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Tonic Inhibition of TRPV3 by Mg2 þ in Mouse Epidermal Keratinocytes Jialie Luo1, Randi Stewart1, Rebecca Berdeaux1 and Hongzhen Hu1

The transient receptor potential vanilloid 3 channel (TRPV3) is abundantly expressed in epidermal keratinocytes and has important roles in sensory biology and skin health. Mg2 þ deficiency causes skin disorders under certain pathological conditions such as type 2 diabetes mellitus. In this study, we investigated the effect of 2 þ 2 þ 2 þ Mg on TRPV3 in primary epidermal keratinocytes. Extracellular Mg ([Mg ]o) inhibited TRPV3-mediated membrane current and influx. TRPV3 activation induced a calcium signaling pathway culminating in 2 þ activation of the cAMP response element binding. TRPV3 inhibition by [Mg ]o, the TRPV3 blocker , or TRPV3 siRNA suppressed this response. In TRPV3-expressing Chinese hamster ovary cells, both extracellular and intracellular Mg2 þ inhibited TRPV3 single-channel conductance, but not open probability. Neutralization of an aspartic acid residue (D641) in the extracellular pore loop or two acidic residues (E679, E682) in the inner pore region significantly attenuated the inhibitory effect of extracellular or intracellular Mg2 þ on TRPV3-mediated signaling, respectively. Our findings suggest that epidermal TRPV3 is tonically inhibited by both extracellular and intracellular Mg2 þ , which act on both sides of the channel pore loop. Mg2 þ deficiency may promote the function of TRPV3 and contribute to the pathogenesis of skin diseases. Journal of Investigative Dermatology (2012) 132, 2158–2165; doi:10.1038/jid.2012.144; published online 24 May 2012

INTRODUCTION Xu et al., 2002; Facer et al., 2007; Kauer and Gibson, 2009), The transient receptor potential vanilloid 3 channel (TRPV3), where it has been shown to have an important role in pain a ‘‘thermoTRP’’, belongs to a large family of Ca2 þ -permeable sensation and mediate psychoactive properties of nonselective cation channels with six transmembrane (TM) (Huang et al., 2008; Moussaieff et al., 2008; Bang domains and a putative pore loop between TM5 and TM6 et al., 2010). (Clapham, 2007). Like other TRP channels, TRPV3 is a Recently, TRPV3 has come to the fore as a key regulator of polymodal cellular sensor that integrates several modalities physiological responses of skin. TRPV3 is required for normal including warm temperature, synthetic small molecules such skin development and function, as TRPV3-deficient mice as 2-aminoethoxydiphenyl borate, and natural compounds have defects in hair morphogenesis and skin barrier function from plant such as , , and (Chung (Cheng et al., 2010). Paradoxically, a recent report revealed et al., 2004a; Hu et al., 2004, 2009; Moqrich et al., 2005; that active TRPV3 expressed in human outer root sheath Vriens et al., 2009). The widespread expression of TRPV3 in keratinocytes inhibits hair growth (Borbiro et al., 2011). neuronal and non-neuronal tissues suggests that TRPV3 may Therefore, maintaining an appropriate balance of TRPV3 have important roles in many cellular and physiological activity is essential for normal function of epidermal functions (Peier et al., 2002; Smith et al., 2002; Xu et al., keratinocytes. Indeed, naturally occurring gain-of-function 2002; Yamada et al., 2010). In fact, activation of TRPV3 in point mutations in TRPV3 (Gly573 to Ser or Cys) have been arterial endothelial cells by the dietary agonist carvacrol linked to autosomal-dominant hairless phenotypes and elicits vasodilation of cerebral arteries in rats (Earley et al., spontaneous dermatitis in DS-Nh mice likely due to their 2010). In mammals, TRPV3 is expressed in dorsal root and constitutive activities (Asakawa et al., 2006; Imura et al., trigeminal ganglia, spinal cord, and brain (Smith et al., 2002; 2007; Xiao et al., 2008b; Yoshioka et al., 2009). Interestingly, these DS-Nh mice also develop severe scratching behavior, suggesting that epidermal TRPV3 may have a causative role 1Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas, USA in inflammation and pruritus (Steinhoff and Biro, 2009; Correspondence: Hongzhen Hu, Department of Integrative Biology and Yoshioka et al., 2009), although much remains elusive regar- Pharmacology, University of Texas Health Science Center at Houston, ding the molecular mechanism underlying these effects. 6431 Fannin Street, Houston, Texas 77030, USA. Mg2 þ is the second most common divalent cation in the E-mail: [email protected] body, and the most abundant intracellular divalent cation. Abbreviations: CHO, Chinese hamster ovary; CREB, cAMP response element Intracellular Mg2 þ ([Mg2 þ ] ) has a pivotal role in structural, binding; TM, transmembrane; TRPV3, transient receptor potential vanilloid 3 i channel catalytic, and regulatory cellular functions. It serves as a Received 15 November 2011; revised 9 March 2012; accepted 29 March cofactor of over 300 enzymes and regulates activity of many 2012; published online 24 May 2012 ion channels (Johnson and Ascher, 1990; Bichet et al., 2003;

2158 Journal of Investigative Dermatology (2012), Volume 132 & 2012 The Society for Investigative Dermatology J Luo et al. Mg2 þ Inhibits TRPV3 Function

Voets et al., 2003; Ahern et al., 2005; Lee et al., 2005; a b 2 þ 75 Obukhov and Nowycky, 2005). Free [Mg ]i undergoes Basal 2 /(nA) dynamic changes due to the chelating effect of ATP and 0 mM 2 mM increases in ischemic neurons (Chinopoulos et al., 2007; 1 Henrich and Buckler, 2008, 2009). Mg2 þ deficiency, also 10 mM 50 ** known as hypomagnesemia, accompanies human pathologi- –100 cal states including type 2 diabetes mellitus (Resnick et al., 2 þ +100

