Evolution of the human cold/menthol receptor, TRPM8.

Genes showing versatile functions or subjected to fast expansion and contraction during the adaptation of species to specific ecological conditions, like sensory receptors for odors, pheromones and tastes, are characterized by a great plasticity through evolution. One of the most fascinating sensory receptors in the family of TRP channels, the cold and menthol receptor TRPM8, has received significant attention in the literature. Recent studies have reported the existence of TRPM8 channel isoforms encoded by alternative mRNAs transcribed from alternative promoters and processed by alternative splicing. Since the first draft of the human genome was accomplished in 2000, alternative transcription, alternative splicing and alternative translation have appeared as major sources of gene product diversity and are thought to participate in the generation of complexity in higher organisms. In this study, we investigate whether alternative transcription has been a driving force in the evolution of the human forms of the cold receptor TRPM8. We identified 33 TRPM8 alternative mRNAs (24 new sequences) and their associated protein isoforms in human tissues. Using comparative genomics, we described the evolution of the human TRPM8 sequences in eight ancestors since the origin of Amniota, and estimated in which ancestors the new TRPM8 variants originated. In order to validate the estimated origins of this receptor, we performed experimental validations of predicted exons in mouse tissues. Our results suggest a first diversification event of the cold receptor in the Boreoeutheria ancestor, and a subsequent divergence at the origin of Simiiformes.

4TM-TRPM8 channels are new gatekeepers of the ER-mitochondria Ca2+ transfer.

Calcium (Ca2+) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca2+ release was associated with the ER Ca2+ release channels, inositol 1,4,5­triphosphate receptor (IP3R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca2+ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca2+ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca2+ concentration ([Ca2+]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca2+ release mechanism distinct from classical Ca2+ release channels.

Organelle membrane derived patches: reshaping classical methods for new targets.

Intracellular ion channels are involved in multiple signaling processes, including such crucial ones as regulation of cellular motility and fate. With 95% of the cellular membrane belonging to intracellular organelles, it is hard to overestimate the importance of intracellular ion channels. Multiple studies have been performed on these channels over the years, however, a unified approach allowing not only to characterize their activity but also to study their regulation by partner proteins, analogous to the patch clamp "golden standard", is lacking. Here, we present a universal approach that combines the extraction of intracellular membrane fractions with the preparation of patchable substrates that allows to characterize these channels in endogenous protein environment and to study their regulation by partner proteins. We validate this method by characterizing activity of multiple intracellular ion channels localized to different organelles and by providing detailed electrophysiological characterization of the regulation of IP3R activity by endogenous Bcl-2. Thus, after synthesis and reshaping of the well-established approaches, organelle membrane derived patch clamp provides the means to assess ion channels from arbitrary cellular membranes at the single channel level.

Cold/menthol TRPM8 receptors initiate the cold-shock response and protect germ cells from cold-shock-induced oxidation.

Testes of most male mammals present the particularity of being externalized from the body and are consequently slightly cooler than core body temperature (4-8°C below). Although, hypothermia of the testis is known to increase germ cells apoptosis, little is known about the underlying molecular mechanisms, including cold sensors, transduction pathways, and apoptosis triggers. In this study, using a functional knockout mouse model of the cold and menthol receptors, dubbed transient receptor potential melastatine 8 (TRPM8) channels, we found that TRPM8 initiated the cold-shock response by differentially modulating cold- and heat-shock proteins. Besides, apoptosis of germ cells increased in proportion to the cooling level in control mice but was independent of temperature in knockout mice. We also observed that the rate of germ cell death correlated positively with the reactive oxygen species level and negatively with the expression of the detoxifying enzymes. This result suggests that the TRPM8 sensor is a key determinant of germ cell fate under hypothermic stimulation.-Borowiec, A.-S., Sion, B., Chalmel, F., Rolland, A. D., Lemonnier, L., De Clerck, T., Bokhobza, A., Derouiche, S., Dewailly, E., Slomianny, C., Mauduit, C., Benahmed, M., Roudbaraki, M., Jégou, B., Prevarskaya, N., Bidaux, G. Cold/menthol TRPM8 receptors initiate the cold-shock response and protect germ cells from cold-shock-induced oxidation.

Targeting of short TRPM8 isoforms induces 4TM-TRPM8-dependent apoptosis in prostate cancer cells.

