Jordt and Julius (15) reported that Y511 is critical for vanilloid sensitivity. Y511 is conserved in TRPV1 from all species reported to date. We have verified that the Y511 is critical for vanilloid sensitivity; rTRPV1-Y511A and hTRPV1-Y511A lost vanilloid sensitivity as shown by electrophysiology ( Figure 3B). In contrast to rTRPV1, 1 or 10 µM capsaicin failed to elicit any current, whereas pH 5 evoked currents were similar to rTRPV1 ( Figure 1D and 3B). In addition, we have tested capsaicin sensitivity of oTRPV1 double mutant containing I550T (gain of function) and Y511A (loss of function), i.e., oTRPV1-Y511A-I550T.
Compared to oTRPV1-I550T, the reduction in capsaicin sensitivity of oTRPV1-Y511A-I550T in 45 Ca 2+ uptake assay is >100 fold. In fact the magnitude in loss of capsaicin sensitivity by Y511A is greater than the gain seen in I550T as represented by the rightward shift of the oTRPV1-Y511A-I550T capsaicin dose response curve beyond the wild type oTRPV1 ( Figure 3A). These studies confirm that Y511 is indeed an important molecular determinant for vanilloid sensitivity of TRPV1.
Interestingly, mutation of rat T550 to the corresponding rabbit I550 (T550I) resulted in the capsaicin dose response curve shifting ten fold to the right whereas it did not appear to reduce RTX sensitivity in the 45 Ca 2+ uptake assay (EC 50 values for rTRPV1 and rTRPV1-T550I are 1.5 + 1.06 nM and 0.93 + 0.69 nM respectively; Figure 3C). However, 3 H-RTX specific binding was significantly reduced in rTRPV1-T550I transfected cells ( Figure 3D). For the first time, we report a difference in the molecular determinants for the functional responses of TRPV1 to capsaicin and RTX. In oTRPV1, which is an insensitive species, replacement of I550 with T550 contributes to both capsaicin and RTX sensitivity (functional responses), whereas replacement of T550 in rat Discrepancy between RTX responses in functional and binding assays has been reported previously (27,28). As discussed elsewhere in detail (29), an emerging plausible explanation is that most of the TRPV1 is internal and only a small proportion is localized to the plasma membrane. The minor subpopulation of TRPV1 at the plasma membrane mediates the 45 Ca 2+ uptake measurements, whereas the binding analysis is dominated by the predominant, internal TRPV1, and these two populations of TRPV1 display different structure-activity relations, presumably reflecting differential modification. Our results described here demonstrate differences in the receptor requirements for RTX in these two assays and suggest the importance of additional residues in TRPV1 affinity for 3 H-RTX. Consequently, to explore the remaining residues that are different between rat and rabbit, we introduced a series of single point mutations into oTRPV1 (M514I, A525V, T526S, H533Q, and L547M) to mimic the residues in rat TRPV1, which has been shown to display the highest RTX binding affinity.

M547 in TRPV1 contributes to RTX affinity as detected by ligand binding
Replacement of oTRPV1 residues individually at M514, A525, T526, and H533 to corresponding residues in rTRPV1 (M514I, A525V, T526S, and H533Q) did not alter the oTRPV1 response to capsaicin or RTX (data not shown). Interestingly, the single residue change L547M in oTRPV1 resulted in a selective gain of approximately 30 fold higher sensitivity to RTX with no apparent change in capsaicin sensitivity in 45 Ca 2+ -uptake assays ( Figure 4A,B H-RTX binding ( Figure 4C). We hypothesized that L547M contributes to RTX sensitivity but requires additional residues such as T550 for sufficient affinity needed for measurable 3 H-RTX binding above the assay background.
To investigate whether M547 contributes to RTX affinity in rTRPV1, a reverse mutation was made (M547L). Wild type rTRPV1 and rTRPV1-M547L showed similar responses to capsaicin and RTX in the functional 45 Ca 2+ -uptake assay ( Figure 4D,E); EC 50 values at rTRPV1 and rTRPV1-M547L were 1.5 + 0.7 nM and 1.7 + 0.8 nM for RTX and 0.013 + 0.002 µM and 0.012 + 0.002 µM for capsaicin, respectively. However, the rTRPV1-M547L mutant showed significantly reduced 3 H-RTX binding compared to wild type rTRPV1, demonstrating a discrepancy between functional assays and binding once again ( Figure 4F) and indicates that M547 indeed contributes to RTX affinity in the binding assays.

