Journal: Redox Biology

Article Title: Menthol evokes Ca 2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

doi: 10.1016/j.redox.2017.10.009

Figure Lengend Snippet: Detection of TRPM8 transcripts and protein in prostate and breast cancer cell lines. A ) Signals for TRPM8 mRNA were found in cell lines derived from prostate cancer-derived (lanes 1–3) and breast cancer-derived (lanes 4–6) cell lines. The normalization with the GAPDH housekeeping control allowed to semi-quantitatively assessing the abundance of the different transcripts present in each sample. B) Signals for TRPM8 protein were detected in all cell lines; the ones for MCF7 and BT-474 were weaker than for the other cell lines (expected mass: 129 kDa). The GAPDH protein signal was used as a loading control. C) Densitometry analysis. The values of TRPM8 expression for mRNA (filled columns) and protein (empty columns) levels were normalized to the values of GAPDH controls.

Article Snippet: Antibodies used and their dilutions were: anti-TRPM8 (1:200; rabbit polyclonal, Alomone Labs #ACC-049) and anti-GAPDH (1∶10,000, rabbit polyclonal, Sigma Aldrich, # SAB2100894) and anti-rabbit-HRP (1:10,000; goat secondary, Sigma-Aldrich, #A5420).

Techniques: Derivative Assay, Expressing

Journal: Redox Biology

Article Title: Menthol evokes Ca 2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

doi: 10.1016/j.redox.2017.10.009

Figure Lengend Snippet: The effect of different Ca 2+ signal modulators on menthol-evoked responses. A)- L) Single-cell (colored traces) and average fluorescence (grey traces) recordings from time-lapse videos show changes in [Ca 2+ ] cyt . Bars represent standard deviations (SD). Each figure represents the results of one representative experiment out of three with similar results. A) ATMB hydrochloride , a TRPM8 blocker administered before menthol had no effect on menthol-evoked Ca 2+ response B ) BCTC, a putative TRPM8 blocker, administered before menthol had no effect on menthol-evoked Ca 2+ responses. C) CapZ, an another putative TRPM8 blocker had no effect on menthol-evoked Ca 2+ responses. D ) Removing the extracellular Ca 2+ ions strongly reduced the menthol-evoked responses E) Cells pre-treated with 50 µM U-73122 did not show oscillations. F) Overexpression of InsP 3 −5-phosphatase hydrolyzing InsP 3 with concomitant BFP expression in transfected cells inhibited menthol-induced oscillations compared to the non-transfected cells. Blue and yellow traces represent average fluorescence recordings from cells with or without InsP 3 −5-phosphatase from the same culture dish, respectively. Asterisks represent significant differences, p < 0.05, student t -test. G-H ) A Du 145 M8KO cell clone responded to menthol ( G ) similar as control Du 145 cells ( H ). I ) Statistical analysis on the integrals of the evoked Ca 2+ responses also showed no significant differences (integrals are from 4 consecutive experiments). J) Icilin did not evoke Ca 2+ oscillations. K ) Suramin, a non-specific purinergic receptor blocker, strongly reduced the menthol-evoked responses. L ) The presence of apyrase in the extracellular milieu also strongly reduced the menthol-evoked responses indicative of a role of extracellular ATP release after menthol treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Article Snippet: Antibodies used and their dilutions were: anti-TRPM8 (1:200; rabbit polyclonal, Alomone Labs #ACC-049) and anti-GAPDH (1∶10,000, rabbit polyclonal, Sigma Aldrich, # SAB2100894) and anti-rabbit-HRP (1:10,000; goat secondary, Sigma-Aldrich, #A5420).

Techniques: Fluorescence, Over Expression, Expressing, Transfection

Journal: Redox Biology

Article Title: Menthol evokes Ca 2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

