Warm Temperatures Activate TRPV4 in Mouse 308 Keratinocytes

Mammalian survival requires constant monitoring of environmental and body temperature. Recently, several members of the transient receptor potential vanilloid (TRPV) subfamily of ion channels have been identified that can be gated by increases in temperature into the warm (TRPV3 and TRPV4) or painfully hot (TRPV1 and TRPV2) range. In rodents, TRPV3 and TRPV4 proteins have not been detected in sensory neurons but are highly expressed in skin epidermal keratinocytes. Here, we show that in response to warm temperatures (>32 °C), the mouse 308 keratinocyte cell line exhibits nonselective transmembrane cationic currents and Ca2+ influx. Both TRPV3 and TRPV4 are expressed in 308 cells. However, the warmth-evoked responses we observe most closely resemble those mediated by recombinant TRPV4 on the basis of their electrophysiological properties and sensitivity to osmolarity and the phorbol ester, 4α-phorbol-12,13-didecanoate. Together, these data support the notion that keratinocytes are capable of detecting modest temperature elevations, strongly suggest that TRPV4 participates in these responses, and define a system for the cell biological analysis of warmth transduction. Mammalian survival requires constant monitoring of environmental and body temperature. Recently, several members of the transient receptor potential vanilloid (TRPV) subfamily of ion channels have been identified that can be gated by increases in temperature into the warm (TRPV3 and TRPV4) or painfully hot (TRPV1 and TRPV2) range. In rodents, TRPV3 and TRPV4 proteins have not been detected in sensory neurons but are highly expressed in skin epidermal keratinocytes. Here, we show that in response to warm temperatures (>32 °C), the mouse 308 keratinocyte cell line exhibits nonselective transmembrane cationic currents and Ca2+ influx. Both TRPV3 and TRPV4 are expressed in 308 cells. However, the warmth-evoked responses we observe most closely resemble those mediated by recombinant TRPV4 on the basis of their electrophysiological properties and sensitivity to osmolarity and the phorbol ester, 4α-phorbol-12,13-didecanoate. Together, these data support the notion that keratinocytes are capable of detecting modest temperature elevations, strongly suggest that TRPV4 participates in these responses, and define a system for the cell biological analysis of warmth transduction. The perception of ambient temperature is a physiological process critical to the maintenance of body temperature and the avoidance of painful or dangerous thermal extremes. Since the identification of the heat-sensing ion channel TRPV1 (1Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7090) Google Scholar), 1The abbreviations used are: TRPV, transient receptor potential vanilloid; TRPM, transient receptor potential ion channel mucolipin; NMDG, N-methyl-d-glucamine; RR, ruthenium red; 4α-PDD, 4α-phorbol-12,13-didecanoate; ANOVA, analysis of variance; HEK, human embryonic kidney; pS, picosiemens; pF, picofarads. which is activated by temperatures above 42 °C, there has been a significant focus on potential thermosensory functions for this and other ion channels of the transient receptor potential family. TRPV2 (2Caterina M.J. Rosen T.A. Tominaga M. Brake A.J. Julius D. Nature. 1999; 398: 436-441Crossref PubMed Scopus (1253) Google Scholar) and TRPV3 (3Peier A.M. Reeve A.J. Andersson D.A. Moqrich A. Earley T.J. Hergarden A.C. Story G.M. Colley S. Hogenesch J.B. McIntyre P. Bevan S. Patapoutian A. Science. 