Neuroscience Letters 397 (2006) 140–144
Ca2+-dependent PKC activation mediates menthol-induced desensitization of transient receptor potential M8 Junji Abe a , Hiroshi Hosokawa a , Yosuke Sawada a , Kiyoshi Matsumura b , Shigeo Kobayashi a,∗ a
Division of Biological Information, Department of Intelligence Science and Technology, Graduate School of Informatics, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan b Faculty of Information Science and Technology, Osaka Institute of Technology, Hirakata, Osaka 573-0196, Japan Received 5 October 2005; received in revised form 2 December 2005; accepted 4 December 2005
Abstract In 1950, Hensel and Zotterman reported cooling-induced desensitization of cold receptors by extracellular discharge recordings of cold fibers. Since then, however, its intracellular mechanism has remained unresolved. We studied menthol-induced desensitization of cold/menthol receptors (TRPM8, transient receptor potential M8) expressed in HEK cells. TRPM8 desensitization depended on extracellular Ca2+ ions, indicating that Ca2+ influx-induced [Ca2+ ]i elevation caused the desensitization. We studied whether Ca2+ -dependent kinase, PKC, mediated TRPM8 desensitization. PMA, a PKC activator, desensitized TRPM8. Inhibitor of Ca2+ -dependent PKC isozymes specifically abolished PMA-induced TRPM8 desensitization. PMA similarly desensitized wild type TRPM8 and mutant TRPM8, in which serine or threonine residues in some putative PKC phosphorylation sites were replaced by alanine. PMA treatment did not induce internalization of TRPM8. As the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated TRPM8 causes Ca2+ -dependent PKC isozymes to desensitize TRPM8 itself. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Cold/menthol receptor; TRPM8; PKC; PMA; Non-selective cation channel
Cold receptors in cold fibers innervating skin are crucial for autonomic and behavioral thermoregulation in homeothermal animals. When temperature of skin decreases from normal (ca. 33–34 ◦ C), afferent discharges appear in cold fibers, leading to heat-production responses such as shivering or cold sensation of the skin in humans. In 1950, Hensel and Zotterman reported that when oral temperature of cats decreased to a constant low temperature (13 ◦ C) in a step, discharge frequency of cold fibers suddenly increased to a peak but decreased gradually with time to a low level [6]. They attributed this transient response of nerve impulses to desensitization of cold receptors expressed in cold fibers [2]. However, intracellular mechanism of the cold receptor desensitization has remained unresolved. Recently, cold/menthol receptor (TRPM8) responding to cooling and menthol has been identified in TRP (transient receptor potential) non-selective cation channel family [9,13]. Menthol is a cooling compound
∗
Corresponding author. Tel.: +81 75 753 9133; fax: +81 75 753 3145. E-mail address:
[email protected] (S. Kobayashi).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.12.005
that elicits cold sensation in humans. In addition, we have clarified that TRPM8 is expressed in nerve endings in rat tongue [1]. These results indicate that TRPM8 acts as cold receptors. Thus, we can use TRPM8 to clarify the mechanism of desensitization of cold receptors. Repeated agonist stimulation desensitizes TRPM8 [9]. This TRPM8 desensitization depends on extracellular Ca2+ , indicating that Ca2+ influx-induced [Ca2+ ]i elevation causes the desensitisation [9]. Ca2+ -dependent protein kinase, such as protein kinase C (PKC), mediates agonist-induced phosphorylation of TRP channels [14]. PKC sensitizes capsaicin-induced current by direct phosphorylation of TRPV1 [11], whereas PKC inhibits activation of TRPC3 and TRPC5 [16]. Here, we hypothesized that Ca2+ -dependent PKC mediates menthol-induced TRPM8 desensitization, and investigated this hypothesis. We purchased l-menthol (abbreviated as menthol) from Wako Pure Chemical (Wako, Osaka, Japan). PMA and 4␣PMA were purchased from Sigma. Go6850 and Go6976 were from Calbiochem. All of the other reagents were analytical grade.