doi: 10.1016/j.redox.2017.10.009

Figure Lengend Snippet: Menthol-evoked responses in HEK-293 cells. A) Time-lapse image series of Ca 2+ waves. The acquisition rate was set to 3 s. Blue and red colors depict lower and higher fluorescence intensities, respectively. A Ca 2+ wave in HEK-293 cells was evoked by administration of 500 µM menthol. Bar represent 100 µm. B) Single-cell (colored traces) and average fluorescence (grey traces) recordings from time-lapse videos show changes in [Ca 2+ ] cyt . Bars represent standard deviations (SD). A Ca 2+ response in HEK-293 cells was evoked by administration of 500 µM menthol. C ) Non-transfected HEK-293 cells didn’t respond to 50 µM menthol, but responded, if cells were transfected with a plasmid encoding the human TRPM8 receptor (yellow trace and green traces, respectively). D) Transfected HEK-293 cells showed only very small responses to 50 µM menthol in the absence of extracellular Ca 2+ ions; see magnification of the traces in absence of extracellular Ca 2+ in the boxed red area. This indicates that essentially TRPM8 PM channels are involved in the Ca 2+ responses. C-D ) Traces show average fluorescence values with SD. Asterisks represent significant differences, p < 0.05, Student t -test. E ) EGFP-TRPM8 (green) and mCherry-ER proteins (red) showed partial co-localization (yellow color on the merged image). Bar represent 10 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Article Snippet: Antibodies used and their dilutions were: anti-TRPM8 (1:200; rabbit polyclonal, Alomone Labs #ACC-049) and anti-GAPDH (1∶10,000, rabbit polyclonal, Sigma Aldrich, # SAB2100894) and anti-rabbit-HRP (1:10,000; goat secondary, Sigma-Aldrich, #A5420).

Techniques: Fluorescence, Transfection, IF-cells, Plasmid Preparation

Journal: Redox Biology

Article Title: Menthol evokes Ca 2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

doi: 10.1016/j.redox.2017.10.009

Figure Lengend Snippet: Effects of cold on TRPM8 channel currents in Du 145 cells with and without TRPM8 (mean ± SD; n = 3 independent experiments). After whole-cell configuration (W.C.), the temperature of the patch chamber was gradually lowered from 27 °C to 15 °C by using a heat-controlled path-chamber and the currents were recorded in the patched cells. A) Recording from a Du 145 M8KO cell with cold stimulation. B) Recording from a Du 145 WT cell with cold stimulation and ACA inhibition. Corresponding I/V- relation (red traces) of currents recorded at the indicated time points 1 and 2. C ) Current densities after cold exposure. Cold-evoked TRPM8 currents were blocked by extracellular ACA (0.025 mM). ( a p < 0.001 vs. the TRPM8 knockout group. b p < 0.001 vs. wild type without ACA treatment). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Article Snippet: Antibodies used and their dilutions were: anti-TRPM8 (1:200; rabbit polyclonal, Alomone Labs #ACC-049) and anti-GAPDH (1∶10,000, rabbit polyclonal, Sigma Aldrich, # SAB2100894) and anti-rabbit-HRP (1:10,000; goat secondary, Sigma-Aldrich, #A5420).

Techniques: Inhibition, Knock-Out

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 1 Rapamycin activates recombinant TRPM8 channels and potentiates cold-evoked responses. (a) Average ± SEM Fura2 ratio time course in HEK293 cells stably expressing rat TRPM8 (CR#1 cells) during application of rapamycin (RAP) at different concentrations. A single dose was applied in individual experiments (n = 38 to 185 cells, eight experiments). (b) Dose– response curve of rapamycin effects. Data shown are means ± SEM obtained from individual peak amplitudes from experiments shown in a. (c) Average ± SEM Fura2 ratio time course in HEK293 cells stably expressing rat TRPM8 during a protocol in which rapamycin responses were monitored in the presence (2.4-mM Ca2+) and in the absence (0 Ca2+) of extracellular calcium (n = 103 cells). (d) Bar histogram summarizing the amplitude ± SEM of rapamycin responses in the presence and absence of extracellular Ca2+ (n = 257 cells, five experiments). Data shown are means ± SEM. *P<0.05, significantly different as indicated; one-way ANOVA for repeated measures followed by Bonferroni post- hoc test. (e) Average ± SEM Fura2 ratio of cold-evoked responses in CR#1 HEK293 cells in control and 30-μM rapamycin (n = 98). (f) Bar histogram summarizing the effect of vehicle or 1-, 10-, 30-μM rapamycin on cold-evoked responses. Individual cold responses have been normalized to the amplitude during the first cold ramp (71 to 92 cells per condition). Ratios >20 have been excluded from the analysis. Data shown are individual values with means ± SEM. *P<0.05, significantly different from vehicle; one-way ANOVA followed by Bonferroni post-hoc test.

