Therapeutic inhibition of keratinocyte TRPV3 sensory channel by local anesthetic dyclonine

The multimodal sensory channel transient receptor potential vanilloid-3 (TRPV3) is expressed in epidermal keratinocytes and implicated in chronic pruritus, allergy, and inflammation-related skin disorders. Gain-of-function mutations of TRPV3 cause hair growth disorders in mice and Olmsted syndrome in humans. Nevertheless, whether and how TRPV3 could be therapeutically targeted remains to be elucidated. We here report that mouse and human TRPV3 channel is targeted by the clinical medication dyclonine that exerts a potent inhibitory effect. Accordingly, dyclonine rescued cell death caused by gain-of-function TRPV3 mutations and suppressed pruritus symptoms in vivo in mouse model. At the single-channel level, dyclonine inhibited TRPV3 open probability but not the unitary conductance. By molecular simulations and mutagenesis, we further uncovered key residues in TRPV3 pore region that could toggle the inhibitory efficiency of dyclonine. The functional and mechanistic insights obtained on dyclonine-TRPV3 interaction will help to conceive therapeutics for skin inflammation.


Introduction 1
Transient receptor potential (TRP) channels belong to a family of calcium-permeable 2 and nonselective cation channels, essential for body sensory processing and local 3 inflammatory development (Clapham, 2003). As a polymodal cellular sensor, TRPV3    8 whether and how TRPV3 could be therapeutically targeted remains to be elucidated. It 9 is thus desirable to identify and understand clinical medications that can selectively 10 target TRPV3 channels. 11 As a clinical anesthetic, dyclonine is characterized by rapid onset of effect, lack of 12 systemic toxicity, and a low index of sensitization (Florestano and Bahler, 1956). Its 13 topical application rapidly relieves itching and pain in patients, by ameliorating 14 inflamed, excoriated and broken lesions on mucous membranes and skin (Morginson 15 et al., 1956). Accordingly, dyclonine is used to anesthetize mucous membranes prior to   5 Here, using a multidisciplinary approach combining electrophysiology, genetic 1 engineering and ultrafast local temperature control, we show that mouse and human 2 TRPV3 channel was selectively targeted by dyclonine. It dose-dependently inhibited 3 TRPV3 currents in a voltage-independent manner and rescued cell death caused by 4 TRPV3 gain-of-function mutation. In vivo, dyclonine indeed suppressed the 5 itching/scratching behaviors induced by TRPV3 channel agonist carvacrol or 6 pruritogen histamine. At single-channel level, dyclonine reduced TRPV3 channel open 7 probability without altering the unitary conductance. We also identified molecular 8 residues that were capable of either eliminating or enhancing the inhibitory effect of 9 dyclonine. These data demonstrate the selective inhibition of TRPV3 channel by 10 dyclonine, supplementing a molecular mechanism for its clinical effects and raising its 11 potential to ameliorate TRPV3-associated disorders.  2 We first examined the effect of dyclonine on TRPV3 activity induced by the TRPV 3 channel agonist 2-APB (100 µM). Whole-cell currents were recorded at a holding 4 potential of -60 mV in HEK 293T cells expressing mouse TRPV3. Because TRPV3 5 channels exhibit sensitizing properties upon repeated stimulation (Chung et al., 2004c), 6 we examined the effect of dyclonine after the response had stabilized following 7 repetitive application of 2-APB (Fig. 1A). The presence of 5 µM and 10 µM dyclonine 8 significantly inhibited TRPV3 currents response to 30 ± 2% and 15 ± 3% of control 9 level, respectively. After washing out of dyclonine, 2-APB evoked a similar response 10 to the control level, indicating the blocking effect of dyclonine is reversible (Fig. 1A-

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B). We repeated the experiments with different doses of dyclonine. The dose-response 12 curve indicates that dyclonine inhibited TRPV3 currents in a concentration-dependent 13 manner with an IC50 of 3.2 ± 0.24 M (n = 6, Fig. 1C). 14 TRPV3 channel in physiological conditions has a low level of response to external 15 stimuli, which is augmented during the sensitization process (i.e., repetitive 16 stimulations, Fig. 1A). In contrast, excessive up-regulation of TRPV3 activity impairs 17 hair growth and increases the incidence of dermatitis and pruritus in both humans and 18 rodents. To determine whether dyclonine affects the process of TRPV3 sensitization,