Both M547 and T550 are required for measurable 3 H-RTX binding in oTRPV1
The oTRPV1-L547M mutant showed an increase in sensitivity to RTX but not to capsaicin in the functional 45 Ca 2+ -uptake assay, while a single I550T mutation resulted in gain of oTRPV1 sensitivity to both vanilloids. However, we were unable to measure 3 H-RTX specific binding in cells expressing either one of these single mutants ( Figure 3D, Figure 5B and Table   2). This demonstrates that M547 and T550, as present in native rTRPV1, are required for measurable 3 H-RTX binding in oTRPV1.
None of the oTRPV1 gain of function mutations (I550T, L547M, and L547M-I550T) displayed significantly altered responses to protons or heat compared to native oTRPV1 ( Figure   5C,D). In agreement with previous literature reports, rTRPV1 was activated by pH 5.5 and below. oTRPV1 showed strong activation at pH 5 and below, slightly different from rat TRPV1.
Changing the residues in oTRPV1 to the corresponding residues in rTRPV1 (I550T and L547M-I550T) resulted in a slight change in sensitivity to pH 5.5. Heat (45°C) induced 45 Ca 2+ uptake by oTRPV1 gain of function mutants did not demonstrate any significant variations from that of the wild type channel ( Figure 5D). These results verify that oTRPV1 and the various mutant proteins were expressed and were able to integrate multiple noxious stimuli despite their differential sensitivity to vanilloids. Of note, PPAHV (a full agonist at rat but not human TRPV1, 18) requires a double mutation of L547M-I550T in oTRPV1 for agonist activity to be observed (Table 2). oTRPV1-I550T

Gain of capsaicin sensitivity parallels sensitivity to other agonists
is similar to hTRPV1 (L547) and no activation by PPAHV is seen, whereas oTRPV1-L547M-I550T is similar to rTRPV1 (M547) and activation is detected, indicating that PPAHV agonism requires both M547 and T550. This suggests that M547 is an important determinant for RTX and RTX-like molecules, which also points out the differences between capsaicinoid and resiniferanoid-like molecules.

Vanilloid sensitivity also confers competitive antagonism
To further explore the pharmacology of oTRPV1 and its vanilloid sensitive mutants we have looked at the ability of a number of different antagonists to block various modes of activation of these channels. A Ca 2+ channel pore blocker ruthenium red blocked pH 5 and capsaicin activation of rTRPV1, hTRPV1, oTRPV1, oTRPV1-I550T and oTRPV1-L547M-I550T with IC 50 values of ~1 µM (Table 3). Since oTRPV1 is not sensitive to capsaicin and oTRPV1-I550T and oTRPV1-L547M-I550T mutants are, we were curious to see if recently reported TRPV1 antagonists inhibit proton activation of wild type oTRPV1, as well as proton and capsaicin activation of its mutants. We chose antagonists that were reported to block both capsaicin and proton activation of human and rat TRPV1, i.e. BCTC and Iodo-RTX (29-31) as well as capsazepine, reported to block human, but not rat TRPV1 responses to low pH (18). of competitive antagonists such as BCTC and Iodo-RTX ( Figure 6 and Table 2).  (Table 3). In summary, BCTC and Iodo-RTX IC 50 values were similar for rat, human and vanilloid-sensitive mutants of rabbit TRPV1 activated with capsaicin or proton. These results clearly demonstrate that T550 is not only critical for vanilloid agonist sensitivity but also for the ability of competitive antagonists to block various modes of TRPV1 channel activation.

As in previous reports
While capsazepine antagonism of capsaicin was similar at both rat, human and vanilloidsensitive oTRPV1 mutants (I550T, L547M-I550T), its antagonism of proton (pH 5) activation indicated an interesting pharmacological difference. Presence of L547 seems to be essential with regard to capsazepine antagonism at proton activation. IC 50 values for capsazepine were similar at proton activated oTRPV1-I550T and hTRPV1 (0.079 + 0.024 and 0.069 + 0.01 µM respectively, Table 3), but capsazepine was inactive up to 40 µM against proton activated oTRPV1-L547M-I550T and native rTRPV1. Both oTRPV1-I550T and hTRPV1 have leucine at position 547 whereas oTRPV1-L547M-I550T and rTRPV1 have methionine. Based on these results we propose that L547 is required for capsazepine antagonism of proton activation of TRPV1, although it is possible that additional residues common to human and rabbit TRPV1 within S505-T550 region (M514, A525, T526 and H533) might also contribute to capsazepine antagonism of proton activated TRPV1.