1993; Tosiello, 1996; Sales and Pedrosa Lde, 2006). Mg V (mV) % Inhibition 25 deficiency induces apoptosis in isolated hepatocytes (Martin –1 et al., 2003) and increases incidence of stroke, hypertension, eclampsia, and atherosclerosis (Saris et al., 2000). Interest- 0 ingly, magnesium deficiency has been widely used in rodents –2 –100 mV +100 mV to experimentally model atopic dermatitis associated with a robust scratching behavior (Neckermann et al., 2000; Biro c 1.5 ** et al., 2005; Akamatsu et al., 2006). However, the mecha- nisms underlying the hypomagnesemia-mediated skin dis- # orders are poorly understood. ** ) Given the association of both TRPV3 and Mg2 þ dysregu- 1.0 –100 I

/ ** lation with skin disorders, we hypothesized that TRPV3 may # # mediate some of the effects of magnesium deficiency in skin. 100 ** In this study, we show that TRPV3 function is inhibited by –( I 0.5 2 þ 2 þ 2 þ both [Mg ]i and extracellular Mg ([Mg ]o), which interacts with acidic residues on both sides of the channel pore loop region. The voltage-dependent inhibition contri- 0.0 2+ butes to the characteristic inward and outward rectification [Mg ]o 2 10 0 2 10 0 (mM) þ 2 2+ of TRPV3 current. The inhibitory effect of Mg was also [Mg ]i 2 10 (mM) translated into inhibition of TRPV3-mediated cell signaling in 2 þ primary mouse epidermal keratinocytes, a well-established Figure 1. Inhibition of TRPV3-mediated current by [Mg ]o in cultured mouse keratinocytes. (a) Representative current traces in response to cell model to study the function of endogenous TRPV3 voltage ramp from 100 to þ 100 mV without (basal) and with the TRPV3 2 þ (Chung et al., 2004b), as evidenced by attenuation of TRPV3- agonist cocktail in the absence (0 mM) and presence of 2 and 10 mM Mg . mediated increase of cAMP response element binding (CREB) (b) Percentage of inhibition of TRPV3 agonist cocktail-activated current 2 þ activity. by 10 mM Mg at holding potential of 100 and þ 100 mV obtained from the voltage ramp shown in a. Please note the different inhibitory effects at RESULTS 100 and þ 100 mV. (c) Rectification ratio [(I100/I100)] with different 2 þ 2 þ concentrations of Mg in the intracellular and extracellular solutions. [Mg ]o suppresses TRPV3-mediated whole-cell currents in 2 þ **Po0.01 compared with 2 mM Mg in the extracellular solution with mouse keratinocytes 2 þ # 2or10mM Mg in the intracellular solution. Po0.05 compared with Whole-cell patch-clamp recordings were performed to 2 þ 2 þ 2mM Mg in the intracellular solution with 0, 2, or 10 mM Mg in the directly measure TRPV3-mediated current in mouse kerati- extracellular solution. TRPV3, transient receptor potential vanilloid 3 nocytes. To activate TRPV3 we used a TRPV3 agonist channel. cocktail, a combination of 100 mM 2-aminoethoxydiphenyl borate and 250 mM carvacrol, as previously described (Cheng et al., 2010). As expected, the TRPV3 agonist cocktail from 2 to 10 mM in the pipette solution significantly activated a nonselective cation current associated with both diminished TRPV3-mediated outward current, evidenced by inward and outward rectification during a voltage ramp from the reduced rectification ratio from 0.90±0.02 to 0.69±0.03 2 þ 100 mV to þ 100 mV, characteristic of TRPV3 current in with 2 mM Mg in the extracellular solution (Po0.05, n ¼ 9; heterologous expression systems (Chung et al., 2004a; Hu Figure 1c). These findings suggest that Mg2 þ suppresses et al., 2004, 2006; Cheng et al., 2010). No current was TRPV3 function in primary epidermal keratinocytes in a observed in keratinocytes from TRPV3-deficient mice, con- voltage-dependent manner on both sides of the cell firming the specificity of the agonist cocktail of TRPV3 in membrane. keratinocytes (Supplementary Figure S1 online). Increasing 2 þ 2 þ [Mg ]o to 10 mM decreased TRPV3-mediated whole-cell [Mg ]o decreases TRPV3 single-channel conductance 2 þ 2 þ current. By contrast, reducing [Mg ]o to 0 mM enhanced the Suppression of TRPV3-mediated current by [Mg ]o could 2 þ current (Figure 1a). The effect of [Mg ]o on inward current result from inhibition of TRPV3 single-channel conductance was more pronounced than the outward current. The and/or open probability. We therefore used single-channel rectification ratio (I þ 100 mV/I100 mV) was increased from recordings on cultured mouse keratinocytes to elucidate the ± ± 2 þ 2 þ 0.78 0.03 to 1.39 0.11 by 10 mM [Mg ]o with 2 mM mechanism underlying [Mg ]o inhibition of TRPV3. Surpris- Mg2 þ in the pipette solution (Po0.01, n ¼ 9; Figure 1b ingly, no single-channel activities could be recorded with dif- and c). On the other hand, an increase of Mg2 þ concentration ferent configurations (cell attached, inside-out or outside-out)

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2 þ a conductance was decreased by 3 mM [Mg ]o from 157.82± ± 2+ +60 mV –60 mV 11.25 to 57.64 9.62 pS at negative membrane potentials [Mg ]o 2 þ (Po0.05, n ¼ 6). By contrast, [Mg ]o did not significantly affect TRPV3 single-channel conductance at positive mem- 0 mM brane potentials (90.35±13.32 and 74.38±11.79 in the 2 þ absence and presence of 3 mM [Mg ]o, P40.05, n ¼ 6; 2þ Figure 2). Therefore, [Mg ]o suppresses TRPV3-mediated current preferably at negative holding potentials (Figure 2). 3 mM 5 pA 2 þ [Mg ]i modulates TRPV3 single-channel activity 200 ms 2 þ [Mg ]i fluctuates with metabolic states, especially during hypoxia when intracellular ATP concentration decreases b 3 (Wilson and Chin, 1991). We therefore investigated the 3 mM Mg2+ 2 þ 2 þ effect of [Mg ]i on TRPV3 activity. Increasing [Mg ]i from 0 mM Mg2+ 2 2to10mM in the recording pipette significantly attenuated the TRPV3-mediated outward current in whole-cell patch-

