Divalent Heavy Metal Cations Block the TRPV1 Ca2+ Channel

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel involved in pain sensation and in a wide range of non-pain-related physiological and pathological conditions. The aim of the present study was to explore the effects of selected heavy metal cations on the function of TRPV1. The cations ranked in the following sequence of pore-blocking activity: Co2+ [half-maximal inhibitory concentration (IC50) = 13 μM] > Cd2+ (IC50 = 38 μM) > Ni2+ (IC50 = 62 μM) > Cu2+ (IC50 = 200 μM). Zn2+ proved to be a weak (IC50 = 27 μM) and only partial inhibitor of the channel function, whereas Mg2+, Mn2+ and La3+ did not exhibit any substantial effect. Co2+, the most potent channel blocker, was able not only to compete with Ca2+ but also to pass with it through the open channel of TRPV1. In response to heat activation or vanilloid treatment, Co2+ accumulation was verified in TRPV1-transfected cell lines and in the TRPV1+ dorsal root ganglion neurons. The inhibitory effect was also demonstrated in vivo. Co2+ applied together with vanilloid agonists attenuated the nocifensive eye wipe response in mice. Different rat TRPV1 pore point mutants (Y627W, N628W, D646N and E651W) were created that can validate the binding site of previously used channel blockers in agonist-evoked 45Ca2+ influx assays in cells expressing TRPV1. The IC50 of Co2+ on these point mutants were determined to be reasonably comparable to those on the wild type, which suggests that divalent cations passing through the TRPV1 channel use the same negatively charged amino acids as Ca2+.


Introduction
Small-diameter sensory neurons in the peripheral nervous system (PNS) express the transient receptor potential/vanilloid receptor subtype 1 (TRPV1). Four identical subunits of this protein form a functional Ca 2+ channel. Similarly to other transient receptor potential channel members, the TRPV1 channel belongs in the large superfamily of cation channels with six transmembrane (TM) segments. Following agonistinduced channel opening, a pore loop between segments TM5 and TM6 serves as a cation filter and entry site [1].
The vanilloid binding site is localized between segments TM3 and TM4 of TRPV1 [5]. The pain signal, however, is generated by opening of the Ca 2+ channel situated between domains TM5 and TM6 of four identical subunits [11,12]. An acidic peptide motif in the pore loop region of TRPV1 (DXEXXEXXD) may serve as a docking site for positively charged ions and channel blockers [13]. However, our sequence comparison and previous in silico modelbuilding efforts have not revealed any obvious homology to other divalent metal ion (M 2+ )-binding structures such as the EF-hand (DXDXDGXXDXXE) or the Excalibur (DXDXDXXXCE) [13].
Various studies have demonstrated that positively charged molecules can act as TRPV1 receptor channel blockers by plugging the pore. Ruthenium Red (RuRed) (a well-known, but non-specific TRPV1 pore blocker) [11], R 4 W 2 (a positively charged hexapeptide) [14] and anti-calmodulins/antipsychotic tricyclics [13] have been shown to be able to bind to the DXEXXEXXD domain of TRPV1, whereby they block the movement of Ca 2+ through the pore region.
We set out to assess the effects of various metal cations at different concentrations on the vanilloid -or heat-induced activity of the TRPV1 channel, focusing on the investigation of the most potent cations in vitro and in vivo. Moreover, in our experiments we aimed to shed light on the characteristics of the gating of the TRPV1 channel in order to improve the understanding of the structure and function of the TRPV1 pore region, which may lead to the development of potentially useful painkiller drugs that modulate the activity of this receptor.

Materials and Methods
Reagents Stock solutions (200 mM) of CoCl 2 , NiCl 2 , ZnSO 4 , CdCl 2 , CuSO 4 , CaCl 2 , CoCl 2 and LaCl 3 were dissolved in water and diluted as required to the working concentrations. To avoid the precipitation of insoluble La(OH) 3 and La (CO 3 ) 3 , the formation of radiocolloids and the loss of La 3+ by adsorption to container surfaces, LaCl 3 solution was prepared fresh daily in polyethylene vials [15]. RuRed and capsazepine (CapZ; Sigma-Aldrich, St. Louis, MO) were dissolved in DMSO. CAPS was dissolved initially as a stock solution of 3 mM in 95 % ethanol. The peptide R 4 W 2 was synthetized in our laboratories and then dissolved in water and used as a 25 mM stock solution. Amitriptyline (AMI), purchased from Sigma-Aldrich, was dissolved in water.