Discussion
It has been reported that membranes derived from rabbit dorsal root ganglia (DRG) lack 3 H-RTX binding in contrast to many species. In an attempt to determine the structural requirements for agonism and antagonism of TRPV1 we cloned and characterized a cDNA from a rabbit DRG cDNA library. The cDNA reported here appears to be an orthologue of human  (20), an alternative model could be proposed by having vanilloid moiety interaction with T550 and the "tail end" hydrophobic group of capsaicin or RTX interacting with Y511 ( Figure 7). Thus, in the present model, Y511 engages in hydrophobic interaction and partly accounts for the observed differences in affinity between ligands owing to the differences in the hydrophobic tails of these molecules i.e., molecules with short aliphatic chain such as zingerone may not interact well with Y511 and hence are weak agonists of TRPV1.
On the other hand, molecules with long aliphatic chains such as arvanil and olvanil may interact with Y511 and other hydrophobic side chains accounts for their strong agonist activity. Vanillyl moiety (methoxy phenol) is common for capsaicin, RTX , arvanil, olvanil, and zingerone; it is reasonable to assume that this part interacts with side chain hydroxyl group of T550. Notably In rabbit TRPV1, mutation of L547 to the corresponding rat M547 resulted in a selective 30-fold higher sensitivity to RTX without a detectable increase in capsaicin sensitivity. RTX is essentially a vanilloid whose marked enhancement of potency, relative to capsaicin, is thought to be brought about by the resiniferanol moiety known as a C-region. The results of the L547M mutation in oTRPV1 imply that perhaps this residue contributes to a structural conformation that is favorable for interaction with RTX. Interestingly, the mutation of the rabbit TRPV1 I550 to T550 markedly enhanced the response to both capsaicin and RTX.
It has become clear from many studies that the measurement of binding to TRPV1, as determined by competition of 3 H-RTX binding, and of calcium uptake reveals distinct structure activity relationships (28). TRPV1 is predominantly expressed at internal membranes in the cell, with only a small fraction at the plasma membrane where it can regulate calcium influx. An attractive explanation for the disparity in structure activity relationships is that the TRPV1 channels present internally are different from those at the plasma membrane, presumably reflecting its differential modulation, whether by phosphorylation (35)(36)(37), interaction with PIP 2 (38), sub-unit conformation or association with other proteins (39). Pharmacological evidence for this assumption was the identification of a vanilloid antagonist that competitively blocked 45 Ca 2+ uptake in response to capsaicin or RTX with high potency (K i of approximately 100 nM) but which inhibited 3 H-RTX binding less than 10% at 30 µM (29). This antagonist likewise was unable to block the release of calcium from internal stores by RTX. The mutational studies presented here complement such pharmacological observations, showing once again partial uncoupling between the assays for 45 human TRPV1, which lacks M547, has about 25-fold lower affinity for 3 H-RTX binding compared to rTRPV1 (28).
We also studied the properties of several published antagonists, of the native and heterologous TRPV1 channels, to the known activators. We and others have shown that BCTC and Iodo-RTX are potent antagonists of capsaicin and proton activation of rat and human TRPV1 (30,32). However, BCTC and Iodo-RTX are ineffective antagonists of oTRPV1 responses to protons (pH 5). Because BCTC and Iodo-RTX are in fact potent antagonists of oTRPV1 gain of function mutants (I550T and L547M-I550T), we believe that T550 is also a critical determinant for competitive antagonist binding. We speculate that Iodo-RTX could inhibit TRPV1 via an additional hydrophobic interaction involving the Iodo group. Overlay of capsazepine with the binding model of capsaicin shows that the polar dihydroxy phenyl part of the former will have similar interactions as the vanillyl moiety of the latter.
Clearly, the ability of capsaicin-site antagonists to block all modes of TRPV1 activation and identification of some of the key molecular determinants (Y511, M547, and T550) confirm this site as a key regulatory site on TRPV1 and may help in designing new antagonists with predictable structure activity relationship. Given reports of the involvement of TRPV1 in inflammatory pain and other sensory neuronal disorders (8,9), antagonists of hTRPV1 may prove useful in the treatment of human diseases such as arthritis, bladder cystitis, and IBS.      Table 2.   Table 3.

Table 3 Inhibition of capsaicin and proton induced activation of TRPV1
Selected antagonists were tested in CHO cells stably expressing rTRPV1, oTRPV1, oTRPV1-L547M, oTRPV1-I550T, or oTRPV1-L547M-I550M. Cells were activated by 0.5µM capsaicin or pH 5.0. IC 50 value for each antagonist was determined using Prism software and is expressed in µM.