2002; 296: 2046-2049Crossref PubMed Scopus (730) Google Scholar, 4Xu H. Ramsey I.S. Kotecha S.A. Moran M.M. Chong J.A. Lawson D. Ge P. Lilly J. Silos-Santiago I. Xie Y. DiStefano P.S. Curtis R. Clapham D.E. Nature. 2002; 418: 181-186Crossref PubMed Scopus (707) Google Scholar, 5Smith G.D. Gunthorpe M.J. Kelsell R.E. Hayes P.D. Reilly P. Facer P. Wright J.E. Jerman J.C. Walhin J.P. Ooi L. Egerton J. Charles K.J. Smart D. Randall A.D. Anand P. Davis J.B. Nature. 2002; 418: 186-190Crossref PubMed Scopus (663) Google Scholar) were first described as heat transducers operative at very hot (>52 °C) and moderately warm (>34 °C) temperatures, respectively. TRPV4, which was originally identified as an osmosensory ion channel (6Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (796) Google Scholar, 7Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar, 8Wissenbach U. Bodding M. Freichel M. Flockerzi V. FEBS Lett. 2000; 485: 127-134Crossref PubMed Scopus (257) Google Scholar, 9Delany N.S. Hurle M. Facer P. Alnadaf T. Plumpton C. Kinghorn I. See C.G. Costigan M. Anand P. Woolf C.J. Crowther D. Sanseau P. Tate S.N. Physiol. Genomics. 2001; 4: 165-174Crossref PubMed Scopus (201) Google Scholar), can also be activated by warm temperatures (>34 °C) (10Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar, 11Watanabe H. Vriens J. Suh S.H. Benham C.D. Droogmans G. Nilius B. J. Biol. Chem. 2002; 277: 47044-47051Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). In addition, two TRP proteins outside of the TRPV subfamily, TRPM8 (12Peier A.M. Moqrich A. Hergarden A.C. Reeve A.J. Andersson D.A. Story G.M. Earley T.J. Dragoni I. McIntyre P. Bevan S. Patapoutian A. Cell. 2002; 108: 705-715Abstract Full Text Full Text PDF PubMed Scopus (1739) Google Scholar, 13McKemy D.D. Neuhausser V.M. Julius D. Nature. 2002; 416: 52-58Crossref PubMed Scopus (1986) Google Scholar) and ANKTM1 (ankyrin repeat/transmembrane-containing ion channel) (14Story G.M. Peier A.M. Reeve A.J. Eid S.R. Mosbacher J. Hricik T.R. Earley T.J. Hergarden A.C. Andersson D.A. Hwang S.W. McIntyre P. Jegla T. Bevan S. Patapoutian A. Cell. 2003; 112: 819-829Abstract Full Text Full Text PDF PubMed Scopus (1943) Google Scholar), have been identified as cold-activated ion channels expressed in sensory neurons. In mammals, the skin is extremely important for the transduction of thermal information and its transmission to the central nervous system. Cutaneous thermosensation has been largely attributed to the sensory nerves that innervate the dermal and epidermal layers of the skin. Recent reports, however, have suggested the possibility that other skin components, most notably keratinocytes, might also participate in temperature sensation. TRPV1 and TRPV2 are highly expressed in distinct subsets of sensory neurons (2Caterina M.J. Rosen T.A. Tominaga M. Brake A.J. Julius D. Nature. 1999; 398: 436-441Crossref PubMed Scopus (1253) Google Scholar). In humans, TRPV3 is also expressed in sensory neurons (5Smith G.D. Gunthorpe M.J. Kelsell R.E. Hayes P.D. Reilly P. Facer P. Wright J.E. Jerman J.C. Walhin J.P. Ooi L. Egerton J. Charles K.J. Smart D. Randall A.D. Anand P. Davis J.B. Nature. 2002; 418: 186-190Crossref PubMed Scopus (663) Google Scholar). In contrast, attempts to detect TRPV4 (9Delany N.S. Hurle M. Facer P. Alnadaf T. Plumpton C. Kinghorn I. See C.G. Costigan M. Anand P. Woolf C.J. Crowther D. Sanseau P. Tate S.N. Physiol. Genomics. 2001; 4: 165-174Crossref PubMed Scopus (201) Google Scholar, 10Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar) and TRPV3 (3Peier A.M. Reeve A.J. Andersson D.A. Moqrich A. Earley T.J. Hergarden A.C. Story G.M. Colley S. Hogenesch J.B. McIntyre P. Bevan S. Patapoutian A. Science. 