Plasmids The C-terminally epsilon-tagged rat TRPV1ε plasmid construct was prepared in the metallothionein (pMTH) plasmid vector as described earlier [9]. To avoid cell loss through the Ca 2+ -excytotoxic mechanism that occurs when TRPV1 is overexpressed at 37°C, only the basal activity of the pMTH promoter was used. The protein kinase C ε epitope tag allowed immunological detection of the TRPV1ε protein, as earlier described [16]. Mutants Y627W, N628W, D646N and E651W were kindly given by Dr. K. J. Swartz (National Institutes of Health, Bethesda, MD 20892, USA) [17] and subcloned into an EF-promoterdriven green fluorescent protein (EGFP)-tagging plasmid vector. The EGFP tag was used for visual determination of the transfection rate by flow cytofluorometry with a FACS-Calibur instrument (Becton Dickinson, San Jose, CA, USA).
Cell Lines Expressing TRPV1 Ectopically The HaCaT keratinocyte cell line was a kind gift of Prof. B. Farkas (Department of Dermatology, University of Cologne, Federal Republic of Germany) [18]. The COS-7 (CRL-1651) and BALB/c-3T3 (CCL-163) cell lines were obtained from ATCC. The 3T3 and HaCaT cell lines permanently expressing the rat TRPV1 channel were prepared as described earlier [19]. COS7 cells were transiently transfected with plasmid containing the sequence of the Y627W, N628W, D646N or E651W TRPV1 mutants or the wild-type TRPV1 channel, by using the Fugene transfection reagent (Roche, Mannheim, Germany). The transfection efficacy was determined by flow cytofluorometry.
Primary Dorsal Root Ganglion Cultures Were prepared from E16 embryonic rats as reported earlier [9]. Briefly, dorsal root ganglions (DRGs) were dissected and then processed in Hank's balanced salt buffer until plated in Dulbecco's Modified Eagle Medium (DMEM). The DMEM contained 20 mM HEPES, pH 7.4, 7.5 % foetal bovine serum, 7.5 % horse serum, 5 mg/ml uridine supplemented with 2 mg/ml 5-fluoro-2′-deoxyuridine and 40 ng/ml nerve growth factor to inhibit cell division and to promote the differentiation of long neuronal processes, respectively. Cells were seeded on 25 mm glass coverslips.
Cobalt Histochemistry Rat DRG cells attached to the coverslips were washed in buffer A (in millimolars: NaCl, 57.5; KCl, 5; MgCl 2 , 2; HEPES, 10; glucose, 12; sucrose, 139; pH 7.4) for 2 min, and then incubated at 37°C for 10 min in Co 2+ -uptake solution (buffer A+5 mM CoCl 2 ) containing 20 μM CAPS. High (20 μM) capsaicin concentration is used in order to obtain a robust and easily detectable Co 2+ signal. Following a brief wash in buffer A, the water-soluble Co 2+ taken up by the cells was precipitated with 0.12 % ammonium polysulphide (Sigma-Aldrich) in buffer A, which resulted in the formation of dark, water-insoluble CoS in TRPV1+ cells. Cells were fixed in 4 % formaldehyde and mounted on glass slides, using Kaiser's glycerol gelatine (Merck, Darmstadt, FRG). Cells were examined under a Nikon light microscope (Melville, NY, USA) and photographed with a SPOT RT-SE™ Digital Camera (Diagnostic Instruments). Pictures of the cells were analysed by means of ImageJ 1.45 s software (National Institutes of Health, USA), and the subsequent statistical analyses were performed with PRISM™ 3.01 software (GraphPad Software, Inc. San Diego, CA, USA).
