The TRPM8 protein is a testosterone receptor: II. Functional evidence for an ionotropic effect of testosterone on TRPM8

Abstract

Testosterone is a key steroid hormone in the development of male reproductive tissues and the regulation of the central nervous system. The rapid signaling mechanism induced by testosterone affects numerous behavioral traits, including sexual drive, aggressiveness, and fear conditioning. However, the currently identified testosterone receptor(s) is not believed to underlie the fast signaling, suggesting an orphan pathway. Here we report that an ion channel from the transient receptor potential family, TRPM8, commonly known as the cold and menthol receptor is the major component of testosterone-induced rapid actions. Using cultured and primary cell lines along with the purified TRPM8 protein, we demonstrate that testosterone directly activates TRPM8 channel at low picomolar range. Specifically, testosterone induced TRPM8 responses in primary human prostate cells, PC3 prostate cancer cells, dorsal root ganglion neurons, and hippocampal neurons. Picomolar concentrations of testosterone resulted in full openings of the purified TRPM8 channel in planar lipid bilayers. Furthermore, acute applications of testosterone on human skin elicited a cooling sensation. Our data conclusively demonstrate that testosterone is an endogenous and highly potent agonist of TRPM8, suggesting a role of TRPM8 channels well beyond their well established function in somatosensory neurons. This discovery may further imply TRPM8 channel function in testosterone-dependent behavioral traits.

Keywords: Androgen; Androgen Receptor; Calcium Channel; Cold and Menthol Receptor TRPM8; Ion Channel; Testosterone; Transient Receptor Potential Channels (TRP Channels).

FIGURE 1.

Testosterone-induced TRPM8 activity in intracellular Ca²⁺ measurements in different cell types. Fluorescence measurements of the intracellular Ca²⁺ concentration were performed on prostate PC3 cells and DRG neurons transiently expressing TRPM8. a–d, fluorescence measurements obtained from PC3 cells that were transiently transfected with the TRPM8 (0.7 μg) and GFP (0.2 μg) constructs. a, representative recording of the menthol-induced activation of TRPM8 channels (n = 6). b, representative recording of the testosterone-induced responses of TRPM8 channels (n = 8). c, TRPM8 channels were also induced by an impermeable variant of testosterone that was conjugated to BSA (BSA-Testosterone), indicating that at least one of the androgen-interacting sites is located on the extracellular side of the TRPM8 protein (n = 6). d, summary of the menthol- and testosterone-induced TRPM8 responses of PC3 cells (error bars, S.E.). e–h, fluorescence measurements obtained from DRG neurons transiently transfected with the TRPM8 (3 μg) and GFP (0.5 μg) constructs. e, representative recording of the menthol-induced activation of TRPM8 channels (n = 4). f, representative recording of the testosterone-induced responses of TRPM8 channels (n = 6). g, the control recording of DRG neurons that were not transfected with TRPM8 shows no response to either testosterone or menthol (n = 3). h, summary of the menthol- and testosterone-induced TRPM8 responses of DRG neurons (error bars, S.E.).

FIGURE 2.

Testosterone-induced TRPM8 activity in intracellular Ca²⁺ measurements in hippocampal neurons. a and b, fluorescence measurements obtained from hippocampal neurons transiently transfected with TRPM8 (3 μg) and GFP (0.5 μg) constructs. a, representative recording of the menthol-induced activation of TRPM8 channels in hippocampal neurons (n = 3); b, representative recording of the testosterone-induced responses of TRPM8 channels in hippocampal neurons (n = 8). c, summary of the menthol- and testosterone-induced TRPM8 responses in hippocampal neurons (error bars, S.E.). d, immunocytochemistry image of the transiently expressed TRPM8 protein in hippocampal neurons, as detected with anti-Myc-IgG. The images were obtained with an Olympus-BX61 confocal microscope with a ×60 objective. Hippocampal neurons endogenously expressing TRPM8 channels respond to both testosterone and menthol. e, representative recording of the testosterone-induced and subsequent menthol-induced responses of TRPM8 channels in hippocampal neurons (n = 5). f, menthol-irresponsive hippocampal neurons are also insensitive to testosterone applications (n = 3). g, summary of the testosterone- and menthol-induced responses of endogenous TRPM8 channels in hippocampal neurons (error bars, S.E.). h, immunocytochemistry image of the endogenous TRPM8 protein in hippocampal neurons detected with anti-TRPM8-IgG. The images were obtained using an Olympus-BX61 confocal microscope with a ×60 objective.