2002; 296: 2046-2049Crossref PubMed Scopus (730) Google Scholar) at the protein level in rodent sensory neurons have been unsuccessful. Rather, immunohistochemical studies of mouse and rat skin have revealed that keratinocytes exhibit the greatest degree of cutaneous TRPV3 (3Peier A.M. Reeve A.J. Andersson D.A. Moqrich A. Earley T.J. Hergarden A.C. Story G.M. Colley S. Hogenesch J.B. McIntyre P. Bevan S. Patapoutian A. Science. 2002; 296: 2046-2049Crossref PubMed Scopus (730) Google Scholar) and TRPV4 (10Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar) expression. In addition, although peripheral sensory neurons cultured from TRPV1 knockout mice exhibit profound deficits in heat-evoked activation, the deficits observed in the intact knockout mice or in skin-nerve explants derived from these animals are considerably more modest (15Caterina M.J. Leffler A. Malmberg A.B. Martin W.J. Trafton J. Petersen-Zeitz K.R. Koltzenburg M. Basbaum A.I. Julius D. Science. 2000; 288: 306-313Crossref PubMed Scopus (2915) Google Scholar, 16Davis J.B. Gray J. Gunthorpe M.J. Hatcher J.P. Davey P.T. Overend P. Harries M.H. Latcham J. Clapham C. Atkinson K. Hughes S.A. Rance K. Grau E. Harper A.J. Pugh P.L. Rogers D.C. Bingham S. Randall A. Sheardown S.A. Nature. 2000; 405: 183-187Crossref PubMed Scopus (1492) Google Scholar). These findings suggest that TRPV3 and/or TRPV4 might mediate warmth detection and, possibly, painful heat detection at the level of the epidermal keratinocyte. The objectives of this study were to determine whether keratinocytes in vitro exhibit responsiveness to innocuous warmth and, if so, whether these responses are mediated by one or more heat-sensitive TRPV family members. We found that in the mouse 308 keratinocyte cell line (17Yuspa S.H. Kulesz-Martin M. Ben T. Hennings H. J. Invest. Dermatol. 1983; 81 (suppl.): 162-168Abstract Full Text PDF PubMed Scopus (42) Google Scholar), mild heating evokes a rise in intracellular calcium ([Ca2+]i) as well as a transmembrane cationic current, with a threshold of ∼33 °C. Moreover, the pharmacological and electrophysiological properties of these responses suggest that they are mediated, at least in part, by the activation of TRPV4. Cell Culture—The 308 cell line is a papilloma-derived keratinocyte cell line from 7,12-dimethylbenz[a]anthracene-treated adult BALB/c mouse skin (17Yuspa S.H. Kulesz-Martin M. Ben T. Hennings H. J. Invest. Dermatol. 1983; 81 (suppl.): 162-168Abstract Full Text PDF PubMed Scopus (42) Google Scholar). 308 cells were maintained at 37 °C in keratinocyte medium, which contains a 3:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum, 5 μg/ml insulin, 0.4 μg/ml hydrocortisone, 5 μg/ml transferrin, 2 × 10–9m 3,3–5′triiodo-l-thyroxine, 10–10m cholera toxin, 10 ng/ml epidermal growth factor, 60 μg/ml penicillin, and 25 μg/ml gentamicin (17Yuspa S.H. Kulesz-Martin M. Ben T. Hennings H. J. Invest. Dermatol. 1983; 81 (suppl.): 162-168Abstract Full Text PDF PubMed Scopus (42) Google Scholar). Cells were treated with 0.05% trypsin with 1 mm EDTA and replated onto glass coverslips 24–32 h prior to each experiment. Medium additives and other chemicals were obtained from Sigma unless otherwise indicated. Ca2 + Imaging—Normal bath solution (290 mOsm) used for Ca2+ imaging contained 130 mm NaCl, 3 mm KCl, 2.5 mm CaCl2, 0.6 mm MgCl2, 10 mm HEPES, 1.2 mm NaHCO3, and 10 mm glucose, adjusted to pH 7.45 with NaOH. To test the effect of osmolarity, 250 mOsm solution was made by reducing the concentration of NaCl to 