1 Relative current clamp recording on mouse keratinocytes (Figure 1c). We further probed the mechanism underlying the inhibitory –100 –80 –60 –40 –20 2 þ effect of [Mg ]i on TRPV3 using inside-out single-channel 2 þ 20 40 60 80 100 recordings. Increasing Mg concentrations (0–3mM) on the V (mV) cytoplasmic side inhibited the outward unitary single- * –1 channel current amplitude in a concentration-dependent 2 þ manner (Figure 3a). [Mg ]i mainly decreased the single- –2 channel conductance of the outward current (165.34±9.52 and 66.38±7.84 pS in the absence and presence of 3 mM –3 Mg2 þ , Po0.05, n ¼ 7), but barely affected the single-channel conductance of the inward current (67.47±7.24 and 2 þ Figure 2. Effect of [Mg ]o on the IV relationship of TRPV3-mediated 61.38±6.35 pS in the absence and presence of 3 mM single-channel current. (a) Representative single-channel current traces Mg2 þ , P40.05, n ¼ 7; Figure 3b). The single-channel NPo activated by 10 mM 2-aminoethoxydiphenyl borate (2APB) at holding 2 þ 2 þ was not significantly affected by the change of [Mg ]i potentials of þ 60 and 60 mV in the absence and presence of 3 mM Mg in (0.25±0.13 and 0.23±0.12 in the presence and absence of the extracellular solution in Chinese hamster ovary (CHO) cells expressing 2 þ 3mM Mg , P40.05, n ¼ 5). The mean open time was not mTRPV3. (b) IV relationships of the TRPV3 single-channel current activated 2 þ by 2APB in CHO cells expressing mTRPV3. Current amplitudes at different significantly affected by [Mg ]i (1 mM) either (not shown, 2 þ 2 þ holding potentials are normalized to that at 60 mV in the presence of 3 mM n ¼ 5). The results indicate that both [Mg ]o and [Mg ]i Mg2 þ in the extracellular solution (marked as asterisk). TRPV3, transient inhibit TRPV3 function by primarily decreasing TRPV3 single- receptor potential vanilloid 3 channel. channel conductance.

Inhibition of TRPV3 by Mg2 þ involves distinct acidic upon stimulation with the TRPV3 agonist cocktail that evoked amino acid residues on both sides of the channel pore loop TRPV3-dependent whole-cell currents (not shown). We then Previous studies have shown that the acidic residue D641 in went on to use Chinese hamster ovary (CHO) cells expressing the extracellular pore loop contributes to inhibition of TRPV3 recombinant TRPV3 to evaluate the impact of Mg2 þ on by extracellular Ca2 þ (Chung et al., 2005; Xiao et al., 2008a). TRPV3 single-channel properties based on the facts that it is We therefore hypothesized that D641 might be part of a 2 þ feasible to record TRPV3 single-channel activities in hetero- common interaction site for divalent cations including Mg. logous expression systems (Xu et al., 2002; Chung et al., To test this possibility, we examined the effect of increasing 2 þ 2 þ 2004a; Hu et al., 2006), and the voltage-dependent Mg [Mg ]o on a mutant TRPV3 (D641N), in which the inhibition of recombinant TRPV3 expressed in CHO cells negatively charged aspartic acid was replaced by neutral recapitulates that of native TRPV3 in mouse keratinocytes asparagine (N). The D641N mutation significantly decreased 2 þ (Supplementary Figure S2 online). the inhibitory effect of 3 mM [Mg ]o on the inward single- We first used outside-out single-channel recordings on channel current amplitude from 54.77±9.24 to 20.78± CHO cells expressing TRPV3. To avoid TRPV3-mediated 8.93% (Po0.05, n ¼ 5; Figure 4a). On the other hand, the 2 þ large macroscopic currents in outside-out membrane inhibitory effect of [Mg ]i on outward single-channel current patches, we used a relatively low concentration of 2-APB amplitude was unaffected (60.37±8.35 vs. 61.41±9.27% (10 mM) to activate single TRPV3 channels (Hu et al., 2006). for wild type and D641N, respectively, P40.05, n ¼ 6). 2 þ Increasing [Mg ]o from 0 to 3 mM in the extracellular side To examine if other negatively charged acidic residues in did not affect 2-APB-evoked single-channel open probability the intracellular side close to the inner pore region con- 2 þ (NPo; 0.21±0.11 and 0.19±0.12 in the presence and absence tribute to the inhibition of [Mg ]i on outward current, 2 þ of 3 mM Mg , P40.05, n ¼ 5). However, single-channel we further screened these residues by mutating each to

2160 Journal of Investigative Dermatology (2012), Volume 132 J Luo et al. Mg2 þ Inhibits TRPV3 Function

2 þ glutamine (Gln or Q), which effectively neutralizes the [Mg ]i (38.23±1.39 vs. 60.13±1.21% in wild type, charge. Out of the six glutamate residues, only neutralization Po0.05, n ¼ 10) on outward current (Figure 4b). As E679 of E682 significantly attenuated the inhibition of TRPV3 by and E682 are the closest negatively charged residues to the inner pore, we mutated both of them into glutamine to a examine if there exists a synergistic action between them. +60 mV –60 mV Remarkably, the inhibitory effect of [Mg2 þ ] was dramatically [Mg2+] i i reduced on the TRPV3 (E679Q/E682Q) mutant to 14.2±3.65% (Po0.01 compared with that on wild type, 0 mM n ¼ 8; Figure 4b). The results indicate that Mg2 þ affects TRPV3 activity by interacting with acidic residues on both 1 mM sides of TRPV3 channel pore loop.