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Dyclonine is a selective inhibitor of TRPV3 channel 16 Next, we compared the inhibitory effect on TRPV3 of dyclonine to its impact on other 17 TRP channels. Mouse TRPV1, TRPV2 and TRPM8 channels were expressed in HEK 18 293T cells and respectively activated by capsaicin, 2-APB and menthol. We observed 19 that 10 M dyclonine exhibited little inhibition on TRPV1, TRPV2 and TRPM8, but 20 potently inhibited TRPV3 channel ( Fig. 2A). The corresponding reduction in current 21 8 amplitude was 2  1% for TRPV1, 6  1% for TRPV2, 3  2% for TRPM8, compared 1 with 84  1% inhibition of TRPV3 current (Fig. 2B). By applying a series of dyclonine 2 concentrations, we derived dose-response curves (Fig. 2C). The corresponding IC50 3 values of dyclonine for inhibiting TRPV1, TRPV2 and TRPM8 channels 4 (337.4 ± 19.4 M, 31.1 ± 2.7 M and 81.8 ± 12.7 M, respectively) were one or two 5 orders of magnitude higher than that for TRPV3 inhibition (3.2  0.24 M), indicating 6 that dyclonine represents a selective inhibitor of TRPV3 channel. Above results were obtained for mouse TRPV3. We further asked whether the   18 To obtain a complete description of the inhibitory effect of dyclonine, we next 19 investigated its voltage dependence using a stepwise protocol (Fig. 3A). We measured 20 membrane currents in TRPV3-expressing HEK 293T cells using a Cs + -based pipette 21 solution that blocks most outward K + channel current but permits measurement of  (Fig. 3A). Addition of dyclonine in the extracellular solution significantly 4 diminished TRPV3-mediated outward and inward currents (Fig. 3A). By contrast, 10 5 M ruthenium red, a broad TRP channel blocker, only inhibited TRPV3-mediated 6 inward currents but enhanced outward currents (Fig. 3A), which is consistent with early   Inhibition of heat-activated TRPV3 currents by dyclonine 14 TRPV3 is a thermal sensitive ion channel and has an activation threshold around 30 to 15 33 °C (Xu et al., 2002). We therefore explored whether the heat-evoked TRPV3 16 currents can be also inhibited by dyclonine. We employed an ultrafast infrared laser 17 system to control the local temperature near single cells; each temperature jump had a 18 rise time of 1.5 ms and lasted for 100 ms (Fig. 4A). TRPV3, expressed in HEK 293T 19 cells, steadily responded to temperature jumps ranging from 30 to 51 °C (Fig. 4B). As 20 in 2-APB experiments, TRPV3 currents were pre-sensitized to stable level by repeated 21 temperature jumps from room temperature to 52 °C. Application of dyclonine 22 appreciably inhibited TRPV3 thermal currents ( Fig. 4B-C). The inhibitory effect of 1 dyclonine was fully reversible, as after its washing out the TRPV3 response recovered 2 to the same level as control condition (Fig. 4C). To determine the concentration 3 dependence of dyclonine inhibition, TRPV3 currents were evoked by a same 4 temperature jump from room temperature to ~52 °C in the presence of 1, 3, 5, 10, 30, 5 and 50 M dyclonine (Fig. 4D). The IC50 of dyclonine on TRPV3 inhibition was 6 assessed to be 14.02  2.5 M with a Hill coefficient of nH = 1.9  0.54, according to 7 the dose-response curve fitting (Fig. 4E). These results thus indicate that dyclonine 8 dose-dependently suppresses heat-evoked TRPV3 currents.

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Dyclonine inhibited hyperactive TRPV3 mutants and rescued cell death 11 It has previously been reported that gain-of-function mutations, G573S and G573C, of 12 TRPV3 are constitutively active and their expression causes cell death (Xiao et al., 13 2008). As TRPV3 channel is effectively inhibited by dyclonine, we explored whether 14 it can rescue the cell death caused by those mutants. We transfected the inducible cDNA 15 constructs encoding respectively the GFP-tagged wild-type TRPV3, G573S, or G573 16 mutant into T-Rex 293 cells and then applied 20 ng/ml doxycycline to induce the gene 17 expression. Cells were exposed to different pharmacological drugs (dyclonine, 2-APB, 18 2-APB and dyclonine, or ruthenium red). Cell death was recognized by the narrow and 14 v.s. 53.60  5.88% for G573S, and 13.85%  2.49% v.s. 47.91  5.54% for G573C after 15 and before addition of dyclonine). Collectively, these results indicate that dyclonine 16 rescues cell death by selectively inhibiting the excessive activity of TRPV3 channel.

