Expression vectors
Human TRPM8 cDNA was a gift from Dr. Ramachandran. TRPM8-YFP was cloned into pEYFP-N1 using AfeI/XhoI restriction sites. Phosphorylation sites were identified using PhosphoSitePlus (https://www.phosphosite.org/homeAction). Mutations of the two TRPM8 serine residues 1040 and 1041 were done using site-directed mutagenesis; the mutations were verified by sequencing and subcloned into pcDNA3 vector to eliminate undesirable mutations elsewhere in the vector. Rat MOR was previously described 20. The PKC-βII-GFP was a gift from Dr. S. Ferguson. The PAR2 was a gift from Dr. M. Hollenberg. CB1, α1AR and α2AR plasmids were obtained from the cDNA resource center (https://www.cdna.org/faq.html). Constructs were confirmed with DNA sequencing (U of Calgary core DNA service).
Cell Culture And Transient Transfection
Human embryonic kidney (HEK) 293 tsA-201 cells were grown to 80% confluence at 37°C (5% CO2) in Dulbecco’s modified Eagle’s medium (+ 10% fetal bovine serum, 200 units/ml penicillin and 0.2 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA)). Cells were dissociated with trypsin (0.25%)-EDTA before plating on glass coverslips (for electrophysiology and calcium imaging) or plastic tissue culture plates (for biochemistry). Cells were transfected with 1.5 µg of TRPM8 and 1 µg of MOR using the calcium phosphate method. For electrophysiology, 0.3 µg of green fluorescent protein (GFP) was added as a transfection marker. Cells were washed 8 hours after transfection; electrophysiological recordings or calcium imaging were conducted 24 hours later while biochemistry was performed 48 hours after transfection.
Isolation Of Drg Neurons
DRG neurons were excised from 6 week old mice and enzymatically dissociated in Hank’s balanced salt solution (HBSS) containing 2 mg/ml collagenase type I and 4 mg/ml dispase (Invitrogen) for 45 min at 37 °C. DRGs were rinsed twice in HBSS and once in Neurobasal A culture medium (Thermo Fisher Scientific) supplemented with 2% B-27, 10% heat-inactivated fetal bovine serum (HI-FBS), 100 µg/ml streptomycin, 100 U/ml penicillin, 100 ng/ml of Nerve Growth Factor (NGF) and 100 ng/ml glial cell-derived neurotrophic factor (GDNF) (all from Invitrogen). Individual neurons were dispersed by trituration through a fire-polished glass Pasteur pipette in 4 ml media and cultured overnight at 37 °C with 5% CO2 in 96% humidity on glass coverslips previously treated 25% Poly-Ornithine and Laminin (both from Sigma).
Biotinylation Assay
Cells were passaged onto 60 mm plate and transfected with TRPM8-YFP. Forty eight hours after transfection, surface proteins were biotinylated using 0.75 mg/ml EZ Link Sulfo-NHS-SS biotin (Invitrogen) in HBSS, then quenched with 100 mM glycine. Cells were then harvested and lysed in RIPA (0.1% SDS, 1% Triton X-100 and 0.5% Na deoxycholate in PBS) with HALT protease inhibitor (Invitrogen) for 45 minutes. Protein concentration of lysates was determined using the Bradford assay and 1 mg of lysate was precipitated on Neutravidin beads (Thermo Fisher) for 2 hours. Beads were washed in HBSS and bound proteins eluted in 4X Laemmli buffer (200 mM Tris, 8% SDS, 40% glycerol, 400 mM beta-mercaptoethanol, and 0.04% Coomassie blue). Biotinylated proteins were then separated by SDS-PAGE, transferred to nitrocellulose membranes and probed for GFP using rabbit GFP antibody (Chromotek; 1:5000). The membrane protein Na+/K + ATPase (Cell Signaling antibody) was used for normalization of TRPM8 signal.
Co-immunoprecipitation And Western Blotting
Cells were harvested 48 hours after transfection with 7.5 µg of TRPM8-HA, PKCβII-GFP and MOR cDNA and lysed in RIPA (0.1% SDS, 1% Triton X-100 and 0.5% Na deoxycholate in PBS) with Halt protease inhibitor (Thermo Scientific) and phosphatase inhibitor (Thermo Scientific) for 20 minutes. Cell lysates were precipitated with the ChromoTek GFP-Trap® coupled to agarose beads and incubated with overnight rotation at 4 °C. Beads and precipitates were washed 3x in RIPA then eluted from beads in 1x Laemmli buffer. Samples were run on 8% tris-glycine gels and transferred to nitrocellulose membranes, then probed with rabbit anti-GFP (Torrey Pines) 1:5000 or the monoclonal anti HA (Covance) followed by ECL-optimized anti-rabbit (GE Healthcare).
RNA Extraction And RT-qPCR
DRGs were harvested after control or prolonged morphine treatment, dissociated using a bullet blender (Next Advance) with SSB02 beads (Next Advance) in RLT buffer (Qiagen, Toronto, Ontario, Canada). Total RNA was extracted using an RNeasy Mini kit (Qiagen), according to the manufacturer's instructions. The quality and quantity of RNA were determined using a Nanodrop 2000c spectrophotometer (Thermo-Fisher Scientific, Montréal, Quebec, Canada). Relative MOR, TRPM8, Potassium channel subfamily K member 4 (TRAAK) and Potassium channel subfamily K member 2 (TREK-1) gene expresion (normalized to Ribosomal Protein Lateral Stalk Subunit P1 (RPLP)) was determined by qPCR using BrightGreen PCR Master Mix (ABMgood) and a StepOnePlus real-time PCR detection system (Applied Biosystems, Burlington, Ontario, Canada). The primers used are included in the Supplementary Table 1 and were designed to amplification of DNA.