Vanilloid-Induced 45 Ca 2+ Uptake Vanilloid-induced 45 Ca 2+ transport was assayed in the HaCaT adherent cell lines ectopically expressing the C-terminally ε-tagged rat TRPV1 (3×10 4 cells/well) and Cos7 cells transiently transfected with rat TRPV1 mutants, seeded in poly-D lysine-coated 96-well plates. Immediately before the transport assay, the cells were washed three times with physiological saline solution at room temperature (20-25°C). We had previously found that TRPV1-transformed cells functioned in the same manner at room temperature as at 30°C (data not shown). 45 Ca 2+ uptake was performed for 10 min with 0.1 μCi 45 Ca 2+ as radioactive tracer in a final volume of 100 μl (1.8 μM). CAPS was diluted from a 3 mM ethanol stock solution to the indicated final concentrations. For the termination of 45 Ca 2+ uptake, cells were rapidly washed four additional times with 0.2 ml PBS solution, and then lysed in 80 μl/well RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 % Triton X-100, 0.5 % deoxycholate, 0.1 % SDS and 5 mM EDTA) for 30 min [8,9,20,21]. Aliquots of the solubilised cell extracts were mixed with 120 μl SuperMiX and counted in a 96-well plate liquid scintillation counter (TopCount-NXT, Packard). To measure the effect of temperature on the TRPV1 function in the 45 Ca 2+uptake assay, TRPV1/HaCaT cells were plated on six-well plates. 45 Ca 2+ uptake was evoked with 10 ml preheated buffer. The procedure was followed as described above.
After the measurement, the data were corrected for the basal activity of TRPV1 and normalized from zero to one, where zero denotes the counts per minute in TRPV1/HaCaT cells without CAPS and one denotes the counts per minute in TRPV1/HaCaT cells with CAPS. The results of three parallel measurements were averaged and evaluated with PRISM™ 3.01 software (GraphPad Software, Inc. San Diego, CA, USA). During curve fitting, the "Analyze/Nonlinear regression (curve fit)/Sigmoidal dose-response" menu of PRISM™ software was applied. The curve-fitting equation was: Y 0Y min + (Y max − Y min )/(1 + 10 (logEC50-X) ), where X0logarithm of concentration and Y0the response.
Eye Wipe Tests Eye wipe tests were performed on CD1 mice. A 100 μM CAPS solution, or a solution containing 100 μM CAPS and 1 mM CoCl 2 , or a solution containing 100 μM CAPS and 5 μM CapZ was dropped into the eye, and the number of defensive wiping movements was then counted.
Statistical Analysis One-way ANOVA followed by Turkey's post-tests was performed with GraphPad Prism version 3.01 software (GraphPad Software, Inc. San Diego, CA, USA).

Results
In the 45 Ca 2+ uptake assay, the EC 50 of CAPS for wild-type TRPV1 was determined to be 0.0860 μM. Approximately 1 μM CAPS caused the full activation (EC 100 ) of TRPV1 at pH 7.5. Channel blocker-screening assays were therefore carried out with 2 μM CAPS (an excess amount of agonist), which does not cause Ca 2+ cytotoxicity during the 10-min incubation period. The interactions of the metal ions with TRPV1 were studied by using a vanilloid-induced 45 Ca 2+uptake assay. Experiments were carried out on the TRPV1/ HaCaT permanent indicator cell line. Channel opening was induced by CAPS in the presence of progressively increasing M 2+ concentrations in the uptake solution. Incubation of the cells in uptake solutions containing both 45 Ca 2+ and Mg 2+ , Mn 2+ or La 3+ (data not shown) resulted in little or no effect, even at the highest concentration (4 mM), whereas Zn 2+ proved to be a weak [half-maximal inhibitory concentrations (IC 50 )027 μM] and only partial inhibitor of the 2 μM CAPS-induced 45 Ca 2+ uptake. The other cations effectively blocked the vanilloid-induced Ca 2+ entry into TRPV1/HaCaT cells, with the following sequence of potency: Co 2+ (IC 50 013 μM) > Cd 2+ (IC 50 038 μM) > Ni 2+ (IC 50 062 μM)>Cu 2+ (IC 50 0200 μM; Fig. 1a).
To assess the effect of Co 2+ , the most potent TRPV1 inhibitor, on the heat-activated TRPV1 channels, the activity of TRPV1 was investigated in the presence either of 2 μM CAPS alone or of 2 μM CAPS+250 μM Co 2+ , at both 37 and 42°C. The negative control did not contain CAPS. In this assay, high temperature activated the TRPV1 channels and also increased the CAPS-evoked 45 Ca 2+ influx. Co 2+ reduced both the heat and CAPS-induced 45 Ca 2+ influx (Fig. 1b).