FIGURE 3.

Testosterone-induced Ca²⁺ signals observed on HEK-293 cells stably expressing TRPM8. Fluorescence measurements of intracellular Ca²⁺ concentration were performed on HEK-293 cells expressing the TRPM8 protein. Shown are the menthol-induced Ca²⁺ response (a) and testosterone-induced (b) and impermeable analog BSA-testosterone-induced (c) TRPM8 activities. A summary is shown in d. Error bars, S.E.

FIGURE 4.

The AR protein exerts inhibitory effects on TRPM8. Fluorescence measurements of intracellular Ca²⁺ obtained from HEK-293-TRPM8 stable cells with co-expressed siRNA for the AR protein. TRPM8 activity was induced with menthol (a), testosterone (b), and BSA-testosterone (c) and showed heterogeneous responses of higher amplitude. A summary is shown in d. HEK-293-TRPM8 stable cells treated with AR inhibitor hydroxyflutamide show recovery of the subsequent menthol-induced responses. In e and f, cells were pretreated with 1 μm hydroxyflutamide for 1 h before application of the agonists. Shown are TRPM8 responses with menthol (e) and testosterone (f) applications. g, co-application of the AR inhibitor hydroxyflutamide (1 μm) with testosterone and menthol. A summary is shown in h. Error bars, S.E. ***, p < 0.005.

FIGURE 5.

Testosterone-induced Ca²⁺ uptake in HEK-293 TRPM8 stable cells cultured under the steroid-deprived conditions. Fluorescence measurements of intracellular Ca²⁺ concentration were performed on HEK-293 cells stably expressing the TRPM8 protein. The cells were cultured in the steroid-deprived medium using 0.25% charcoal and 0.0025% dextran for up to 38 h. Menthol-induced (a), testosterone-induced (b), and BSA-testosterone-induced (c) TRPM8 activity is shown. A summary is presented in d. Error bars, S.E.

FIGURE 6.

Effect of cholesterol on TRPM8 channel activity. Application of cholesterol (1 pm; 1 nm and 1 μm) does not induce Ca²⁺ uptake in HEK-293 cells stably (a; n = 51) or transiently expressing TRPM8 channel (b; n = 16). c, summary of the cholesterol- and menthol-induced responses of TRPM8. Error bars, S.E.

FIGURE 7.