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TRPV3 is highly expressed in skin keratinocytes, whose hyperactivity causes pruritic 20 dermatitis and scratching behavior. We next examined in vivo the therapeutic effect of 21 dyclonine on TRPV3 hyperactivity-caused scratching behavior in mouse model.  Effects of dyclonine on single TRPV3 channel activity 1 We then examined the functional and molecular mechanisms underlying the inhibition 2 of TRPV3 by dyclonine. To distinguish whether such inhibition arises from the changes 3 in channel gating or conductance, we measured single-channel activity. Single-channel 4 recordings were performed in an inside-out patch that was derived from HEK 293T 5 cells expressing the mouse TRPV3 (Fig. 7). Currents were evoked by 10 M 2-APB in 6 the absence and presence of dyclonine (30 M) after sensitization induced by 300 M

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Statistical analysis, however, revealed that dyclonine had no effect on single TRPV3  Dyclonine is an open-channel blocker 17 In order to understand the molecular mechanism underlying the blockade of TRPV3 by 18 dyclonine, we utilized the molecular docking approach to model their interaction.  (Fig. 8F). These data suggest that dyclonine interacts with 19 the pore cavity of TRPV3 channel, thereby preventing ion passage and resulting in 20 channel inhibition.  Accordingly, dyclonine efficiently blocked the excessive activation of TRPV3 mutants 20 and prevented cell death. Single-channel recordings revealed that dyclonine effectively 21 suppresses the channel open probability without changing single-channel conductance.

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These data not only supplement a molecular mechanism for the therapeutic effect of 1 dyclonine, but also suggest its application to curb TRPV3-related disorders. Using 2 mouse model, we indeed observed that dyclonin ameliorates the TRPV3 hyperactivity-3 caused itch/scratching behaviors, indicating its therapeutic inhibition effect being 4 maintained in vivo. As TRPV3 responds to moderate temperatures (30 -40 °C), 5 dyclonine may thus be used to alleviate skin inflammations persisted in physiological 6 temperatures. Also, as a clinical drug dyclonine has been widely used and thus has 7 shown its safety to human body. In addition, as a selective and potent inhibitor, 8 dyclonine can also be a research tool to dissect the physio-and pathological 9 characteristics of TRPV3 channel. 10 The current data also unraveled the molecular residues within TRPV3 channel pore 11 that regulate the effect of dyclonine. Dyclonine interacts likely with L655 and F666 12 located in the pocket A and C, and thus regulates the ion-conducting pathway. In reality, 13 since binding domains A and C are arranged one by one along the ion-conducting 14 pathway, it is unlikely that dyclonine can occupy these two sites at the same time.     Ultrafast temperature jump achievement 6 Rapid temperature jumps were generated by the laser irradiation approach as described 7 previously (Yao et al., 2009). In brief, a single emitter infrared laser diode (1470 nm) 8 was used as a heat source. A multimode fiber with a core diameter of 100 m was used 9 to transmit the launched laser beam. The other end of the fiber exposed the fiber core 10 was placed close to cells as the perfusion pipette is typically positioned. The laser diode 11 driven by a pulsed quasi-CW current powder supply (Stone Laser, Beijing, China).

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Pulsing of the controller was controlled from computer through the data acquisition 13 card using QStudio software developed by Dr. Feng Qin at State University of New 14 York at Buffalo. A blue laser line (460 nm) was coupled into the fiber to aid alignment. 15 The beam spot on the coverslip was identified by illumination of GFP-expressing cells 16 using the blue laser during experiment.

Evaluation of scratching behavior in mice 19
To assess itch-scratching behaviors, the hair of the rostral part of the C57B/6 mouse 20 right neck was shaved using an electric hair clipper 24 hours before the start of 14 Molecular docking 15 The molecular docking approach was used to model the interaction between dyclonine 16 and TRPV3 channel protein (PDB ID code: 6DVZ). The 3D structure of dyclonine was 17 generated by LigPrep (Gadakar et al., 2007). Induce-Fit-Docking (IFD) was employed 18 to dock dyclonine into the potential binding pocket using default parameters (Sherman 19 et al., 2006). During this docking process, the protein was fixed while dyclonine was    The authors declare that they have no conflict of interest.    Dose dependence of dyclonine effects on TRPV3 currents in cultured keratinocytes. 10 The solid line corresponds to a fit by Hill's equation with IC50 = 5.2 ± 0.71 M and nH 11 = 2.4 ± 0.75 (n = 6).   (Inset) The inhibition effects of dyclonine and RR on TRPV3 currents at negative 12 holding potentials is magnified and displayed on the right. Note that dyclonine had an 13 inhibitory effect on TRPV3 currents at both positive and negative potentials, but RR 14