To compare the potency of Co 2+ with those of the other positively charged channel blockers, we measured the IC 50 values of RuRed, AMI and R 4 W 2 , which are known to have a docking site in the pore loop of TRPV1. The inhibitor potentials of these pore blockers were measured via the CAPS-induced 45 Ca 2+ uptake. All of them inhibited CAPS-activated TRPV1, with the following IC 50 values: RuRed01 μM, AMI020 μM and R 4 W 2 0100 μM (Fig. 1c).
For a better understanding of the inhibition kinetics of Co 2+ on TRPV1, increasing concentrations of both Co 2+ and CAPS were applied in the vanilloid-induced 45 Ca 2+ -uptake assays. The Ca 2+ uptake of TRPV1/HaCaT cells was inhibited by the simultaneous presence of Co 2+ in a dosedependent manner. However, increasing concentrations of Co 2+ decreased only the maximal response of efficacy (E max ) of Ca 2+ entry; the affinity of CAPS for TRPV1 did not change. The inflection point in the CAPS dose-response curves in each of the Co 2+ co-incubation studies was found at ∼0.08 μM (i.e. EC 50 ), independently of the Co 2+ concentration. The Co 2+ inhibition patterns unequivocally indicated channel blocking kinetics (Fig. 2a).
By varying the concentrations of Co 2+ and Ca 2+ and measuring the radioactive 45 Ca 2+ influx, we assessed whether there was a competition between Co 2+ and Ca 2+ . The effect of dilution on the amount of accumulated 45 Ca 2+ did not appear at extracellular cold Ca 2+ concentrations below 1 mM (Fig. 1a) , showing that the effect of Co 2+ on Ca 2+ entry mainly depends on the competition for entry sites. These results suggest that the Co 2+ entry through the TRPV1 channel is slower, and the Co 2+ displacing the Ca 2+ from the pore region of TRPV1 slows down or inhibits the Ca 2+ uptake (Fig. 2b).
The prolonged agonist stimulation of TRPV1 has been reported to result in an increased permeability to larger cations [22] or small molecules [23], due to conformational changes in the open state of the TRPV1. Thus, we analysed the kinetics of the channel-blocking activity of Co 2+ by employing different CAPS concentrations. An anticipated shift in the IC 50 of Co 2+ would be evidence supporting the idea that Co 2+ entry depends on the TRPV1 open stages. We indeed observed a shift in the IC 50 of Co 2+ , which decreased with increasing CAPS concentration (Fig. 2c). Consequently, increasing agonist concentration enhances the blocking ability of Co 2+ . To investigate this phenomenon, we plotted the IC 50 values as a function of CAPS concentration. Curvefitting analysis confirmed a strong interrelationship between IC 50 and the CAPS dose applied (Fig. 2d), suggesting that the increased efficiency of inhibition correlates with the different open-state conformations of the TRPV1 channel.
We traced Co 2+ upon vanilloid induction in sensory neuron cultures prepared from DRGs of rat embryos. To test Co 2+ -accumulation patterns, cells were co-incubated with 20 μM CAPS in Co 2+ -containing Ca 2+ -uptake medium, and the Co 2+ was then localized by means of NH 4 S histochemistry. These experiments revealed that Co 2+ not only competes with Ca 2+ but also enters into the cytosol of specific PNS sensory neurons. Functionally responsive vanilloid-sensitive neurons (i.e. TRPV1 + ) exhibited darkbrown Co 2+ precipitates inside the rounded neuronal bodies (Fig. 3e). As expected from previous studies, TRPV1 is endogenously expressed in approximately one third of the cultured neurons [24][25][26][27]. Without CAPS, no intracellular Co 2+ accumulation was observed (data not shown). Similar experiments were carried out on rTRPV1/HaCaT and rTRPV1/3T3 cell lines. The accumulation of Co 2+ was blocked by RuRed, a channel blocker of heat and vanilloid pain signalling. Moreover, the dose-dependent inhibition of the cellular entry of Co 2+ was determined by the coapplication of 5 μM CapZ, a long-known competitive antagonist of pungent vanilloids. After analysis of the photographs of the cells with the ImageJ program, statistical analysis of the data further confirmed our findings: the mean gray values of CAPS-exposed, CAPS-free and CapZexposed cells proved to be significantly different (CAPS without Co 2+ , 72.05±12. 38 Fig. 3). The gray values were measured on the negatives of the images: the darker the cells, the higher the gray values. Analysis of the pictures in Fig. 4, showing TRPV1-expressing HaCaT cells, resulted in the same outcome. Following ANOVA, the groups were compared by using t tests. Each t test except that involving CAPS without Co 2+ vs. CAPS+Co 2+ +100 μM CapZ indicated a significant difference between the pairs of groups (P<0.05; Fig. 4). No substantial staining could be observed on 3T3 cells (Fig. 5a-e). Statistical analysis of the gray values of the cells indicated no significant darkening in the absence of TRPV1 in the cell membrane.