105 mm. Mannitol was added to achieve 290 or 330 mOsm. Osmolarity was measured using a vapor pressure osmometer (Wescor, Logan, UT). For Ca2+-free solution, CaCl2 was replaced with 10 mm EGTA. Cells were loaded with 10 μm Fura-2/AM (Molecular Probes, Inc., Eugene, OR) in normal bath solution containing 0.02% pleuronic acid (Molecular Probes, Inc.) at 32 or 24 °C for 40 min. Coverslips containing Furaloaded cells were continuously superfused with bath solution unless otherwise indicated. Ratiometric Ca2+ imaging was performed using an inverted fluorescence microscope (Nikon, Melville, NY), excitation filter changer (Sutter, Novato, CA), and cooled CCD camera (Roper, Tucson, AZ). Paired images (340- and 380-nm excitation, 510-nm emission) were collected every 2 s with RatioTool software (ISee Imaging, Raleigh, NC). Fura ratios (emission at 340 nm excitation/emission at 380 nm excitation) were converted to absolute [Ca2+]i following system calibration (18Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Heat stimuli were delivered using an in-line heater (Warner Instruments, Hamden, CT), monitored with a thermocouple (Physitemp, Clifton, NJ) placed within 4 mm of the microscopic field, and recorded using pClamp software (Axon Instruments, Union City, CA). Drugs were applied by perfusion using an automated valve manifold (Warner Instruments). Data Analysis—[Ca2+]i was calculated for ∼40–100 randomly selected cells/coverslip, with equal numbers of cells selected in each matched set of control and experimental coverslips. To compare responses among treatment groups, we calculated the [Ca2+]i change by subtracting background [Ca2+]i (average of 5 [Ca2+]i measurements immediately prior to heat stimulation) from all observations. For determination of the fraction of responding cells, a [Ca2+]i change histogram generated using a given heat stimulation protocol was used to calculate a "response level" in each 2 °C bin from 28 to 40 °C. Cells showing a [Ca2+]i change greater than the response level, defined as the level two S.D. values above the mean of the nonresponsive cell peak in each temperature bin, were classified as responsive. Throughout this work, data are expressed as means ± S.E. (n = number of observations). Unless otherwise indicated, statistical comparisons were made with an unpaired Student's t test or one-way ANOVA. PCR Analysis—308 cells were grown to 50% confluence and scraped into TRIZOL reagent (Invitrogen) for total RNA isolation according to the manufacturer's instructions. Reverse transcription was performed in a 20-μl reaction containing 1 μg of total RNA, 500 ng of oligo(dT) primer, 1 nmol of each dNTP, 200 nmol of dithiothreitol, 2 units of RNasin (Promega, Madison, WI), and 200 units of SuperScript II RNase H (Invitrogen) at 42 °C for 50 min, followed by enzyme inactivation at 70 °C for 15 min. TRPV mRNA species were amplified via PCR by denaturation (95 °C, 30 s), annealing (53 °C, 30 s), and elongation (72 °C, 20 s) over 30 cycles. Primers used for PCR were as follows: TRPV1, 5′-ACACCAACGTGGGCATCATC-3′ and 5′-TGGTTAGATTCACAGCTCGCTTC-3′; TRPV2, 5′-GGATGGTATACCTGATGAGC-3′ and 5′-GCAAAATTCCCTACTCTACCCTGC-3′; TRPV3, 5′-ACGGTGGAGAACGTCTCC-3′ and 5′-TGTCCGTCTTATGGGTCC-3′; and TRPV4, 5′-ACCAGTACTATGGCTTCTCC-3′ and 5′-AATTCCCTACTCTACCCTGC-3′. Products were separated by electrophoresis on agarose gels, stained with ethidium bromide, and sequence-verified. Molecular Biology—Mouse TRPV1 and TRPV2 cDNAs were amplified using the reverse transcription-polymerase chain reaction on polyadenylated RNA purified from mouse dorsal root ganglion