3 mM 2 þ 2 þ [Mg ]o inhibits TRPV3-mediated [Ca ]i increase in mouse 10 pA keratinocytes 200 ms TRPV3 activation evokes calcium influx, which is involved in many cellular functions of keratinocytes (Peier et al., 2002; b 6 Smith et al., 2002; Xu et al., 2002; Chung et al., 2004a, b; 0 mM Mg2+ Ansari et al., 2008; Savignac et al., 2011). To examine the 2+ 2 þ 3 mM Mg effect of [Mg ]o on TRPV3-mediated calcium influx, we 4 used ratiometric calcium imaging. The TRPV3 agonist cock-

Relative current tail evoked a rapid increase of intracellular-free calcium 2 þ ([Ca ]i) in Fura-2-loaded primary keratinocytes (Figure 5a). 2 þ The agonist cocktail failed to increase [Ca ]i when calcium- 2 free extracellular buffer was used (not shown). This calcium response was also abolished in keratinocytes isolated from TRPV3 knockout mice (Cheng et al. (2010) and Supplemen- –100 –80 –60 –40 –20 tary Figure S1 online), which confirms that the agonist 20 40 60 80 100 cocktail-evoked calcium influx is mediated exclusively by * V (mV) TRPV3. Interestingly, repetitive application of the TRPV3 agonist cocktail gradually caused desensitization of the –2 2 þ [Ca ]i response (Figure 5), similar to TRPV3-mediated 2 þ Figure 3. Effect of [Mg ]i on the IV relationship of TRPV3-mediated responses evoked by monoterpenoids in a human keratino- single-channel current. (a) Representative single-channel current traces cyte-derived cell line (HaCaT cells; Sherkheli et al., 2009). 2 þ activated by 10 mM 2-aminoethoxydiphenyl borate (2APB) at holding When 2 or 10 mM Mg was added to the extracellular potentials of þ 60 and 60 mV in the absence and presence of 1 and 3 mM buffer, the calcium responses activated by the second and 2 þ Mg in the intracellular solution in Chinese hamster ovary (CHO) cells third applications of TRPV3 agonist cocktail were further expressing mTRPV3. (b) IV relationships of the TRPV3 single-channel suppressed by 24.89±3.56 and 40.17±4.25% (P 0.05, current activated by 2APB in CHO cells expressing mTRPV3. Current o amplitudes at different holding potentials are normalized to those at 60 mV n ¼ 6), respectively (Figure 5b). The results indicate that 2 þ 2 þ in the presence of 3 mM Mg in the intracellular solution (asterisk). [Mg ]o tonically inhibits TRPV3 in epidermal keratinocytes 2 þ TRPV3, transient receptor potential vanilloid 3 channel. contributing to intracellular Ca homeostasis.

ab(i) b(ii) 75 Wt E679Q E682Q Wt + * E679Q+E682Q E682Q * D641N E736Q 50 50 E702Q E689Q E687Q * E682Q 25 25 * % Inhibition

% Inhibition E679Q E641N Wt 0 0 –60 +60 0 10 20 30 40 50 60 70 0.0 1.0 2.0 3.0

Voltage (mV) % Inhibition Mg2+ (mM)

2 þ 2 þ Figure 4. Amino acids required for Mg inhibition of TRPV3. (a) Bar graph illustrating the inhibitory effect of 3 mM [Mg ]o on TRPV3-mediated single- 2 þ channel current amplitude with D641N mutation at holding potentials of þ 60 and 60 mV. (bi) Bar graph illustrating the inhibitory effects of 3 mM [Mg ]i on wild-type (wt) and TRPV3 mutants with neutralizing mutations of acidic residues on the intracellular side near the channel pore. (bii) Graph illustrating 2 þ the concentration-dependent inhibition of [Mg ]i on the wild-type and TRPV3 mutants at concentrations ranging from 0 to 3 mM,*Po0.05, **Po0.01. TRPV3, transient receptor potential vanilloid 3 channel.