Testosterone activates the TRPM8 channel in planar lipid bilayers. a, TRPM8 channel openings induced by testosterone application. Representative single-channel current recordings of TRPM8 were obtained with different testosterone concentrations, as indicated above the current traces. The TRPM8 channel was incorporated in planar lipid bilayers formed from POPC/POPE (3:1) in n-decane between symmetric bathing solutions of 150 mm KCl, 0.2 mm MgCl₂, and 1 μm CaCl₂ in 20 mm HEPES buffer, pH 7.4, at 22 °C, in the presence of 2.5 μm DiC₈-PIP₂. The TRPM8 protein, at a concentration of 20 ng/ml, was incorporated into POPC/POPE micelles, which were added to the cis compartment (ground) with gentle stirring. The clamping potential was +60 mV. The horizontal line on the left of the traces and the letter c delineate the closed state of the channel. b, dose responses of testosterone and DHT as a function of the open probability of the TRPM8 channel obtained at +60 mV. Testosterone activation of TRPM8 has an EC₅₀ of ∼64.9 pm (n = 20), and DHT has an EC₅₀ of ∼21.4 nm (n = 9). Error bars, S.E. c, d, inhibition of testosterone-evoked TRPM8 channel activity with TRPM8 antagonists. c, representative current traces of TRPM8 channels activated with 500 pm testosterone and subsequently inhibited with 1 μm M8-B, a specific TRPM8 inhibitor. The addition of M8-B to only one side of the channel, presumably intracellular, did not inhibit the TRPM8 channel activity, whereas the addition to the other side, presumably extracellular, momentarily inhibited the channel openings. Before the addition of M8-B, TRPM8 channels were recorded for more than 2.5 h, where the channels were constantly gating. After the addition of M8-B, TRPM8 channels were inhibited for the rest of the recording (∼2 h) and did not show any openings with different voltages. d, graph demonstrates inhibition of open probability for the TRPM8 channels activated with 500 pm testosterone and inhibited with 10 μg/ml polylysine (polyK) (n = 7), 10 μm capsazepine (n = 3), and 1 μm M8-B (n = 4) (error bars, S.E.). P_o values were obtained at 100 mV. e, control measurements were performed in the planar lipid bilayers in the absence of the TRPM8 protein. Conditions were the same as described in a. Testosterone concentrations were in the micromolar range (beginning from 1 μm and up to 5 μm), and the bilayers were challenged at various voltages for several h. No single event of channel-like behavior was observed. The control experiments were repeated four times.

FIGURE 8.

Comparison of the TRPM8 single channel activity evoked by icilin, menthol, and testosterone in planar lipid bilayers. a, representative single-channel current recordings and current-voltage relationship of TRPM8 obtained with icilin (1 μm), menthol (25 μm), and testosterone (10 pm). Experimental conditions are the same as described in the legend to Fig. 7; details are indicated on the traces. b, current (I)-voltage (V) relationship and mean slope conductance values of TRPM8 activated with icilin, menthol, and testosterone at the concentrations indicated in a, showing linear I-V curves for the icilin- and testosterone-evoked TRPM8 channels and the rectifying I-V curve for menthol-induced current. Mean slope single-channel conductance for icilin-activated TRPM8 is 73.5 ± 6.1 pS; menthol-activated outward conductance is 71.9 ± 3.5 pS; menthol-activated inward conductance is 42.9 ± 1.6 pS; testosterone-activated conductance is 37.1 ± 8.5 pS. c, testosterone activates TRPM8 in the presence of PIP₂ and Ca²⁺. Representative current trace recordings demonstrate that no channel activity can be observed with TRPM8 and testosterone (500 pm) in planar lipid bilayers in the absence of PIP₂. The addition to the bilayers of 2.5 μm DiC₈-PIP₂ resulted in full opening of the channel (n = 10). The subsequent addition of 1 mm EGTA inhibits TRPM8 activity (n = 4). A summary is represented on the right.

FIGURE 9.

Single channel activity of TRPM8 obtained under non-saturated conditions, activated with 10 pm testosterone, in planar lipid bilayers. a, representative current traces and all-points histograms of TRPM8 activated with 10 pm testosterone obtained at different voltages, as indicated on the traces. The experimental conditions are the same as indicated in the legend to Fig. 7a. Shown are the current-voltage relationship (b) and shows open probability values (c) of TRPM8 activated with 10 pm testosterone obtained at different voltages.

FIGURE 10.