In order to rule out the possibility that Co 2+ can enter the cells through VGCCs, 3T3 cells were challenged with 50 mM extracellular KCl. These cells did not show any VGCC activity: the high extracellular KCl concentrationinduced depolarization that opens the VGCC channels did not cause 45 Ca 2+ accumulation in the 45 Ca 2+ -uptake assay.
Moreover, the VGCC blocker nisoldipine did not decrease the CAPS-induced TRPV1-mediated 45 Ca 2+ accumulation (data not shown). No Co 2+ staining was observed in the presence of 50 mM extracellular KCl (Fig. 5f-j). ANOVA indicated no significant differences among the groups (P00.9150). These results confirm that the CAPSinduced Ca 2+ and Co 2+ influx in TRPV1/3T3 cells is due exclusively to the TRPV1 channel activity.
Besides the in vitro demonstration of Co 2+ antagonism, we further validated this Co 2+ inhibition phenomenon in tests of eye wiping in response to pungent vanilloids [28]. Co 2+ again decreased the frequency of vanilloid-evoked defending movements. Inhibition experiments with CapZ cross-validated and confirmed our earlier findings (Fig. 6a).
To validate that Co 2+ inhibition is a consequence of competition with Ca 2+ for M 2+ -chelating sites in the pore loop domain, we prepared several point mutants in this region of TRPV1, and determined the channel kinetics through 45 Ca 2+uptake experiments in 3T3 cells expressing the mutant channels. Some of these mutants had been partially characterized earlier in the context of spider venom channel inhibitors [17], and these residues proved to have an important role in the binding of various previously tested channel blockers (RuRed, etc.). Mutated sites are illustrated schematically in Fig. 6b RuRed on TRPV1 mutants were therefore analysed with 4 μM CAPS. The neutralization of D646 reduced the sensitivity of TRPV1 to RuRed inhibition [11]. Our 45 Ca 2+ -influx studies on TRPV1 point mutants revealed the following IC 50 data for RuRed: IC 50 (D646N)012.8 μM>IC 50 (N628W)0 1.33 μM>IC 50 (E651W) 00.94 μM>IC 50 (wild type) 0 0.87 μM>IC 50 (Y627W)00.26 μM (Fig. 6c), which findings correspond with published results [17]. Likewise, Co 2+ inhibited the 4 μM CAPS-evoked 45 ; Fig. 6d). Based on these findings, inhibition seems to be a consequence of competition with Ca 2+ for M 2+ -chelating sites in the pore loop domain.

Discussion
Testing the effects of various metal cations on the vanilloidinduced activity of the TRPV1 channel, we demonstrated that Mg 2+ , Mn 2+ or La 3+ caused little or no decrease in It was reported by Nilius et al. [29] that Co 2+ reduced the inward Ca 2+ current through ECaC1 (TRPV5), a close relative of TRPV1, sharing around 30 % homology with it. Fast and reversible recovery of the current upon washout of the inhibitor was detected during their experiments. Furthermore, they identified other M 2+ -s as effective inhibitors of the Ca 2+ influx. Their results indicated the following overall blocking sequence: Pb 2+ 0Cu 2+ 0Gd 3+ >Cd 2+ >Zn 2+ >La 3+ > Co 2+ >Fe 2+ >>Fe 3+ . Zeng et al. found Cu 2+ to be a potent inhibitor of the whole-cell current evoked by intracellular ADP-ribose through TRPM2, another member of the TRP group. The inhibitory effect of Cu 2+ was irreversible, and occurred only if Cu 2+ was administered in outside-out patches, suggesting that the action site is located extracellularly. The TRPM2 current was also blocked by Hg 2+ , Pb 2+ , Fe 2+ and Se 2+ [30].