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The cDNA encoding rat TRPM8 (pcDNA3TRPM8) was kindly gifted from Dr. D. Julius. We made the deletion mutant lacking amino acid 9–22 (9–22) and single amino-acid substitution mutants (S221A, S286A, T312A, S319A, S541A, and 556A) by site-directed mutagenesis using QuickChange II sitedirected mutagenesis kit (Stratagene, La Jolla, CA, USA). All mutants were verified by DNA sequencing. Flp-In TREx293 cells (Invitrogen) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and containing penicillin (100 g/ml), streptomycin (50 g/ml), blasticidin (15 g/ml), and hygromycin B (100 g/ml) in a humidified atmosphere containing 5% CO2 . They were passaged twice a week in a 1:5 ratio to keep an exponentially growing state. To obtain tetracycline (TET)—inducible stable cell lines, wild type and mutant TRPM8 were subcloned into pcDNA5/FRT/TO (invitrogen) and were introduced into Flp-In TREx293 cells (Invitrogen) with pOG44 (Invitrogen). Transfection was performed using lipofectoamine 2000 (Invitrogen) according to manufacture’s instruction. The expression of TRPM8 and TRPM8 mutants was induced by incubation with medium containing doxycycline (1 g/ml) for 12–24 h before experiments. Intracellular Ca2+ measurement was performed by Fura2 method using CAF-110 fluorospectrometer (Nihon Bunko, Tokyo, Japan). Cells were washed with PBS, harvested with PBS containing 1 mM EGTA, and resuspended with Krebs’ Hepes buffered solution (KHB:135 mM NaCl, 5 mM KCl, 2 mM CaCl2 , 2 mM MgCl2 , 10 mM D(+)-glucose, 10 mM HEPES, pH 7.4 adjusted with NaOH). Cells were loaded with 2 M Fura-2 AM (Dojin, Tokyo, Japan) at 37 ◦ C for 45 min. After washing with KHB solution, cells were resuspended (nearly 1 × 106 cells/ml) with KHB containing 0.1% bovine serum albumin. [Ca2+ ]i measurements finished within 40 min after the start of measurement. Each cuvette contained 0.5 ml (1 × 105 cells) cell suspension with constant stirring at 800 rpm. [Ca2+ ]i was estimated by following equation: [Ca2+ ]i = Kd β(R − Rmin )/Rmax − R. Here, R is the ratio of Fura-2 fluorescence at 340 nm excitation to that at 380 nm excitation; Kd , dissociation constant, was assumed to be 224 nM; Rmax , R at a saturating concentration of Ca2+ , was obtained by adding Triton X-100; Rmin , minimum value of R, was obtained by 20 mM EDTA; β is the ratio of Ca2+ -free Fura2 fluorescence to Ca2+ -bound Fura-2 fluorescence at 380-nm excitation. Cells were stimulated by KHB with or without 100 nM PMA for 30 min at 37 ◦ C. After washing the cells, cell surface membrane protein was biotin-labeled by 2 mM sulfo-NHS-LC-biotin (Pierce) with manufacture’s instruction. To purify biotin-labeled cell surface protein, cells were homogenized in RIPA buffer (150 mM NaCl, 10 mM Tris (pH 7.2), 0.1% SDS, 1% TritonX100, 1% deoxycholate, and 5 mM EDTA). After removal of cell debris, cell lysate was incubated with 45 l of streptavidin agarose beads (50% slurry; Sigma) for 3 h at 4 ◦ C. Beads were washed three times with RIPA buffer and eluted by boiling in 45 l of x2 SDS sampling buffer.
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Whole cell lysate was used for western blotting with antiTRPM8 antibody. Cells were harvested in PBS and lysed by sonication. Protein samples were prepared with lammeli’s SDS sampling buffer, separated by 8% SDS–PAGE, and transferred to nitrocellulose membrane. Wild type and mutant TRPM8 were detected by immunoblotting with anti-TRPM8 antibody. The absorbed antibody was detected by HRP-conjugated anti-rabbit antibody (Pierce) and chemiluminescence Supersignal West Pico (Pierce) as substrate, according to manufacture’s instruction. Whole-cell patch-clamp recordings were performed as described previously [12]. KHB or Ca2+ -free KHB were used as extracellular solutions. For Ca2+ -free KHB, 2 mM CaCl2 was removed from KHB. Patch electrodes were filled with pipette solution (140 mM KCl and 10 mM HEPES, pH 7.4 adjusted with KOH). Electrode resistance was 5–10 M. Before recordings, coverslips were washed to remove culture medium and transferred to a recording chamber. The chamber was continuously perfused with extracellular solution at 1 ml/min by gravity. Seals (> 10 G) were achieved prior to rupture for whole-cell recordings. Currents were recorded