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a 2.4 10 mM Mg2+ Scrambled siRNA control had no effect (Figure 6a). µ µ 2 þ 200 M 2APB+300 M carvacrol Moreover, increasing [Mg ]o suppressed TRPV3-induced 2.0 CREB luciferase activity in a concentration-dependent manner (Figure 6b). These data present evidence that activation of TRPV3 enhances CREB activity in mouse 1.6 2 þ keratinocytes and [Mg ]o inhibits the TRPV3-mediated cell signaling. 1.2 DISCUSSION Fura-2 ratio (340/380) 2 þ 0.8 Hypomagnesemia and intracellular Mg deficiency are 0 360 720 1,080 1,440 found in many pathological conditions associated with skin Time (sec) lesions and hypersensitivity, such as type 2 diabetes mellitus and neuropathic pain (Resnick et al., 1993; Xiao and Bennett, b 1.0 1994; Tosiello, 1996; Sales and Pedrosa Lde, 2006). Hairless Control rats receiving Mg2 þ -deficient diet develop a long-lasting Mg2+ inflammatory dermatosis of unknown mechanism (Saurat et al., 1983; Ponvert et al., 1984). Interestingly, a gain-of- 0.8 function TRPV3 mutation also causes ‘‘no hair’’ phenotype, dermatitis, and pruritus in rodents (Asakawa et al., 2006; Imura et al., 2007; Xiao et al., 2008b; Yoshioka et al., 2009). As TRP channels are known to be regulated by pathophysio- 0.6 * logical concentrations of cations (Voets et al., 2003; Ahern et al., 2005; Lee et al., 2005; Obukhov and Nowycky, 2005), Relative response TRPV3 may mediate some of the Mg2 þ deficiency–induced 0.4 ** skin inflammation. This is also supported by the finding that TRPV3 function is directly enhanced by many inflammatory mediators (Hu et al., 2006). 0 2100 2 þ 2+ Our findings show that increasing [Mg ]o suppresses Mg (mM) TRPV3-mediated intracellular calcium response, membrane 2 þ 2 þ Figure 5. Inhibition of TRPV3-mediated [Ca ]i increase by [Mg ]o current, and CREB activity in primary epidermal keratino- in cultured mouse keratinocytes. (a) Averaged response of 200 cells in cytes. Therefore, our data support a model in which TRPV3 time-lapse images from a representative coverslip illustrating the inhibitory function is activated under conditions of Mg2 þ deficiency. 2 þ effect of 10 mM Mg on TRPV3 agonist cocktail-induced increase of 2 þ Hyperactivated TRPV3 may, in turn, promote release of ATP, [Ca ]i. Horizontal bars indicate the time course of chemical applications. (b) Red bars are summarized data illustrating the responses to four prostaglandins, nitric oxide, and nerve growth factor from consecutive applications of TRPV3 agonist cocktail in the absence (first epidermal keratinocytes, which are known to sensitize and fourth response) and presence of 2 mM (second response) and 10 mM nociceptors and initiate neurogenic inflammation and pain (third response) Mg2 þ . All responses are normalized to the first response. (Huang et al., 2008; Mandadi et al., 2009; Miyamoto et al., Black bars serve as non-treatment control showing the four consecutive 2011). 2 þ responses without Mg application in the control group, *Po0.05, At the single-channel level, extracellular Mg2 þ inhibits **Po0.01. 2APB, 2-aminoethoxydiphenyl borate; TRPV3, transient receptor TRPV3 mainly by reducing the single-channel conductance potential vanilloid 3 channel. without significantly affecting the open probability. More- over, we have identified an aspartic acid residue D641, neutralization of which significantly attenuated the inhibitory 2 þ 2 þ [Mg ]o attenuates TRPV3-mediated CREB signaling in mouse effect of [Mg ]o on TRPV3, suggesting that this aspartic acid 2 þ keratinocytes is required for [Mg ]o action on TRPV3. By contrast, 2 þ Calcium/CREB signaling is critical for epidermal keratinocyte [Mg ]o sensitizes and gates TRPV1 in the primary afferent proliferation (Ansari et al., 2008; Rozenberg et al., 2009) and neurons and initiates pain (Ahern et al., 2005). Although the inflammatory pain mediated by TRPV1 in neurons (Nakanishi mechanism underlying the difference between TRPV1 and 2 þ et al., 2010). To investigate whether TRPV3 activation is TRPV3 remains unknown, it seems that the effects of [Mg ]o involved in the regulation of CREB activity, we prepared on both channels are mediated by the negatively charged primary epidermal keratinocytes from transgenic mice that acidic residues in the extracellular pore loop (D641 for contain a genetically encoded CREB-dependent luciferase TRPV3 and E600/E648 for TRPV1). Interestingly, D641 is also (CRE-luc) reporter gene (Song et al., 2010). We found that the involved in the interaction between extracellular Ca2 þ and TRPV3 agonist cocktail produced a 2.5-fold increase of TRPV3 (Xiao et al., 2008a), and neutralization of this amino CREB-dependent luciferase activity in the primary keratino- acid in TRPV3 severely attenuates the blocking effect of cytes (Figure 6a). The effect of TRPV3 agonist cocktail was ruthenium red and extracellular calcium, similar to TRPV1 at nearly completely abolished by a TRPV blocker ruthenium the equivalent position D646 (Garcia-Martinez et al., 2000 red or transfection of TRPV3-specific siRNA (Figure 6a). and data not shown). As ruthenium red is also an organic

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300 a # # Using site-directed mutagenesis we screened negatively * * charged acidic residues located in the inner pore loop and 250 identified two glutamate residues between the sixth TM domain and the TRP box, E679 and E682. Neutralization 200 of these two residues nearly abolished the inhibitory effect 2 þ 2 þ of [Mg ]i. Interestingly, an increase of Mg concentration 150 in the intracellular and extracellular sides selectively depressed the outward and inward currents, respectively. 100 Moreover, change of the ratio of intracellular and extra- cellular Mg2 þ altered the rectification ratio of TRPV3 current,

Luciferase activity (%) 2 þ 50 suggesting that Mg -induced voltage-dependent inhibition contributes to the characteristic rectification of TRPV3 current (Chung et al., 2005). 0 RR Scramble TRPV3 In summary, our study has revealed the molecular Control siRNA mechanism underlying the modulation of TRPV3 by both 2 þ 2APB, 100 µM+ carvacrol, 250 µM extracellular and intracellular Mg in mouse epidermal keratinocytes. The data favor a pore-blocking model, in b 300 which Mg2 þ interacts with negatively charged acidic residues on both sides of the channel pore loop. Enhanced 250 TRPV3 function in hypomagnesemia resulting from the disinhibition by Mg2 þ may be involved in the pathogenesis 200 of magnesium deficiency–induced atopic dermatitis and pruritus, in which intracellular calcium signaling and 150 * CREB-dependent transcription are activated (Supplementary Figure S3 online). 100 MATERIALS AND METHODS Luciferase activity (%) 50 Mouse epidermal keratinocyte culture Primary epidermal keratinocytes were prepared as described 0 previously with minor modifications (Chung et al., 2004b; Miyamoto 3 10 et al., 2011) with approval by The University of Texas Health Control Mg2+(mM) Science Center at Houston Animal Welfare Committee. In brief,