In accord with the above-mentioned findings, we also observed ion influx-inhibitory effects of M 2+ -s on TRP channel. Depending on the TRP channel type, differences of the orders of blocking potency could be detected. Furthermore, the blocking effects of the individual cations could be reversible or irreversible, depending on the channel type. The three channels are close relatives and share high degree of sequence and structural homology with one another, which explains the similar responses to M 2+ -s. Having diverged from a common ancestor, TRP channels operate on uniform principles. However, during evolution TRP superfamily has evolved for various specialized functions. TRPV1 and TRPV5, for example, belong to different subgroups of the TRPV family; TRPV1-4 are non-ionselective, whilst TRPV5-6 are highly Ca 2+ -selective. Hence, this functional adaptation may cause the differences in the M 2+ -evoked responses.
As concerns our own results, Co 2+ reduced not only CAPS-induced but also heat-induced 45 Ca 2+ influx. When increasing concentrations of both Co 2+ and CAPS were applied, the Co 2+ inhibition patterns indicated channelblocking kinetics. Our dose-response and Co 2+ accumulation experiments revealed a competition for binding sites and a co-entry mechanism. We presume that Co 2+ inhibits TRPV1 through its ability to bind to the ion selectivity filter of the channel: it passes through the ion channel much more slowly than Ca 2+ . Ca 2+ entry is also slowed down by the binding of Co 2+ , which occupies the appropriate amino acid residues of the ion selectivity filter. This hypothesis seems to be further supported by the findings of Sajadi [31], who determined the stability constants of the 1:1 complexes formed between M 2+ and L-tryptophan and other amino acids. The sequence obtained in the case of tryptophan was Ca 2+ <Mg 2+ <Mn 2+ <Co 2+ <Ni 2+ <Cu 2+ > Zn 2+ , which follows the Irving-Williams sequence [32]. The order of the stability constants was similar in the cases of methionine, alanine, leucine, valine and glycine. The amino acid sequence of the putative pore region is STSHRWRGPACRPPDSSYNSLYSTCLELFKFTIGMGD (Q8NER1, UniProt), containing all the tested amino acids but valine. The stability constants formed between M 2+ and tryptophan were Ca 2+ 2+ and Cu 2+ can be characterized by much higher complex-forming strength, elucidating the elevated TRPV1 blocking potency. Interestingly, for these three M 2+ -s, an unexpected relationship can be observed between the stability constant and the TRPV1 blocking potency. The stronger the bond, the weaker the TRPV1 inhibition potency is. The ionic radii (in picometres) of these cations are Ca 2+ , 100; Mg 2+ , 72; Mn 2+ , 67; Co 2+ , 65; Ni 2+ , 69; Cu 2+ , 73 and Zn 2+ , 74 [33]. Ca 2+ is likely to have the ideal ionic radius and stability constant in its reactions with amino acids in order to be effectively passed along the carbonyl groups of the peptide backbone in the ion selectivity filter and the pore loop. Co 2+ has a medium stability constant and the smallest ionic radius, which is probably not adequate for efficient transport. These two parameters seem to be equally involved in the proper ion influx. The stronger the M 2+ -amino acid complex and the smaller the ionic radius is, the more probable it is that M 2+ will block the Ca 2+ influx through the TRPV1 channel. As another interesting finding, in our experiments, Zn 2+ seemed to be only weak and partial inhibitor of the ion current. Its stability constant is almost as high as that of Co 2+ , suggesting a strong TRPV1-blocking ability, whereas its ionic radius is much larger than that of Co 2+ . Interestingly, the IC 50 of Zn 2+ is the second lowest exceeding only that of Co 2+ , but Zn 2+ can achieve a decrease of merely 30 % of the maximal ion influx.
The TRPV1 channel is a non-selective cation channel, and still shows preference for Ca 2+ . The sequence of permeability is Ca 2+ >Mg 2+ >Na + 0K + 0Cs + [3]. In addition to all these, TRPV1 also conducts protons [34]. Following prolonged exposure to agonists, TRPV1 becomes permeable even to larger organic cations, including dyes such as YO-PRO1 and FM1-43 [22] and a lidocaine derivative QX-314 [23]. Increasing agonist concentration enhances the blocking ability of Co 2+ , suggesting a correlation between the increased efficacy of inhibition and the different open-state conformations of the TRPV1 channel. Further experiments (involving patch-clamp recordings) would be needed to clarify the inhibitory effect of Co 2+ on the fluxes of the other cations or molecules mentioned above. However, no channel blocker or antagonist of TRPV1 has been reported that is able to block the flux of only one specific ion, and antagonists seem to block all these influxes. For example, CapZ blocks the influxes of both Ca 2+ and Na + [35]. Overall, we presume that Co 2+ can also block the ion currents mentioned above.