in whole-cell voltage-clamp mode using EPC7 amplifier (HEKA), with filter cut-off at 2 kHz and holding potential was −60 mV. Current output was continuously sampled with MacLab (AD Instruments, Hastings, UK). We applied menthol by bath perfusion. Experiments were performed at room temperatures (20–23 ◦ C). To keep whole-cell recordings, we treated cells with PMA for 5 min. Values were displayed as mean ± S.D. Statistical significance was analyzed using unpaired t-test (p < 0.05 was considered significant). In whole-cell voltage-clamp (−60 mV) recordings, menthol caused inward current in KHB solution containing 2 mM Ca2+ in TRPM8-expressing HEK293 cells. Menthol did not induce currents in mock-trasnfected cells (data not shown). These results indicated that menthol specifically activated TRPM8 channels expressed in HEK cells. When menthol applications were repeated, amplitude of the inward current decreased, indicating gradual desensitization of TRPM8 activity (Fig. 1A, top). This desensitization was inhibited by removal of extracellular Ca2+ ions (Fig. 1A, bottom), consistent with previous study [9]. Thus, Ca2+ influx-induced [Ca2+ ]i elevation caused desensitization of TRPM8. Intracellular Ca2+ -dependent enzymes regulate activity of some cation channels, including TRP channel family [11,18]. We hypothesized that Ca2+ -activated enzymes such as conventional PKC mediated desensitization of TRPM8. We tested effects of PKC activator, phorbol 12-myristate 13-acetate (PMA), on TRPM8 desensitization. Under extracellular nominal Ca2+ free condition (Fig. 1B, top), menthol-induced inward currents decreased after 5 min PMA (100 nM) treatment. These results implied that conventional Ca2+ -dependent PKC desensitized TRPM8. We quantitatively analyzed PMA-induced desensitization with Fura-2 fluorometry of [Ca2+ ]i by using CAF110 spectrophotometer (Fig. 2). Numerous TRPM8-expressing cells in a cuvette were treated with 100 nM PMA, 100 nM 4␣-PMA (a non-functional analog of PMA), or vehicle (DMSO) for 20 min. Menthol-induced (20 M) [Ca2+ ]i elevation in PMA-treated
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Fig. 1. Ca2+ - and PMA-dependent desensitization of TRPM8 in whole-cell voltage-clamp (−60 mV) recordings. (A) Ca2+ -dependent desensitization of TRPM8. Menthol (100 M)-induced currents decreased when Ca2+ ions (2 mM) were present in extracellular solution (top), whereas menthol-induced current did not decrease when Ca2+ ions were absent in extracellular solution (bottom). (B) PMA-dependent desensitization of TRPM8. PMA treatment (top) for 5 min reduced menthol-induced currents (n = 4). DMSO treatment (bottom) for 5 min did not reduce menthol-induced currents (n = 5).
cells (232.8 ± 56.4 nM) was significantly lower than that in DMSO-treated cells (842.3 ± 85.8 nM). There was not significant difference in menthol-induced [Ca2+ ]i elevation between 4␣-PMA- and DMSO-treated cells (Fig. 2A and B). These results suggested that PMA-sensitive Ca2+ -dependent PKC was involved in TRPM8 desensitization. PMA activates conventional PKC (␣, I , II , and ␥) and novel PKC (␦, , , , and ) [10]. Because TRPM8 desensitization depends on extracellular Ca2+ , conventional PKC isozymes are candidates for TRPM8 desensitization. To study which PKC inactivates TRPM8, we examined effects of PKC inhibitors on PMA-induced TRPM8 desensitization (Fig. 2C). Conventional and novel PKC inhibitor, Go6850, inhibits ␣, I , II , ␥, ␦ and isozymes of PKC [4]. When Go6850 was applied, PMA-induced TRPM8 desensitization disappeared (725.9 ± 117.5 nM). Conventional PKC inhibitor, Go6976, inhibits PKC␣ and PKCI [8]. Go6976 similarly inhibited PMA-induced TRPM8 desensitiza-
Fig. 2. PMA-dependent desensitization of TRPM8 in [Ca2+ ]i -elevation assay. (A) Time course of 20 M menthol-induced [Ca2+ ]i elevation after treatment for 20 min with DMSO (white), PMA (mesh) and 4␣-PMA (slash). [Ca2+ ]i level at 30 s after menthol application was plotted in B and C. (B) PMA (mesh) treatment for 20 min decreased menthol-induced response (n = 4). DMSO (white) or 4␣PMA (slash) treatment for 20 min did not reduce menthol-induced response (n = 5, * p < 0.05). (C) Go6850 or Go6976 treatment for 20 min inhibits PMAdependent desensitization of menthol-induced [Ca2+ ]i elevation (n = 4).