2APB, 100 µM+ carvacrol, 250 µM newborn mouse pups (P0–P2) were killed and soaked in 10% povidone-, 70% , and phosphate-buffered saline for Figure 6. Suppression of TRPV3-mediated increase of CREB transcriptional 5 min, respectively. The back skin was removed and placed in a Petri activity by [Mg2 þ ] . (a) The CRE-Luc activity in control groups is set to 100% o dish containing 2.5% dispase II (Life Technologies, Grand Island, for a and b, and treated groups are normalized to this value (mean±SEM from 1 15 animals). Summarized data showing TRPV3 agonist cocktail-induced CRE- NY) and incubated at 4 C overnight. Epidermis was then separated Luc activity in the absence or presence of ruthenium red (RR) in transgenic from subcutaneous tissues. Keratinocytes were dissected by gentle mouse keratinocytes as well as transgenic mouse keratinocytes transfected scraping and flushing with CnT-07 medium (CELLnTEC Advanced with TRPV3 siRNA and control siRNA. *Po0.05 compared with control; Cell Systems, Bern, Switzerland). Harvested cells were plated on # Po0.05. (b) TRPV3 agonist cocktail-induced CRE-Luc activity in the absence coverslips coated with collagen IV in serum-free, fully supplemented or presence of different concentrations of [Mg2 þ ] in keratinocytes from o keratinocyte medium CnT-07. The culture medium was replaced transgenic mice, *Po0.05. 2APB, 2-aminoethoxydiphenyl borate; CREB, cAMP response element binding; CRE-luc, CREB-dependent luciferase; every 2 days and cells were used 48 hours later. TRPV3, transient receptor potential vanilloid 3 channel. Luciferase activity measurement Epidermal keratinocytes were cultured as described above from cation, this conserved acidic residue might be part of an individual transgenic newborn pups, containing a genomically extracellular cation sensor for both TRPV3 and TRPV1. encoded CREB-driven luciferase reporter transgene (CRE-Luc; Song 2 þ In addition to fluctuations of [Mg ]o under some et al., 2010). Copy number was maintained as hemizygous by 2 þ pathological conditions, [Mg ]i also undergoes dynamic intercrossing CRE-luciferase transgenic males with wild-type albino changes following changes of cellular metabolic state. Our C57Bl6/J-Tyrc females (The Jackson Laboratory, Bar Harbor, ME). 2 þ results show that [Mg ]i inhibited outward single-channel Newborn pups were genotyped by PCR within the luciferase gene conductance more potently than the inward single-channel (forward: 50-GCTGGGCGTTAATCAGAGAG-30; reverse: 50-TTTTCC current without affecting the NPo. This result is consistent GTCATCGTCTTTCC-30). Confluent cells (495%) were treated as with whole-cell recording data showing that Mg2 þ inhibited described in the figure legends for 4 hours and then lysed with 2 þ TRPV3 in a voltage-dependent manner, suggesting that Mg Reporter Lysis Buffer containing: 25 mM gycyllglycine (GlyGly), likely interacts with TRPV3 in an area experiencing the TM 15 mM MgSO4,4mM EGTA, 1 mM dithiothreitol, and 1% Triton electric field. X-100. Lysates were mixed 1:1 with 2 luciferase assay buffer:

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25 mM GlyGly, 15 mM K2PO4,2mM ATP, 2 mM dithiothreitol and and plotted using Clampfit 10 (Molecular Devices, Sunnyvale, assayed immediately after injection of D-luciferin (0.1 mM in 25 mM CA). Single-channel events were identified on the basis of the GlyGly, 4 mM EGTA) using a BioTek Synergy II plate reader (BioTek half-amplitude threshold-crossing criteria. P(open) was determined Instruments, Winooski, VT). Luciferase relative light units were from idealized traces as the ratio of the sum of all open durations normalized to total protein concentration determined using a BCA to the total trace duration. Values are given as the means±SEM; protein assay kit (Thermo Scientific, Rockford, IL). Data are n represents the number of measurements. expressed as fold change above baseline. Data analysis Ratiometric measurement of intracellular free calcium All data are the means±SEM for n independent observations, and Primary keratinocytes were loaded with 4 mM Fura-2 AM (Life statistical analyses were performed using Student’s t-test. Po0.05 is Technologies) in culture medium at 37 1C for 60 minutes. Cells were considered statistically significant. then washed three times and incubated in Hank’s buffered salt solution at room temperature for 30 minutes before use. Fluores- CONFLICT OF INTEREST cence at 340 and 380 nm excitation wavelengths was recorded on The authors state no conflict of interest. an inverted Nikon Ti-E microscope equipped with 340, 360, and 380 nm excitation filter wheels using NIS-Elements imaging software ACKNOWLEDGMENTS (Nikon Instruments, Melville, NY). Fura-2 ratios (F340/F380) reflects We thank Hai-Tang Li for technical support and Feng Qin for help with single- 2 þ channel analysis. We are grateful to our colleagues for comments and changes in [Ca ]i upon stimulation. Values were obtained from discussions. This project is partly supported by grants from the Texas Medical 100 to 250 cells in time-lapse images from each coverslip. Center Digestive Diseases Center (to HH, 0008355) and Mission Connect/ TIRR Foundation (to HH, 011-101), the University of Texas Health Science Center (to HH and RB), and the National Institutes of Health National Institute Molecular biology, CHO cell culture, and transfection of Diabetes and Digestive and Kidney Diseases (R01 DK092590 to RB). CHO cells were grown as a monolayer using passage numbers o30 and maintained in DMEM (Life Technologies), supplemented with SUPPLEMENTARY MATERIAL 10% fetal bovine serum (Life Technologies), 100 units ml1 peni- Supplementary material is linked to the online version of the paper at http:// cillin, 100 mgml1 streptomycin in a humidified incubator at 37 1C www.nature.com/jid with 5% CO2. The cells were transiently transfected with cDNA for mouse TRPV3 (mTRPV3) in pIRES2-EGFP (Clontech Laboratories, REFERENCES Mountain View, CA) using FuGENE 6 (Roche Applied Science, Ahern GP, Brooks IM, Miyares RL et al. (2005) Extracellular cations sensitize Indianapolis, IN) with a ratio of 1.2:5. Following transfection, the and gate receptor TRPV1 modulating pain signaling. cells were maintained in DMEM at 37 1C for 24 hours before use. J Neurosci 25:5109–16 All mutants of mTRPV3 were made using the QuikChange XL Akamatsu H, Makiura M, Yamamoto N et al. (2006) The effect of mutagenesis kit (Agilent Technologies, Santa Clara, CA) according to fexofenadine on pruritus in a mouse model (HR-ADf) of atopic the manufacturer’s directions. All mutations were confirmed by dermatitis. J Int Med Res 34:495–504 DNA sequencing. Ansari KM, Rundhaug JE, Fischer SM (2008) Multiple signaling pathways are responsible for prostaglandin E2-induced murine keratinocyte prolifera- Patch-clamp recording tion. Mol Cancer Res 6:1003–16 Whole-cell and single-channel patch-clamp recordings were per- Asakawa M, Yoshioka T, Matsutani T et al. (2006) Association of a mutation in TRPV3 with defective hair growth in rodents. J Invest Dermatol formed using an EPC 10 amplifier (HEKA Elektronik, Lambrecht, 126:2664–72 1 Germany) at room temperature (22–24 C) on the stage of an inverted Bang S, Yoo S, Yang TJ et al. (2010) Farnesyl pyrophosphate is a novel phase-contrast microscope equipped with an appropriate filter set for pain-producing molecule via specific activation of TRPV3. J Biol Chem green fluorescence protein visualization. Pipettes pulled from 285:19362–71 borosilicate glass (BF 150-86-10; Sutter Instrument Company, Bichet D, Haass FA, Jan LY (2003) Merging functional studies with structures Novato, CA) with a Sutter P-97 pipette puller had resistances of of inward-rectifier K(+) channels. Nat Rev Neurosci 4:957–67 2–4 and 8–10 MO for whole-cell and single-channel recordings, Biro T, Ko MC, Bromm B et al. (2005) How best to fight that nasty itch—from respectively, when filled with pipette solution containing 140 mM new insights into the neuroimmunological, neuroendocrine, and 1 neurophysiological bases of pruritus to novel therapeutic approaches. CsCl, 2 mM EGTA, and 10 mM HEPES with pH 7.3 and 315 mOsm l Exp Dermatol 14:225–40 in osmolarity. The extracellular solution for whole-cell recording Borbiro I, Lisztes E, Toth BI et al. (2011) Activation of transient receptor contains 140 mM NaCl, 5 mM KCl, 2 mM CaCl2,10mM , potential Vanilloid-3 inhibits human hair growth. J Invest Dermatol and 10 mM HEPES (the pH was adjusted to 7.4 with NaOH, and 131:1605–14 1 the osmolarity was adjusted to o340 mOsm l with ). Cheng X, Jin J, Hu L et al. (2010) TRP channel regulates EGFR signaling in hair A symmetrical solution with the same components as that in the morphogenesis and skin barrier formation. Cell 141:331–43 pipette solution was used in single-channel recordings. Various Chinopoulos C, Connor JA, Shuttleworth CW (2007) Emergence of a spermine-sensitive, non-inactivating conductance in mature hippo- concentrations of MgCl2 were included in the intracellular or extracellular solutions, as stated in the figure legends. The whole-cell campal CA1 pyramidal neurons upon reduction of extracellular Ca2+: dependence on intracellular Mg2+ and ATP. Neurochem Int membrane currents were recorded using voltage ramp from 100 to 50:148–58 þ 100 mV during 500 ms at holding potential of 0 mV. Data were Chung MK, Guler AD, Caterina MJ (2005) Biphasic currents evoked by acquired using Patchmaster software (HEKA Elektronik). Currents chemical or thermal activation of the heat-gated ion channel, TRPV3. were filtered at 2 kHz and digitized at 10 kHz. Data were analyzed J Biol Chem 280:15928–41