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Recombinant, Stable Transfection, Expressing, Control

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 2 Rapamycin activates mouse and human TRPM8 orthologues and the menthol-insensitive mutant. (a) Average ± SEM Fura2 ratio time course in HEK293 cells expressing mouse TRPM8. Three different behaviours were observed; cells which responded to a first cold ramp in control conditions (blue trace), cells which did not respond to this cold ramp in control conditions but responded to cold in the presence of rapamycin (RAP) or menthol (Ment; green trace), and cells which did not show any response to the applied stimuli (black trace). (b) Bar histogram summarizing the individual and mean responses of the cold-sensitive HEK293 cells (n = 162, five experiments). Data shown are individual values with means ± SEM. *P<0.05, significantly different as indicated; one-way ANOVA followed by Bonferroni post-test. (c) Average ± SEM Fura2 ratio time course in HEK293 cells expressing human TRPM8. The different traces represented the three behaviours described in a. (d) Bar histogram summarizing the responses of the cold-sensitive HEK293 cells (n = 178, five experiments) transfected with human TRPM8. Data shown are individual values with means ± SEM. *P<0.05, significantly different as indicated; one-way ANOVA followed by Bonferroni post-test. (e) Average ± SEM Fura2 ratio time course in HEK293 cells expressing mouse TRPM8-Y745H. The different traces represented the three behaviours described in a. (f) Bar histogram summarizing the responses of the cold-sensitive HEK293 cells (n = 182, five experiments) transfected with mouse TRPM8-Y745H. Data shown are individual values with means ± SEM. *P<0.05, significantly different as indicated, n.s., not significant; one-way ANOVA followed by Bonferroni post-test.

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Mutagenesis, Expressing, Control, Transfection

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 3 Rapamycin (RAP) activates TRPM8 currents. (a) Representative time course of whole-cell current at 100 and +100 mV in a HEK293 cell transiently transfected with mTRPM8 during the sequential application of different agonists. The bottom trace shows the simultaneous recording of the bath temperature. (b) Current–voltage (I-V) relationship obtained by a 400-ms voltage ramp from 100 to +150 mV during the experiment shown in (a). The colour of individual traces matches the colour at each particular time point in (a). (c) Bar histogram of current density values at 100 and +100 mV evoked by the different stimuli shown in (a), with the same colour code (n = 9 cells). Data shown are individual values with means ± SEM. *P<0.05, significantly different as indicated; one-way ANOVA, followed by Bonferroni's post-hoc test. (d) V1/2 values calculated from fitting individual I-V curves to a linearized Boltzmann equation. Data shown are individual values with means ± SEM. *P<0.05, significantly different from control, &P<0.05, significantly different as indicated; one-way ANOVA, followed by a Bonferroni's post-hoc test. (e) Representative whole-cell current at 100 and +100 mV in a HEK293 cell transiently transfected with mTRPM8 during a protocol in which the effect of AMTB on rapamycin response was explored. Bottom trace corresponds to the simultaneous recording of bath temperature. (f) Current–voltage relationship (I-V) obtained with a 400 ms voltage ramp from 100 to +150 mV of the responses plotted in e. the colour of the I-V curves matches the coloured time points in e. (g) Bar histogram of individual and mean ± SEM current density values at +100 mV evoked by the different stimuli shown in ( e). Data shown are individual values with means ± SEM; n = 5 cells. *P<0.05, significantly different from control, &P<0.05, significantly different as indicated; one-way ANOVA, followed by Bonferroni's post-hoc test.

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Transfection, Control

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 4 Rapamycin activates single TRPM8 channels. (a) Single channel recordings at +100 mV in the cell-attached configuration in HEK293 cells transfected with mTRPM8. The same patch in control (i.e., high K+), rapamycin (RAP; 30 μM) and WS-12 (10 μM). The coloured ticks mark the expanded traces shown below. (b) All-point histograms of current amplitudes for the different recording conditions. The histograms have been fitted with the sum of two Gaussians (red line). (c) Single channel amplitudes of individual patches at different membrane potentials. The dotted lines represent linear fits to the data. (d) Open probability of channel current at different potentials. The dotted lines are fits to the Boltzmann function.