and brain, respectively (Trizol reagent; Invitrogen) and subcloned into pCDNA3 (Invitrogen). Oligonucleotide primers used were as follows: mouse TRPV1, 5′-CACTTGCTCCATTCGGAGTGTG-3′ and 5′-TGGTTAGATTCACAGCTCGCTTC-3′; mouse TRPV2, 5′-GGACGATACAGAGAAAGCTACGGC-3′ and 5′-GCAAAATTCCCTACTCATACCCTGC-3′. Mouse TRPV3 cDNA was amplified from DRG and spinal cord polyadenylated RNA using a rapid amplification of cDNA ends strategy (GeneRacer; Invitrogen). A full-length TRPV3 cDNA was obtained by the ligation of rapid amplification of cDNA ends products and subcloned into pCDNA3. The mouse TRPV4 cDNA was a generous gift of Veit Flockerzi, (Universität des Saarlandes, Homburg, Germany). Immunoblot and Immunofluorescence Analysis—Human embryonic kidney (HEK) 293 cells were cultured and transfected using LipofectAMINE 2000 (Invitrogen) as described previously (10Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar). 24 h after transfection, cells were washed once with PBS and solubilized in SDS-PAGE loading buffer. 308 cells were washed with PBS, scraped, collected by centrifugation, and homogenized in ice-cold PBS containing 1% Triton X-100 and protease inhibitor mixture (Sigma) and incubated for 2 h on ice. Solubilized proteins were separated by centrifugation (5 min, 20,000 × g). 37.5 μg of supernatant was loaded in each well. Proteins were resolved by SDS-PAGE and transferred to polyvinylidine difluoride membranes (Millipore Corp., Bedford, MA). TRPV1, TRPV2, and TRPV4 carboxyl-terminal rabbit antibodies have been described (2Caterina M.J. Rosen T.A. Tominaga M. Brake A.J. Julius D. Nature. 1999; 398: 436-441Crossref PubMed Scopus (1253) Google Scholar, 10Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar, 19Tominaga M. Caterina M.J. Malmberg A.B. Rosen T.A. Gilbert H. Skinner K. Raumann B.E. Basbaum A.I. Julius D. Neuron. 1998; 21: 1-20Abstract Full Text Full Text PDF PubMed Scopus (2586) Google Scholar). An antiserum specific for the C terminus of mouse TRPV3 was generated by conjugating the following peptide: NH2-KTTLYAFDELDEFPETSV-COOH to keyhole limpet hemocyanin, followed by immunization of rabbits (Strategic Biosolutions, Newark, DE) and affinity purification on an antigenic peptide column. Membranes were immunoblotted with anti-TRPV1, anti-TRPV2, anti-TRPV3, or anti-TRPV4 at 1:1000 dilutions. Horseradish peroxidase-conjugated donkey anti-rabbit IgG secondary antibodies (Amersham Biosciences) were used at a dilution of 1:5000. For control, the TRPV3 and TRPV4 antibodies were blocked by preincubating with antigenic peptide at a concentration of 20 μg/ml. Protein concentrations were measured using bicinchoninic acid (Pierce). For immunostaining, 308 cells were fixed with 3.7% formaldehyde in phosphate-buffered saline, blocked with 10% normal goat serum, and treated with the appropriate affinity-purified anti-TRPV antibodies in the presence of 0.3% Triton X-100, followed by Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) as described (2Caterina M.J. Rosen T.A. Tominaga M. Brake A.J. Julius D. Nature. 1999; 398: 436-441Crossref PubMed Scopus (1253) Google Scholar). Staining specificity was confirmed by signal ablation after incubation of antibodies with antigenic peptide or peptide-conjugated resin. Samples were imaged using an Ultraview laser-scanning confocal microscope (Nikon). Electrophysiology—For whole-cell recording, the recording pipette was filled with internal solution containing 120 mm cesium aspartate, 10 mm CsCl, 1 mm MgCl2, 5 mm EGTA, and 10 mm HEPES (pH 7.4 adjusted with CsOH, 295 mOsm adjusted with mannitol). Cells were