Before the exploration of TRPV1 protein, Co 2+ histochemistry was a very useful tool for the identification of vanilloid-sensitive primary afferent neurons with C-and Aδ fibres after in vivo experiments. Co 2+ uptake and the postmortem determination of Co 2+ deposits quite accurately identified C-and Aδ afferents, the neuronal subset that can be activated by treatment with a vanilloid agonist, CAPS [36,37]. Likewise, as previously documented in a subpopulation of pseudo-unipolar neurons [38], we have now demonstrated selective vanilloid-induced Co 2+ accumulation in the cytosol of DRG primary cultures and TRPV1-transfected HaCaT and 3T3 cells. The accumulation of Co 2+ could be blocked by RuRed or CapZ in a dose-dependent manner. The CAPS-induced Ca 2+ and Co 2+ influxes in TRPV1/3T3 cells proved to be due exclusively to the TRPV1 channel and not to VGCC activity.
Co 2+ inhibited the pain-evoked defensive movements in eye wipe tests in response to pungent vanilloids. However, not only TRPV1 but also some other calcium channels of the sensory neurons, such as VGCCs, can be blocked by Co 2+ [39]. VGCCs share structural homology with TRPV1 channel. The α1 subunit of voltage-gated calcium channels is organized in four homologous domains (I-IV), with six transmembrane segments (S1-S6) in each [40]. There is an additional region (H5) between S5 and S6, which forms a part of the pore region of the channel [41]. Within each H5 region, there exist conserved glutamate residues, significantly homologous to conventional EF-hand motifs [42], acting as the selectivity filter [43]. Mn 2+ , Ni 2+ and Cd 2+ in contrast with Co 2+ are known to be stronger blockers of the VGCCs. Cu 2+ , Mn 2+ and Co 2+ blocked high-voltage activated currents conducted by Ba 2+ with IC 50 of 920, 58, and 65 μM, respectively [44,45]. All of these ions exert their effects through high-affinity docking to the cation-binding site at the IIIS5-H5 pore region of the VACCs [46,47]. Since Co 2+ has higher IC 50 for VGCCs (≈65 μM) than for TRPV1 (≈15 μM), we can conclude that decrease in the number of eye wipes might be due, at least partially, to an inhibitory effect of Co 2+ on TRPV1.
Positively charged tricyclics, K/R-rich basic peptides and RuRed dock to the DXEXXEXXD motif at the channel orifice [13]. Although our point mutants overlap with the RuRed docking site [11,14,45], our point mutant studies suggest that Co 2+ has a different binding site. Accordingly, the D646N point mutant, which is crucial for RuRed binding, does not change the kinetic parameters of Co 2+ inhibition in cells ectopically expressing the D646N mutant TRPV1. We carried out vanilloid-induced 45 Ca 2+ -uptake experiments with channel point mutants of TRPV1 in which the agonist binding site remained intact. Interestingly, no significantly decreased efficacy of Co 2+ inhibition was found when 646 aspartate was replaced by asparagine. Based on these results, Co 2+ is supposed to evoke its effect at a different site on the pore loop region than RuRed, or to use the same negatively charged amino acids passing through the TRPV1 channel as Ca 2+ . This line of research on functional point mutants should be continued to determine whether Co 2+ has a specific binding site on the channel orifice or not.
Most painkiller drugs are competitive agonists and target the CAPS-binding domain [48]. As the 646DLEFTENYD acidic tetrad sequence of the TRPV1 receptor is unique among Ca 2+ -binding proteins, this permits the design of painkillers targeting the channel orifice of TRPV1 and acting as channel blockers. A better understanding of the structural background and dynamics of the competition of Ca 2+ with other M 2+ for entry may result in the discovery of novel channel blocker painkillers. Furthermore, our data can contribute to a better understanding of the structures and functions of all TRP superfamily members. The specific effect of the selected M 2+ -s on the given ion channel pore region can serve as a valuable constraint during in silico modelling of the pore region. By comparing the different cation action profiles of pore regions, the model can be finetuned. The mechanism of Co 2+ -mediated inhibition provides screening for adjuvant therapeutics with higher selectivity than that of AMI, an approved drug currently used in clinical practice, but with only limited efficacy and with serious side effects.