Extracellular PHBylated peptide is one of the testosterone-binding sites. a, IP performed with the WT TRPM8 and PHB mutants, 5S-G and Y826G. b, summary of the relative binding to testosterone for the WT TRPM8 and PHB mutants. c, d, PHB mutants of TRPM8 show a dramatic decrease in the channel colocalization with testosterone on plasma membrane. c, immunocytochemistry was done with the HEK-293 cells transiently expressing WT TRPM8 and PHB mutants 5S-G and Y826G. After fixing with 2% paraformaldehyde for 30 min and BSA blocking, nonpermeabilized cells were treated with 1 nm testosterone for 1 h following incubation with anti-Myc-IgG for the protein and anti-DHT/testosterone-IgG for testosterone detection. Cell images were obtained with Olympus BX61 confocal microscope and were analyzed with ImageJ software. d, the Pearson coefficients for colocalization of testosterone with the WT TRPM8 protein or PHB mutants were calculated using the Colocalization Finder plugin of the ImageJ software. e, model of the TRPM8 protein demonstrates testosterone binding to PHBylated peptide on the extracellular side.

FIGURE 11.

PHB-deficient mutants of TRPM8 are irresponsive to testosterone stimuli. Fluorescence measurements of intracellular Ca²⁺ concentration were performed on WT TRPM8 and PHB mutants, 5S-G and Y826G, transiently expressed in HEK-293 cells cultured under the steroid-deprived conditions. Bottom, summary graph. Error bars, S.E.

FIGURE 12.

Bioinformatics analysis of the TRPM8 and the AR proteins. The protein sequence alignment for TRPM8 and AR reveals three peptides that share some similarity. Interestingly, two of the peptides from the AR protein belong to the androgen-binding domain, and two peptides of TRPM8 are modified by PHB, including intracellular and extracellular modifications (15).

FIGURE 13.

Testosterone elicits a cooling sensation upon acute topical skin application. Sixteen healthy volunteers (eight female and eight male) between the ages of 18 and 55 years were recruited for the single-blind test. Testosterone was applied in different concentrations and orders on the skin surface, and the durations of the responses were recorded. The volunteers described their sensation as a feeling of cold and, for the higher agonist concentrations, as mixed feelings of cold and stinging. The duration of the cold-evoked responses was significantly longer in the female volunteers than in the males at the following concentrations of testosterone: 1 pm, p = 0.928; 1 nm, p = 0.559; 1 μm, p = 0.190; 10 μm, p = 0.031 (*); 100 μm, p = 0.039 (*); 1 mm, p = 0.014 (*); 50 mm, p = 0.136; for 50 mm menthol, p = 0.324. Females demonstrated significant difference in the cold duration compared with ethanol: 1 pm, p = 0.282; 1 nm cold response, p = 0.015 (*); 1 nm pain response, p = 0.0036 (***); 1 μm cold response, p = 0.024 (*); 1 μm pain response, p = 0.016 (*); 10 μm cold response, p = 0.033 (*); 10 μm pain response, p = 0.002 (***); 100 μm cold response, p = 0.015 (*); 100 μm pain response, p = 0.0019 (***); 1 mm cold response, p = 0.0139 (*); 1 mm pain response, p = 3.4E−5 (***); 50 mm cold response, p = 0.1001; 50 mm pain response, p = 0.027 (*); 50 mm menthol/cold response, p = 0.050. Males demonstrated significant difference in the cold duration compared with ethanol: 1 pm, p = 0.124; 1 nm cold response, p = 0.0027 (**); 1 μm cold response, p = 0.054; 10 μm cold response, p = 0.048 (*); 100 μm cold response, p = 0.021 (*); 100 μm pain response, p = 0.0126 (*); 1 mm cold response, p = 0.004 (***); 50 mm cold response, p = 0.0003 (***); 50 mm menthol/cold response, p = 0.065. The graph shows dark cyan asterisks for statistical difference between female cold/pain responses and female ethanol responses and blue asterisks for statistical difference between male cold/pain responses and male ethanol responses; black asterisks show significant difference between two genders. Error bars, S.E.

FIGURE 14.

Schematic representation of testosterone-induced TRPM8 channel activity. The scheme represents TRPM8 as an ionotropic testosterone receptor, which has higher affinity for testosterone than for DHT. The figure also depicts the genomic androgen-dependent regulation of TRPM8 expression.