Homozygous TRPV3-deficient (TRPV3−/−) mice were provided by Dr. Patapoutian. Wild-type (WT) and TRPV3−/− mice of C57BL/6 background were maintained under SPF conditions in a controlled environment (12-h light/dark cycle with free access to food and water, 25 °C, and 50–60% humidity). Seven weeks old male mice were used. All procedures were approved by the Institutional Animal Care and Use Committee of the National Institute of Natural Sciences and carried out according to the National Institutes of Health and National Institute for Physiological Sciences guidelines.
Reagents and antibodies
T16Ainh-A01 (T16A, Calbiochem) and Ani9 (Sigma-Aldrich) were used as an Ano1 inhibitor. Dyclonine (MedChemExpress) was used as a TRPV3 inhibitor. The following antibodies were used: rabbit anti-ANO1 antibody (Abcam, ab53213, 1:5 for Western blotting), (Abcam, ab53212, 1:100 for immunoprecipitation), rabbit anti-phospho-ERK (extracellular signal-related kinase) antibody (Cell Signaling Technology, #4370, 1:1000), rabbit anti-phospho-p38 antibody (Cell Signaling Technology, #4511, 1:1000), rabbit anti-phospho-JNK (c-Jun N-terminal Kinase) antibody (Cell Signaling Technology, #4668, 1:1000), rabbit anti-ERK antibody (Cell Signaling Technology, #4695, 1:1000), rabbit anti-p38 antibody (Cell Signaling Technology, #8690, 1:1000), rabbit anti-JNK antibody (Cell Signaling Technology, #9252, 1:1000), mouse anti-β-actin antibody (Abcam, ab6276, 1:2500), rabbit anti-TRPV3 antibody (Cell Signaling Technology, #3484, 1:1000 for Western blotting; 1:50 for immunoprecipitation), anti-rabbit-HRP antibody (Cell Signaling Technology, #7074, 1:2000) and anti-mouse-HRP antibody (Cell Signaling Technology, #7076, 1:2000).
HEK293T cells (ATCC CRT-3216) were maintained at 37 °C in 5% CO2 in Dulbecco’s modified Eagle’s medium (Wako) containing 10% fetal bovine serum (BioWest), 50 units/mL penicillin, 50 μg/mL streptomycin (Life Technologies), and 2 mM/L glutamine (GlutaMAX, Life Technologies). Normal human epidermal keratinocytes (NHEK, Adult, KURABO) were maintained at 37 °C in 5% CO2 in Humedia-KG2 (KURABO). Custom MCDB153 medium lacking NaCl (Research Institute for Functional Peptides) was used for low chloride experiments. Custom MCDB153 medium was used by adding 130 mM NaCl or 130 mM sodium aspartate and 0.1 mM O-phosphorylethanolamine (Sigma), 0.1 mM ethanolamine (Sigma), 0.5 μg/mL hydrocortisone (Sigma), 5 ng/mL epidermal growth factor (EGF, Miltenyi Biotec), and 5 μg/mL insulin (Sigma).
Primary mouse keratinocyte isolation and culture
Skin keratinocytes were isolated based on the previous description with minor modifications53. Briefly, mice tails were used for preparing the keratinocytes. The dissociated tails were incubated overnight at 4 °C in 4 mg/mL DISPASE II (Wako, 383-02281) in customized MCDB153 medium (CSR) containing 5 ug/mL insulin (Sigma, I1882), 0.4 ug/mL hydrocortisone (Sigma, H0888), 10 ug/mL transferrin (Sigma, T8158), 14.1 ug/mL phosphorylethanolamine (Sigma, P0503), 10 ng/mL epidermal growth factor (Sigma, E4127), 25 ug/mL gentamicin (Gibco, 15710064), 50 units/mL penicillin, 50 ug/mL streptomycin and 40 ug/mL bovine pituitary extracts (Kyokuto, 20200). After 16 h of incubation, the epidermis was detached and incubated with 0.25% trypsin (Gibco, 15050065) for 20 min at room temperature with the basal layer down. Keratinocytes were next mechanically dissociated with dissecting forceps and filtered through a 100-um cell strainer. Cells were next seeded on coverslips for patch-clamp experiments. All recordings were performed on day 3 and day 4 after isolation.