tion (690.2 ± 153.4 nM). Thus, PKC␣ or PKCI was involved in TRPM8 desensitization. PKC is Ser/Thr kinase and recognizes consensus sequences such as (Ser/Thr)-(X)-(Arg/Lys), where X represents any amino acid (AA) [17]. Nine putative Ser or Thr residues (Ser9 , Ser12 , Ser20 , Ser221 , Ser286 , Ser312 , Ser319 , Ser541 , and Thr556 ) in TRPM8 intracellular region were candidates for PKCdependent phosphorylation. We analyzed which residue was PKC-dependent phosphorylation site, crucial for PMA-induced TRPM8 desensitization. To examine the involvement of Ser9 , Ser12 , and Ser20 residues, we made TRPM8 deletion mutant (9–22), in which Ser9 –Ser22 was deleted. To examine the
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Fig. 4. Lack of PMA-dependent internalization of TRPM8. Cell surface protein was obtained from biotin-labeled (lanes 1 and 2) and non-labeled (lane 3) cell surface. TRPM8 protein of cell surface and total lysate was detected by immunoblotting using anti-TRPM8 antibody. PMA treatment did not reduce the amount of cell-surface TRPM8 protein (lane 2). Fig. 3. PKC-dependent phosphorylation sites crucial for PMA-induced desensitization of TRPM8. (A) PMA-dependent desensitization of menthol-induced [Ca2+ ]i responses in wild type (WT) and mutant TRPM8s. White bar: mentholinduced [Ca2+ ]i elevation; bar (mesh): PMA-dependent desensitization of menthol-induced [Ca2+ ]i response. (B) Normalized PMA-dependent TRPM8 desensitization in WT and mutant TRPM8s, obtained from data as shown in (A). (C) WT and mutant TRPM8s were detected by immunoblotting (IB) with anti-TRPM8 antibody.
involvement of Ser221 , Ser286 , Thr312 , Ser319 , Ser541 , and Thr556 , we made TRPM8 mutants, in which each residue was mutated to alanine (S221A, S286A, T312A, S319A, S541A, and T556A). Menthol-induced (20 M) [Ca2+ ]i response was measured in cells expressing wild type TRPM8 or mutant TRPM8s. Mentholinduced activation in 9–22-, S221A-, and S286A-expressing cells was similar to that in wild type TRPM8-expressing cells. Menthol-induced activation decreased in T312A-, S319A, S541A-, and T556A-expressing cells (Fig. 3A). However, PMA (100 nM) treatment for 20 min similarly caused desensitization of TRPM8 in wild type TRPM8 and all of TRPM8 mutants (Fig. 3B). These results denied a view that one particular phosphorylation site in TRPM8 was crucial for PKC-dependent desensitization. PKC may indirectly inactivate TRPM8. In addition, we investigated whether PMA caused internalization of TRPM8 from surface plasma membrane to intracellular region, which might cause PMA-dependent desensitization of TRPM8. After PMA treatment for 30 min, biotinylated cell surface proteins were purified with streptavidin-agarose, and separated by SDS–PAGE. After proteins were transferred to nitrocellulose membrane, we probed TRPM8 proteins with anti-TRPM8 antibody (Fig. 4). PMA treatment did not reduce TRPM8 proteins on cell surface, indicating that PMA-dependent internalization of TRPM8 did not happen. In 1950, Hensel and Zotterman showed that a step-cooling induced desensitization of cold receptors. Since then, however, the intracellular mechanism of desensitization has not been elucidated. Here, we clarified that Ca2+ -dependent PKC was involved in the intracellular process of the desensitization by using TRPM8-expressing cells.
Pharmacological assay showed that ␣ and/or I subtype in conventional Ca2+ -dependent PKC mediated menthol-induced TRPM8 desensitization. However, PKC-induced phosphorylation on putative phosphorylation sites of TRPM8 was not involved in the desensitization. On the other hand, PKC did not cause internalization of TRPM8 from plasma membrane. These results suggested that PKC indirectly mediated menthol-induced TRPM8 desensitization. We did not identify the final targets of PKC for TRPM8 desensitization. Recent reports show that a decrease in plasma membrane PIP2 content causes TRPM8 inactivation and PLC mediates this response [7,15]. Hence, PKC might regulate PLC activity [3,5]. Further studies will be needed to clarify this point. Acknowledgement This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] J. Abe, H. Hosokawa, M. Okazawa, M. Kandachi, Y. Sawada, K. Yamanaka, K. Matsumura, S. Kobayashi, TRPM8 protein localization in trigeminal ganglion and taste papillae, Brain Res. Mol. Brain Res. 136 (2005) 91–98. [2] E.D. Adrian, The Basis of Sensation, Christophers, 1928. [3] A.M. Boon, B.J. Beresford, A. Mellors, A tumor promoter enhances the phosphorylation of polyphosphoinositides while decreasing phosphatidylinositol labelling in lymphocytes, Biochem. Biophys. Res. Commun. 129 (1985) 431–438. [4] V. Gekeler, R. Boer, F. Uberall, W. Ise, C. Schubert, I. Utz, J. Hofmann, K.H. Sanders, C. Schachtele, K. Klemm, H. Grunicke, Effects of the selective bisindolylmaleimide protein kinase C inhibitor GF 109203X on P-glycoprotein-mediated multidrug resistance, Br. J. Cancer 74 (1996) 897–905. [5] S.P. Halenda, M.B. Feinstein, Phorbol myristate acetate stimulates formation of phosphatidyl inositol 4-phosphate and phosphatidyl inositol 4,5-bisphosphate in human platelets, Biochem. Biophys. Res. Commun. 124 (1984) 507–513.
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