initially superfused with a solution containing 130 mm sodium aspartate, 6 mm NaCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm glucose, 10 mm HEPES (pH 7.4 adjusted with NaOH, 305 mOsm adjusted with mannitol). After the establishment of whole-cell mode, the solution was exchanged with a recording solution containing 130 mm sodium aspartate, 10 mm NaCl, 1 mm MgCl2, 10 mm glucose, 10 mm HEPES (pH 7.4 adjusted with NaOH, 305 mOsm adjusted with mannitol). To minimize contamination from Ca2+-activated currents and other background currents, we omitted Ca2+ from the external solution and reduced Cl– concentration on both sides of the membrane. For the evaluation of cationic selectivity, a solution containing 120 mm N-methyl-d-glucamine (NMDG)-aspartate, 12 mm NMDG-Cl, 10 mm HEPES, 10 mm glucose (pH 7.4 adjusted with NMDG, 305 mOsm adjusted with mannitol) was used. To test Ca2+ permeability, we used a solution containing 5 mm CaCl2, 1 mm MgCl2, 30 mm calcium aspartate, 10 mm glucose, 10 mm HEPES (pH 7.4 adjusted with aspartic acid, 305 mOsm adjusted with mannitol). For single-channel recordings, pipette contained a solution composed of 130 mm sodium aspartate, 12 mm NaCl, 10 mm HEPES, 10 mm glucose (pH 7.4 with NaOH, 295 mOsm with mannitol). Cells were superfused with a solution containing 130 mm potassium aspartate, 10 mm KCl, 1 mm MgCl2, 10 mm glucose, 10 mm HEPES (pH 7.4 with KOH, 305 mOsm with mannitol) in cell-attached recordings. This perfusate was changed to internal solution (described above for whole-cell recording) just prior to patch excision for inside-out recordings. Because of their extremely flat morphology, cells were treated briefly with trypsin-EDTA just before the experiment to facilitate seal formation and measurement of single-cell membrane capacitance in whole-cell recordings. Cell-attached and inside-out recordings were performed without prior trypsinization. An Axopatch 200B amplifier (Axon Instruments, Union City, CA) connected to a Digidata 1322A analog/digital converter (Axon Instruments) was used. pClamp 8.0 software was used to acquire and analyze data. Patch electrodes were fabricated from borosilicate glass and had a tip resistance of 1.5–2 megaohms for whole-cell measurement and 2–5 megaohms for single-channel recording when filled with pipette solution. We recorded the whole-cell current only when the series resistance was less than 5 megaohms without compensation. An agar salt bridge containing 3 m KCl or NaCl was used throughout the experiment. Data were corrected for liquid junction potential calculated using pClamp. Membrane capacitance was measured using a capacitance neutralization circuit. Data were low pass-filtered at 2 kHz and digitized at 5 kHz in single-channel recordings and 3.3 kHz in whole-cell recordings. Warm Temperatures Evoke a Rise in [Ca2 + ] i in Mouse 308 Keratinocytes—We performed microscopic Ca2+ imaging on the mouse 308 keratinocyte cell line to determine whether these cells would respond to a heat stimulus with a change in [Ca2+]i. Rapid but modest increases in bath temperature (from 25 to 38°C in 10 s) (Fig. 1A) elicited substantial increases in [Ca2+]i (5.7 ± 1.2-fold, from 38 ± 5 nm to 189 ± 17 nm, p < 10–6, n = 6 coverslips) in 27% of cells. Over the entire population, the mean [Ca2+]i increased by 2.8 ± 0.4-fold (from 33 ± 3 to 87 ± 8 nm, p < 0.001, n = 6). In nearly all responsive cells, restoration of bath temperature to 25 °C caused [Ca2+]i to return to values at or near the initial baseline within 30–60 s. By applying extracellular bath solution at various peak temperatures to