Total RNA was purified from NHEKs using Sepasol-RNA I Super G (Nacalai Tesque) or RNeasy Micro (QIAGEN). Reverse transcription was performed using Super Script III reverse transcriptase (Invitrogen) for 50 min at 50 °C. For investigation of mRNA expression of transient receptor potentials (TRPs), anoctamins (ANOs), and Cation-Chloride-Cotransporters (CCCs) in NHEKs, DNA fragments were amplified using EmeraldAmp PCR Master Mix (TAKARA) with PCR primers shown in Table 1. The PCR products were confirmed by electrophoresis on a 2% agarose gel containing ethidium bromide.
Proteins were extracted from NHEKs treated with 10 μM T16A or control medium for 13 h. The cells were washed with cold PBS and lysed by treatment with lysis buffer (1% Triton X-100 contained 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, 1 mM Na3VO4, and protease inhibitor cocktail, cOmplete (Roche), pH 7.5). Following centrifugation at 10,000×g for 30 s, the supernatants were denatured by treatment with SDS buffer containing 0.5 M Tris-HCl, 10% sodium dodecyl sulfate, 6% β-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue, 100 mM dithiothreitol, at 90 °C for 5 min. The protein samples were used in SDS-PAGE.
Transient transfection of HEK293 cells was achieved with Lipofectamine Transfection Reagent (Life Technologies), PLUS Reagent (Life Technologies), and Opti-MEM I Reduced Serum Medium (Life Technologies). Plasmid DNAs (hTRPV6/pcDNA3.1, hTRPV3/pcDNA3, hANO1/pcDNA3.1, or pcDNA3.1) were transfected with pGreen Lantern 1 into HEK293T cells, and the transfected cells were used for patch-clamp recording and immunoprecipitation 16–30 h after transfection.
NHEKs on coverslips were incubated at 37 °C for 30 min in Humedia-KG2 containing 5 μM Fura-2-acetoxymethyl ester (Molecular Probes). The coverslips were washed with a standard bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM d-glucose at pH 7.4, adjusted with NaOH. A calcium-free bath solution was prepared by omitting 2 mM CaCl2 from the standard bath solution and adding 5 mM EGTA. Fura-2 was excited with 340- and 380-nm wavelength lights and the emission was monitored at 510 nm with a CCD camera, Cool Snap ES (Roper Scientific/Photometrics) at room temperature. Data were acquired using IP lab software (Scanalytics) and analyzed with ImageJ software (National Institutes of Health). Ionomycin (5 μM, Sigma-Aldrich) was applied to confirm the maximal response of each cell. The high K+ bath solution contained 65 mM NaCl, 80 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM d-glucose at pH 7.4, adjusted with NaOH.
Transfected HEK293T cells, NHEKs, or isolated mouse keratinocytes were used for whole-cell recordings. Patch pipettes were made from borosilicate glass (type 8250, King Precision Glass) with a five-step protocol using a P-2000 (Sutter Instrument). The pipette resistance was 3–8 MΩ. Currents were recorded at 10 kHz using an Axopatch 200B amplifier (Molecular Devices) and filtered at 5 kHz with a low-pass filter. Currents were digitized with a Digidata 1440 A or 1550 (Axon Instruments). Data acquisition was achieved with pCLAMP 10 software (Axon Instruments). Four extracellular solutions for whole-cell recording were as follows: (1) a standard bath solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM d-glucose at pH 7.4, adjusted with NaOH); (2) an NMDG-Cl bath solution (140 mM NMDG, 140 mM HCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM d-glucose at pH 7.4, adjusted with HCl); (3) a calcium-free NMDG-Cl bath solution that was prepared by omitting 2 mM CaCl2 from the NMDG-Cl bath solution and adding 5 mM EGTA, and (4) an NMDG-aspartate bath solution that was prepared by using l-aspartic acid instead of HCl. The pipette solutions were as follows: (1) a standard pipette solution (140 mM KCl, 5 mM EGTA, 2 mM MgCl2, and 10 mM HEPES at pH 7.3, adjusted with KOH) or (2) an NMDG-Cl pipette solution (140 mM NMDG, 140 mM HCl, 5 mM BAPTA, 2 mM MgCl2, and 10 mM HEPES at pH 7.3, adjusted with HCl). CaCl2 was added to the pipette solution so that the free calcium concentration was 100 or 500 nM. Free calcium concentrations were calculated with the MAXC program at Stanford University.