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Transfection, Control, Membrane

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 6 TRPM8 is the principal determinant of rapamycin responses in mouse DRG neurons. (a) Representative traces of Fura2 ratio fluorescence in a Trpm8EGFPf/+ DRG culture. Consecutive applications of cold, rapamycin (RAP, 30 μM), menthol (100 μM), AITC (100 μM), capsaicin (100 nM) and high K+ (30 mM) were used to phenotype each neuron. The green trace corresponds to a EGFPf(+) neuron, which responds to cold, rapamycin, menthol and capsaicin. The other two traces (blue and red) correspond to EGFPf() neurons. Bottom trace shows the simultaneous recording of bath temperature. (b) Venn diagram summarizing the responses to rapamycin in EGFPf(+) and EGFPf() neurons in Trpm8EGFPf/+ DRG cultures. (c) Bar histograms of the amplitude of the responses to different agonists in EGFPf(+) rapamycin-sensitive neurons (n = 20, five experiments). Data shown are individual values with means ± SEM. *P<0.05, significantly different as indicated; one-way ANOVA for repeated measures followed by a Bonferroni's post-hoc test. (d) Correlation between the amplitude of the cold-evoked response and the rapamycin response (n = 20, six experiments). The horizontal dotted line marks the threshold level for rapamycin sensitivity. Green circles represent the three EGFPf(+), cold-sensitive, rapamycin-insensitive neurons recorded. (e) Representative traces of Fura2 ratios in cultured neurons from Trpm8 KO mouse. The protocol was the same as in (a). Orange trace corresponds to a EGFPf(+) neuron that was rapamycin- sensitive. Green trace represents a EGFPf(+) neuron which was not sensitive to any of the agonists tested and the red and blue traces are examples of two EGFPf() neurons. Bottom trace corresponds to the simultaneous recording of the bath temperature during the experiment. (f) Venn diagram summarizing the responses to rapamycin in EGFPf(+) and EGFPf() neurons in Trpm8EGFPf/EGFPf DRG cultures (eight experiments).

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Fluorescence, Cell Culture

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 7 Rapamycin (RAP) increases the excitability of cold-sensitive DRG neurons. (a) Representative whole-cell recording in the voltage- clamp configuration (Vhold = 60 mV) of a TRPM8-expressing, cold-sensitive DRG neuron identified by EYFP expression. Bottom trace corresponds to the simultaneous recording of bath temperature. Rapamycin activates a small inward current and potentiates the response to cold. (b) Temperature dependence of the cold-evoked current in the same neuron in control conditions and in the presence of 30 μM rapamycin. (c) Bar histogram of peak inward current density values evoked by cold, rapamycin and cold in the presence of rapamycin (n = 12). Data shown are individual values with means ± SEM. #P<0.05, significantly different as indicated; one-way ANOVA for repeated measures followed by Bonferroni's post-hoc test. (d) Representative recording of a cold-sensitive neuron in the whole-cell current-clamp configuration. Cold and rapamycin elicited the firing of action potentials. The combined application of cold and rapamycin led to faster firing, followed by a strong depolarization and the blockade of spikes. (e) Bar histogram of responses, measured as average firing frequency, during the different stimuli applied (n = 11). Firing frequency for cold was the average from the first to the last spike during the cooling ramp. Firing frequency in control conditions was calculated during the 60 s previous to rapamycin application. Rapamycin-evoked firing was calculated from the first spike during rapamycin application to the start of the cold ramp. *P<0.05, significantly different from control, #P<0.05, significantly different as indicated; one- way ANOVA for repeated measures followed by Bonferroni's post-hoc test.

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Expressing, Control

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 9 Mathematical modelling of the effects of rapamycin (RAP) on cold thermoreceptor activity. (a) Membrane potential (blue trace), temperature (red trace), and action potential firing frequency (blue dots), as a function of time. The green bar indicates the period of application of agonist (i.e. a change of -100 mV in V1/2 in this example). The red dots in the temperature trace indicate where the temperature threshold has been reached. The black dashed lines in the lower panel indicate the value of the average firing frequency, and the time range where it has been calculated (i.e. the duration of the temperature ramps). (b) Firing temperature threshold as a function of the shift in V1/2. Each trace corresponds to a cell with different parameters, which have been selected in order to encompass a wide range of thresholds under control conditions. The larger dot corresponds to the cell and conditions simulated in panel (a). (c) Same as in panel B for the average firing frequency during cold stimulation. (d) TRPM8 current density (blue trace) as a function of time, and changes in temperature (red trace) in voltage-clamp simulations. The green horizontal bar represents the application of agonist, i.e. a shift of -100 mV in V1/2 as in (a). (e) TRPM8 current density as a function of temperature with (green trace) or without agonist (blue trace). (f) Peak cold-evoked current as a function of the shift in V1/2 for the same cells as shown above with the corresponding colours. Values for V1/2 = 0 correspond to the cold-evoked current in the absence of agonist. The magenta line corresponds to a neuron with very small TRPM8 currents that did not fire action potentials and thus is not shown in the current-clamp panels above. Color code is the same for panels b, c, e and f. The set numbers in panel f correspond to the row numbers within the table found in Extended Data Figure 11-1 of Rivera et al. (2021), detailing the simulation parameter values.