parallel sets of cells (Fig. 1B), we determined that these responses were temperature-dependent and that their threshold was ∼34 °C, regardless of whether it was calculated on the basis of percentage of responsive cells or mean response amplitude. Application of slower temperature ramps from 25 to 40 °C over 2 min (Fig. 1C) also produced reversible increases in [Ca2+]i in many cells. Despite the monotonic shape of these temperature ramps, responses appeared only once the ambient temperature exceeded 32 °C (a threshold just slightly lower than that observed in the rapid heating experiments) and increased in prevalence and size thereafter. At 39 °C, 46 ± 6% of cells exhibited a significant change in [Ca2+]i, with an increase among responders from 33 ± 2 to 140 ± 10 nm and an overall population increase from 31 ± 1 to 108 ± 6 nm. Sustained heat stimuli (from 25 to 37 ± 1 °C within 10 s, followed by a 50-s plateau) (Fig. 2A) produced prolonged [Ca2+]i responses, many of which commenced during the first 20–30 s of the heat stimulus, with others appearing later. The averaged population response decreased only slightly during the sustained phase of heating. However, some cells exhibited marked desensitization of heat-evoked [Ca2+]i responses during this period. In addition, when we applied several brief heat stimuli at intervals of 3 min (Fig. 2B), responses observed during the second and third stimuli were diminished in amplitude by ∼40 and ∼60%, respectively.Fig. 2Responses of 308 cells to prolonged or repetitive heat stimuli. A, kinetically complex [Ca2+]i responses during prolonged heat stimulation. Left, representative traces of heat-evoked [Ca2+]i change (bottom) with temperature plot (top). Right, population-averaged [Ca2+]i during protracted heat stimulation (bottom, open circles, n = 7 coverslips) with superimposed temperature traces (top). B, desensitization of [Ca2+]i responses to brief, repetitive heat stimuli. Left, representative [Ca2+]i traces evoked by three consecutive heat stimuli to 38 °C delivered every 3 min. Right, [Ca2+]i change (n = 5 independent coverslips) during each stimulus (*, p < 0.05, one-way ANOVA).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In order to determine the source of the heat-evoked rise in 308 cell [Ca2+]i, we performed heat stimulation after removal of extracellular Ca2+ from the bath solution. Under these conditions (Fig. 3A), the heat-evoked change in [Ca2+]i was extremely small (19% of control, n = 6, p < 10–4), suggesting that Ca2+ influx, rather than Ca2+ release from intracellular stores, was primarily responsible for the effects we observed. Further support for this notion comes from the observation that depletion of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores by pretreatment of cells with the endoplasmic reticulum Ca2+ pump inhibitor, thapsigargin (1 μm, 15 min) failed to inhibit the heat-evoked Ca2+ responses (data not shown). Together, these results demonstrate that warm temperatures evoke Ca2+ influx in 308 keratinocytes. Mouse Keratinocytes Express Both TRPV3 and TRPV4 — Given the results described above and previous demonstrations of TRPV channel expression in keratinocytes in vivo (3Peier A.M. Reeve A.J. Andersson D.A. Moqrich A. Earley T.J. Hergarden A.C. Story G.M. Colley S. Hogenesch J.B. McIntyre P. Bevan S. Patapoutian A. Science. 2002; 296: 2046-2049Crossref PubMed Scopus (730) Google Scholar, 10Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar, 20Inoue K. Koizumi S. Fuziwara S. Denda S. Inoue K. Denda M. Biochem. Biophys. Res. Commun. 