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Chung MK, Lee H, Mizuno A et al. (2004a) 2-Aminoethoxydiphenyl borate inhibited by systemic or topical SDZ ASM 981. Br J Dermatol activates and sensitizes the heat-gated ion channel TRPV3. J Neurosci 142:669–79 24:5177–82 Obukhov AG, Nowycky MC (2005) A cytosolic residue mediates Mg2+ block Chung MK, Lee H, Mizuno A et al. (2004b) TRPV3 and TRPV4 mediate and regulates inward current amplitude of a transient receptor potential warmth-evoked currents in primary mouse keratinocytes. J Biol Chem channel. J Neurosci 25:1234–9 279:21569–75 Peier AM, Reeve AJ, Andersson DA et al. (2002) A heat-sensitive TRP channel Clapham DE (2007) SnapShot: mammalian TRP channels. Cell 129:220 expressed in keratinocytes. Science 296:2046–9 Earley S, Gonzales AL, Garcia ZI (2010) A dietary agonist of transient receptor Ponvert C, Galoppin L, Burtin C et al. (1984) Noninvolvement of histamine potential cation channel V3 elicits endothelium-dependent vasodilation. and prostaglandins in the dermatosis of magnesium-deficient hairless Mol Pharmacol 77:612–20 rats. Pharmacology 28:235–40 Facer P, Casula MA, Smith GD et al. (2007) Differential expression of the Resnick LM, Altura BT, Gupta RK et al. (1993) Intracellular and extracellular capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 magnesium depletion in type 2 (non-insulin-dependent) diabetes and TRPM8 in normal human tissues and changes in traumatic and mellitus. Diabetologia 36:767–70 diabetic neuropathy. BMC Neurol 7:11 Rozenberg J, Rishi V, Orosz A et al. (2009) Inhibition of CREB function in Garcia-Martinez C, Morenilla-Palao C, Planells-Cases R et al. (2000) mouse epidermis reduces papilloma formation. Mol Cancer Res Identification of an aspartic residue in the P-loop of the vanilloid 7:654–64 receptor that modulates pore properties. J Biol Chem 275:32552–8 Sales CH, Pedrosa Lde F (2006) Magnesium and diabetes mellitus: their Henrich M, Buckler KJ (2008) Effects of anoxia, aglycemia, and acidosis on relation. Clin Nutr 25:554–62 2+ cytosolic Mg , ATP, and pH in rat sensory neurons. Am J Physiol Cell Saris NE, Mervaala E, Karppanen H et al. (2000) Magnesium. An update on Physiol 294:C280–94 physiological, clinical and analytical aspects. Clin Chim Acta 294: Henrich M, Buckler KJ (2009) Acid-evoked Ca2+ signalling in rat sensory 1–26 neurones: effects of anoxia and aglycaemia. Pflugers Arch 459:159–81 Saurat JH, Chavez P, Ponvert C et al. (1983) Skin inflammation induced by Hu H, Grandl J, Bandell M et al. (2009) Two amino acid residues determine 2- hypomagnesaemia in the rat. Br J Dermatol 109(Suppl 25):106–10 APB sensitivity of the ion channels TRPV3 and TRPV4. Proc Natl Acad Savignac M, Edir A, Simon M et al. (2011) Darier disease: a disease model Sci USA 106:1626–31 of impaired calcium homeostasis in the skin. Biochim Biophys Acta Hu HZ, Gu Q, Wang C et al. (2004) 2-Aminoethoxydiphenyl borate is a 1813:1111–7 common activator of TRPV1, TRPV2, and TRPV3. J Biol Chem Sherkheli MA, Benecke H, Doerner JF et al. (2009) Monoterpenoids induce 279:35741–8 agonist-specific desensitization of transient receptor potential vanilloid-3 Hu HZ, Xiao R, Wang C et al. (2006) Potentiation of TRPV3 channel function (TRPV3) ion channels. J Pharm Pharm Sci 12:116–28 by unsaturated fatty acids. J Cell Physiol 208:201–12 Smith GD, Gunthorpe MJ, Kelsell RE et al. (2002) TRPV3 is a temperature- Huang SM, Lee H, Chung MK et al. (2008) Overexpressed transient receptor sensitive vanilloid receptor-like protein. Nature 418:186–90 potential vanilloid 3 ion channels in skin keratinocytes modulate pain Song Y, Altarejos J, Goodarzi MO et al. (2010) CRTC3 links catecholamine sensitivity via prostaglandin E2. J Neurosci 28:13727–37 signalling to energy balance. Nature 468:933–9 Imura K, Yoshioka T, Hikita I et al. (2007) Influence of TRPV3 mutation on hair Steinhoff M, Biro T (2009) A TR(I)P to pruritus research: role of TRPV3 in growth cycle in mice. Biochem Biophys Res Commun 363:479–83 inflammation and itch. J Invest Dermatol 129:531–5 2+ Johnson JW, Ascher P (1990) Voltage-dependent block by intracellular Mg Tosiello L (1996) Hypomagnesemia and diabetes mellitus. A review of clinical of N-methyl-S-aspartate-activated channels. Biophys J 57:1085–90 implications. Arch Intern Med 156:1143–8 Kauer JA, Gibson HE (2009) Hot flash: TRPV channels in the brain. Trends Voets T, Janssens A, Prenen J et al. (2003) Mg2+-dependent gating and strong Neurosci 32:215–24 inward rectification of the cation channel TRPV6. J Gen Physiol Lee J, Cha SK, Sun TJ et al. (2005) PIP2 activates TRPV5 and releases its 121:245–60 2+ inhibition by intracellular Mg . J Gen Physiol 126:439–51 Vriens J, Appendino G, Nilius B (2009) Pharmacology of vanilloid transient Mandadi S, Sokabe T, Shibasaki K et al. (2009) TRPV3 in keratinocytes receptor potential cation channels. Mol Pharmacol 75:1262–79 transmits temperature information to sensory neurons via ATP. Pflugers Wilson JE, Chin A (1991) Chelation of divalent cations by ATP, studied by Arch 458:1093–102 titration calorimetry. Anal Biochem 193:16–9 Martin H, Richert L, Berthelot A (2003) Magnesium deficiency induces Xiao R, Tang J, Wang C et al. (2008a) Calcium plays a central role in the apoptosis in primary cultures of rat hepatocytes. J Nutr 133:2505–11 sensitization of TRPV3 channel to repetitive stimulations. J Biol Chem Miyamoto T, Petrus MJ, Dubin AE et al. (2011) TRPV3 regulates nitric oxide 283:6162–74 synthase-independent nitric oxide synthesis in the skin. Nat Commun Xiao R, Tian J, Tang J et al. (2008b) The TRPV3 mutation associated with the 2:369 hairless phenotype in rodents is constitutively active. Cell Calcium Moqrich A, Hwang SW, Earley TJ et al. (2005) Impaired thermosensation in 43:334–43 mice lacking TRPV3, a heat and camphor sensor in the skin. Science Xiao WH, Bennett GJ (1994) Magnesium suppresses neuropathic pain 307:1468–72 responses in rats via a spinal site of action. Brain Res 666:168–72 Moussaieff A, Rimmerman N, Bregman T et al. (2008) Incensole acetate, an Xu H, Ramsey IS, Kotecha SA et al. (2002) TRPV3 is a calcium-permeable component, elicits psychoactivity by activating TRPV3 channels temperature-sensitive cation channel. Nature 418:181–6 in the brain. FASEB J 22:3024–34 Yamada T, Ueda T, Ugawa S et al. (2010) Functional expression of transient Nakanishi M, Hata K, Nagayama T et al. (2010) Acid activation of Trpv1 leads receptor potential vanilloid 3 (TRPV3) in corneal epithelial cells: to an up-regulation of calcitonin gene-related peptide expression in involvement in thermosensation and wound healing. Exp Eye Res dorsal root ganglion neurons via the CaMK-CREB cascade: a potential 90:121–9 mechanism of inflammatory pain. Mol Biol Cell 21:2568–77 Yoshioka T, Imura K, Asakawa M et al. (2009) Impact of the Gly573Ser Neckermann G, Bavandi A, Meingassner JG (2000) Atopic dermatitis- substitution in TRPV3 on the development of allergic and pruritic like symptoms in hypomagnesaemic hairless rats are prevented and dermatitis in mice. J Invest Dermatol 129:714–22

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