Calcium Imaging
Transfected HEK cells or isolated DRGs were loaded with Fura-2-AM (0.5 µM for 30 min, Invitrogen) before imaging. The recordings were performed at 37ºC. We perfused either menthol (100 µM, Sigma) or extracellular solution at 25ºC, at a rate of ~ 1 ml/min, to examine TRPM8 activity in control conditions or in cells treated with morphine (500 nM) for 16 hrs. Images of cell-field were continuously recorded every 100 ms using 340 and 380 nm excitation wavelengths with emission measured at 520 nm with a microscope based imaging system (Olympus IX73) on CellSense software. Images were processed using ImageJ by drawing discrete regions of interest around cells that responded to menthol or cold. We expressed a change in fluorescence as a percentage change from the amplitude of the first response to menthol or cold.
Behavior Analysis
6-week old C57BL/6 male mice were obtained from Jackson Laboratory (USA) and were acclimated for 2 days prior to behavioral experiments. Mice were housed with free access to food and water, with a 12/12 light dark cycle. All experiments were conducted on age-matched animals, under protocols approved by the University of Calgary Animal Care Committee and in accordance with the international guidelines for the ethical use of animals in research and guidelines of the Canadian Council on Animal Care.
Behavioral assessment of cold sensitivity in mice was done using the cold plate test. Mice received intraperitoneal injection of escalating doses of morphine (from 10 mg/kg to 50 mg/kg) or saline, twice daily for 5 days. Cold sensitivity was measured using the cold plate test (Bioseb). Briefly, mice were placed on the surface of a metal plate cooled at 0° C (with an ambient temperature of 21° C). The time taken for each mouse to show the first nociceptive response (paw withdrawal, shaking, liking of the rear paw, or jumping to try to escape) was monitored on the first and last day of the morphine injection. An experimenter blinded to the treatments performed the behavioral assessment. Data points represent each individual mouse.
Electrophysiological Measurements
Electrophysiological recordings were conducted using an external solution containing (in mM): 140.0 NaCl, 1.5 CaCl2, 2.0 MgCl2, 5.0 KCl, 10.0 HEPES, 10.0 D-glucose, pH 7.4 adjusted with NaOH. HEK cells expressing the transfected TRPM8 channel and MOR were identified via GFP fluorescence using an inverted epi-fluorescence microscope (Olympus IX51, Olympus America Inc., USA). DRG neurons were recorded based on size, knowing that TRPM8 is expressed in a subpopulation of small neurons 21. Membrane currents were measured using conventional whole-cell patch clamp and action potentials were recorded using current clamp. Borosilicate glass (Harvard Apparatus Ltd., UK) pipettes were pulled and polished to 2–5 MΩ resistance with a DMZ-Universal Puller (Zeitz-Instruments GmbH., Germany). For the voltage clamp experiments pipettes were filled with an internal solution containing (in mM): 120.0 CsCl, 10.0 EGTA, 10.0 HEPES, 3.0 MgCl2, 2.0 ATP Na2, 0.5 GTP, pH 7.2 adjusted with CsOH whereas for the current clamp experiments the internal solution contained (in mM): 140.0 KCl, 5.00 NaCl, 1 CaCl2, 1.0 EGTA, 10.0 HEPES, 1.0 MgCl2, 3.0 ATP Na2, pH 7.3 adjusted with KOH. All solutions were prepared and used at room temperature (22 ± 2ºC) and their osmolarity adjusted to 310 mOsm. Data obtained with different types of internal solutions were not pooled. For the current clamp experiments the spontaneous activity of the DRG neurons was recorded at room temperature (~ 22⁰C) for three minutes before application of the first cold (10⁰C) or menthol challenge (applied to the bath at ~ 1000 µm from the cell at a rate of 500 µl/min). Only the neurons in which the resting membrane potential was more negative than − 40 mV were used. Recordings were performed using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). Voltage- and current-clamp protocols were applied using pClamp 10.4 software (Axon Instruments). Data were filtered at 1 kHz (8-pole Bessel) (whole cell voltage clamp) and 5 kHz (current clamp) and digitized at 10 kHz with a Digidata 1550 A converter (Axon Instruments). Average DRG neurons capacitance was 11.26 ± 0.69 pF for naive and 10.59 ± 0.98 pF for morphine-treated animal. HEK cells had an average capacitance of 26.78 ± 2.15 pF. Only the cells that exhibited a stable voltage control throughout the recording were used for analysis.
For prolonged morphine treatment, neurons were incubated with a low concentration of morphine (500 nM) for 16 hours.
Chemicals And Drugs
Menthol, DAMGO, morphine sulfate and Y27632 were obtained from Sigma-Aldrich. The PKC inhibitor GF109203X, and TRPM8 antagonist AMTB (N-(3-Aminopropyl)-2-[(3-methylphenyl) methoxy]-N-(2-thienylmethyl) benzamide hydrochloride) and H89 were purchased from Tocris Bioscience. The specific PKCβ blocker Enzastaurin (LY317615) was purchased from Selleckchem and Naloxone from Santa Cruz Biotechnology.
Statistics
Data analysis and offline leak subtraction were completed in Clampfit 10.4 (Axon Instruments), and all electrophysiology curves were fitted using Origin 7.0 analysis software (OriginLab, Northampton, MA, USA). The menthol and cold dose-response relationships were fitted using the Hill equation. All averaged data are plotted as mean ± SEM and numbers in parentheses reflect the number of cells (n). Statistical analyses were completed with Origin 7.0 software using unpaired t-tests when data were compared from two independent groups of cells. One-way analysis of variance (ANOVA) followed by the Bonferroni or Sidak’s post hoc test was used for multiple comparisons, with the criterion for statistical significance set at p < 0.01.