2002; 291: 124-129Crossref PubMed Scopus (247) Google Scholar, 21Southall M.D. Li T. Gharibova L.S. Pei Y. Nicol G. Travers J. J. Pharmacol. Exp. Ther. 2003; 304: 217-222Crossref PubMed Scopus (258) Google Scholar), we explored whether any of the four known heat-sensitive TRPV subtypes were expressed in 308 cells. After isolation of poly(A)+ RNA, we performed reverse transcription-PCR using primers specific for mouse TRPV1–4 (Fig. 4A). DNA bands of the appropriate size and sequence for each subtype could be amplified from these samples, indicating the expression of all four at the mRNA level. Immunofluorescence microscopy using affinity-purified polyclonal peptide antibodies specific for each subtype (Fig. 4B) revealed that TRPV3 and TRPV4 proteins were both expressed at readily detectable levels in 308 keratinocytes. Curiously, the distribution of immunoreactivity for both TRPV3 and TRPV4 was most prominent in apparently intracellular compartments, especially structures in the perinuclear area. However, TRPV4 immunoreactivity was also detected on or near the plasma membranes of many cells. In contrast, immunoreactivity for TRPV2 or TRPV1 was much less convincing, despite the presence of mRNA encoding these species; whereas very weak TRPV2 immunoreactivity could be detected in some cells, no clear evidence of specific TRPV1 immunoreactivity was observed (data not shown). To verify the specificity of the TRPV3 and TRPV4 antibodies used in these experiments and their ability to recognize the corresponding mouse orthologs, we performed immunoblot analysis on whole-cell extracts from HEK293 cells transfected with cDNAs encoding mouse TRPV1, TRPV2, TRPV3, or TRPV4 (Fig. 4C). Both antibodies recognized the correct recombinant mouse protein, whereas neither exhibited cross-reactivity with other subtypes. Similarly, antibodies against mouse TRPV1 and TRPV2 recognized only the appropriate subtype (not shown). Finally, we sought to confirm the expression of TRPV3 and TRPV4 in 308 cells by immunoblot analysis (Fig. 4D). Both anti-TRPV3 and anti-TRPV4 antibodies recognized protein bands with apparent molecular weights similar to those of recombinant TRPV3 and TRPV4. The high molecular weight band marked with an asterisk in the TRPV3 blot may represent a multimeric form of this protein. The TRPV4 antibody recognized a doublet in the 308 cell extracts. A similar TRPV4 doublet has been described by others, with the higher molecular weight band proposed to represent a glycosylated form (22Xu H. Zhao H. Tian W. Yoshida K. Roullet J.B. Cohen D.M. J. Biol. Chem. 2003; 278: 11520-11527Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). TRPV4 Probably Mediates the Heat-evoked [Ca2 + ] i Increase Observed in Mouse Keratinocytes—To further examine whether one or more member(s) of the TRPV family might be responsible for the influx of Ca2+ evoked by exposure to warmth, we tested the effect of ruthenium red (RR), a channel blocker that has been shown to potently inhibit heat responses mediated by all four TRPV subtypes (1Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7090) Google Scholar, 2Caterina M.J. Rosen T.A. Tominaga M. Brake A.J. Julius D. Nature. 1999; 398: 436-441Crossref PubMed Scopus (1253) Google Scholar, 3Peier A.M. Reeve A.J. Andersson D.A. Moqrich A. Earley T.J. Hergarden A.C. Story G.M. Colley S. Hogenesch J.B. McIntyre P. Bevan S. Patapoutian A. Science. 2002; 296: 2046-2049Crossref PubMed Scopus (730) Google Scholar, 10Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar). The [Ca2+]i increase evoked by a 37 °C heat stimulus in the presence of RR (1 μm) was sig