Proteins were extracted from HEK293T cells after transfection. The cells were lysed as described in Western blotting. Following centrifugation at 16,100×g for 30 min, the supernatants were incubated in a rotator for 2 h with protein G Mag Sepharose (GE Healthcare). After the removal of magnetic beads, the supernatants were incubated in a rotator overnight with an anti-TRPV3 antibody or anti-ANO1 antibody. After incubation, protein G Mag Sepharose was added, and the solutions were incubated in a rotator for 2 h. After incubation, the magnetic beads were rinsed with washing buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5). The proteins were denatured by SDS buffer at 95 °C for 5 min. The protein samples were assessed by SDS-PAGE. Blotting was done with anti-TRPV3 and anti-rabbit-HRP antibodies.
Culture insert assay
NHEKs were seeded confluently in two-well culture inserts (ibidi) on glass-bottom dishes (Matsunami). Culture inserts were removed after overnight incubation, followed by washing with PBS. Cells were then cultured in Humedia-KG2, MCDB153 medium, or low chloride MCDB153 medium. After 12 or 24 h of cultivation, calcein-AM (Dojindo) was added to the culture medium to visualize the cells. ImageJ software was used for data analysis. For time-lapse analysis, cells were cultured in a Stage Top Incubator (TOKAI HIT) on a confocal laser scanning microscope (IX83 Olympus) and images were captured every 10 min.
NHEKs were seeded on 96-well plates (Falcon). Cells were cultured in a control medium, 10, 5, or 2.5 μM T16A-containing medium for 24 or 48 h. After culture, MTT assays were done using an MTT Cell Proliferation Assay Kit (Cayman). The absorbance of the formazan was measured with a microplate reader (Multiskan Spectrum, Thermo Fisher) at 570 nm.
NHEKs were seeded on glass-bottom dishes (Matsunami). Cells were incubated with 10 mM MQAE (chloride ion-quenched fluorescent indicator, Dojindo) for 60 min at 37 °C in a Stage Top Incubator (TOKAI HIT) on a confocal laser scanning microscope (LSM 510META, Carl Zeiss). MQAE was excited at 780 nm using a two-photon excitation laser system (Mai Tai, Spectra-Physics), and emission was at 458–479 nm.
The 0 mM chloride calibration solution contained 10 mM NaNO3, 140 mM KNO3, 0.5 mM Ca(NO3)2, 0.5 mM Mg(NO3)2, 10 mM HEPES, and 5 mM d-glucose at pH 7.4, adjusted with CsOH. The 100 mM chloride calibration solution contained 10 mM NaNO3, 100 mM KCl, 40 mM KNO3, 0.5 mM Ca(NO3)2, 0.5 mM Mg(NO3)2, 10 mM HEPES, and 5 mM d-glucose at pH 7.4, adjusted with CsOH. The 50 mM chloride calibration solution was made by mixing 0 and 100 mM chloride calibration solution 1:1. Each calibration solution was used by adding nigericin (monovalent cation ionophore), valinomycin (potassium ionophore) and tributyltin (chloride ionophore) so that final concentrations were 5, 10, and 10 μM, respectively. All experiments were done at 37 °C.
Cell cycle assay
NHEKs were seeded in the same way as the wound-healing assay. After removing the culture insert at 12 h, cells were stained with a Cell-Clock Cell Cycle Assay Kit (Biocolor). ImageJ software was used for data analysis. The distribution of cell cycle phases was defined as the threshold color. G2/M phase (dark blue) cells were defined as Hlu 0–255, Saturation 40–255, Brightness 0–90. S phase (green) cells were defined as Hlu 70–255, Saturation 40–255, Brightness 90–255. G0/G1 phase (yellow-green) cells were defined as Hlu 0–70, Saturation 40–255, Brightness 90–255.
Statistics and reproducibility
Data were expressed as means ± SEM. Statistical analysis was performed using the two-tailed Mann–Whitney test or one-way ANOVA to calculate the significance of differences between the two groups. Bonferroni correction or Dunnett’s test was used to calculate the significance of differences between multiple comparisons. p < 0.05 was considered to be significant.
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