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Activity Assay, Membrane, Control

Journal: British journal of pharmacology

Article Title: The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

doi: 10.1111/bph.16402

Figure Lengend Snippet: FIGURE 10 Rapamycin stimulates tearing by a TRPM8-dependent mechanism. Effect of topical solutions of rapamycin (RAP; 1%) or vehicle on tearing in mice. Tearing is represented as the length of staining in the threads (in mm) placed in the eye. Each dot corresponds to the tearing measured in an eye of an individual mouse (15 WT and 15 Trpm8 KO). Data shown are individual values of evoked tearing, with means ± SEM. *P < 0.05, significantly different as indicated; n.s., not significant; Mann– Whitney test).

Article Snippet: When necessary, we co-transfected the cells with 1 μg of TRPM8 channel plasmid (from different species) and 0.5 μg of EGFP or mCherry plasmid (Addgene_84329) (van Unen et al., 2016).

Techniques: Staining, MANN-WHITNEY

Journal: Neurology: Genetics

Article Title: Trigeminal Neuralgia TRPM8 Mutation

doi: 10.1212/NXG.0000000000000550

Figure Lengend Snippet: Average changes in the Fura-2 ratio of human embryonic kidney 293 cells transfected with wild-type TRPM8 (A) and R30Q mutant (B), in the continued presence of menthol (100 μM; arrow indicates time of addition). (C) Average increase in Fura-2 ratio in response to menthol (100 μM) in nontransfected cells (NT), wild-type TRPM8, and R30Q transfected cells (n = 10 experiments, 4 transfections). * p < 0.05, unpaired Student's t test. (D) Basal fura-2 ratio in wild-type TRPM8 and R30Q mutant transfected cells (n = 15 experiments, 4 transfections). * p < 0.05, unpaired Student t test. TRPM8 = transient receptor potential melastatin 8.

Article Snippet: The wild-type TRPM8 plasmid was obtained from GenScript, in which the insert was cloned in-frame with 2A-GFP at the C-terminus of the channel in the vector pcDNA3.1.

Techniques: Transfection, Mutagenesis

Journal: Neurology: Genetics

Article Title: Trigeminal Neuralgia TRPM8 Mutation

doi: 10.1212/NXG.0000000000000550

Figure Lengend Snippet: (A) Representative whole-cell current traces through wild-type TRPM8 and R30Q transfected cells, in response to the indicated voltage step protocol. (B) Steady state current-voltage relationships of the basal whole-cell currents for wild-type TRPM8 and R30Q mutant (n = 12, ** p < 0.01, *** p < 0.001, 2-way analysis of variance with Bonferroni post hoc test). (C) Steady-state activation curves of wild-type TRPM8 and R30Q transfected cells. The normalized conductance (G/Gmax) was plotted against voltage and fitted with a Boltzmann function, giving rise to V 1/2 and slope factor as follows: TRPM8, 153 ± 8 mV and 30 ± 3 mV (n = 12 cells, 3 transfections); R30Q, 115 ± 5 mV and 28 ± 2 mV (n = 12 cells, 3 transfections). ** p < 0.01, unpaired Student's t test. (D) Representative time courses (left) recorded at +80 mV and −80 mV (black and grey curves, respectively) and I-V traces (right) of whole cell currents through wild-type TRPM8 transfected cells, in the presence of 100 μM menthol, at the indicated time intervals. (E) Same as D), except that time courses and I-V traces are recorded from R30Q transfected cells. (F) Pooled data of whole-cell current (at +80 mV and −80 mV) evoked by 100 μM menthol, from wild-type TRPM8 and R30Q transfected cells. Each column represents mean ± SEM of n = 8 cells, 3 independent experiments. * p < 0.05 (unpaired Student's t -test). TRPM8 = transient receptor potential melastatin 8.

Article Snippet: The wild-type TRPM8 plasmid was obtained from GenScript, in which the insert was cloned in-frame with 2A-GFP at the C-terminus of the channel in the vector pcDNA3.1.

Techniques: Transfection, Mutagenesis, Activation Assay