Since its cloning a decade ago, TRPM8 channel has emerged as a promising prognostic marker and a putative therapeutic target in prostate cancer (PCa). However, recent studies have brought to light the complexity of TRPM8 isoforms in PCa. Consequently, the respective role of each TRPM8 isoform needs to be deciphered prior to considering TRPM8 as an attractive therapeutic target. Full-length (6 transmembrane (TM)-domain) TRPM8 channel is overexpressed in early PCa and repressed in advanced prostate tumors whereas the localization of the truncated, 4TM-TRPM8 channel (4 transmembrane (TM)-domain), in the membranes of endoplasmic reticulum (ER) is independent of the pathogenic status of epithelial cells. In the same line, expression of non-channel cytoplasmic small TRPM8 isoforms (namely sM8) is conserved in cancer cells. In this study, we identify sM8s as putative regulator of PCa cell death. Indeed, suppression of sM8 isoforms was found to induce concomitantly ER stress, oxidative stress, p21 expression and apoptosis in human epithelial prostate cancer cells. We furthermore demonstrate that induction of such mechanisms required the activity of 4TM-TRPM8 channels at the ER-mitochondria junction. Our study thus suggests that targeting sM8 could be an appropriate strategy to fight prostate cancer.

Fine-tuning of eTRPM8 expression and activity conditions keratinocyte fate.

Recently, we reported the cloning and characterization of short isoform of the icilin-activated cold receptor TRPM8 channel in keratinocytes, dubbed eTRPM8. We demonstrated that eTRPM8 via fine tuning of the endoplasmic reticulum (ER) - mitochondria Ca(2+) shuttling regulates mitochondrial ATP and superoxide (O2(•-)) production and, thereby, mediates control of epidermal homeostasis by mild cold. Here, we provide additional information explaining why eTRPM8 suppression and TRPM8 stimulation both inhibit keratinocyte growth. We also demonstrate that stimulation of eTRPM8 with icilin may give rise to sustained oscillatory responses. Furthermore, we show that ATP-induced cytosolic and mitochondrial Ca(2+) responses are attenuated by eTRPM8 suppression. This suggests positive interplay between eTRPM8 and purinergic signaling pathways, what may serve to facilitate the ER-mitochondria Ca(2+) shuttling. Finally, we demonstrate that cold (25°C) induces eTRPM8-dependent superoxide-mediated necrosis of keratinocytes. Altogether, these results are in line with our model of eTRPM8-mediated cold-dependent balance between keratinocyte proliferation and differentiation.

Functional and Modeling Studies of the Transmembrane Region of the TRPM8 Channel.

Members of the transient receptor potential (TRP) ion channel family act as polymodal cellular sensors, which aid in regulating Ca(2+) homeostasis. Within the TRP family, TRPM8 is the cold receptor that forms a nonselective homotetrameric cation channel. In the absence of TRPM8 crystal structure, little is known about the relationship between structure and function. Inferences of TRPM8 structure have come from mutagenesis experiments coupled to electrophysiology, mainly regarding the fourth transmembrane helix (S4), which constitutes a moderate voltage-sensing domain, and about cold sensor and phosphatidylinositol 4,5-bisphosphate binding sites, which are both located in the C-terminus of TRPM8. In this study, we use a combination of molecular modeling and experimental techniques to examine the structure of the TRPM8 transmembrane and pore helix region including the conducting conformation of the selectivity filter. The model is consistent with a large amount of functional data and was further tested by mutagenesis. We present structural insight into the role of residues involved in intra- and intersubunit interactions and their link with the channel activity, sensitivity to icilin, menthol and cold, and impact on channel oligomerization.

Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a cold-dependent manner.

Deviation of the ambient temperature is one of the most ubiquitous stimuli that continuously affect mammals' skin. Although the role of the warmth receptors in epidermal homeostasis (EH) was elucidated in recent years, the mystery of the keratinocyte mild-cold sensor remains unsolved. Here we report the cloning and characterization of a new functional epidermal isoform of the transient receptor potential M8 (TRPM8) mild-cold receptor, dubbed epidermal TRPM8 (eTRPM8), which is localized in the keratinocyte endoplasmic reticulum membrane and controls mitochondrial Ca(2+) concentration ([Ca(2+)]m). In turn, [Ca(2+)]m modulates ATP and superoxide (O2(·-)) synthesis in a cold-dependent manner. We report that this fine tuning of ATP and O2(·-) levels by cooling controls the balance between keratinocyte proliferation and differentiation. Finally, to ascertain eTRPM8's role in EH in vivo we developed a new functional knockout mouse strain by deleting the pore domain of TRPM8 and demonstrated that eTRPM8 knockout impairs adaptation of the epidermis to low temperatures.

Remodeling of channel-forming ORAI proteins determines an oncogenic switch in prostate cancer.

ORAI family channels have emerged as important players in malignant transformation, yet the way in which they reprogram cancer cells remains elusive. Here we show that the relative expression levels of ORAI proteins in prostate cancer are different from that in noncancerous tissue. By mimicking ORAI protein remodeling observed in primary tumors, we demonstrate in in vitro models that enhanced ORAI3 expression favors heteromerization with ORAI1 to form a novel channel. These channels support store-independent Ca(2+) entry, thereby promoting cell proliferation and a smaller number of functional homomeric ORAI1-based store-operated channels, which are important in supporting susceptibility to apoptosis. Thus, our findings highlight disrupted dynamic equilibrium of channel-forming proteins as an oncogenic mechanism.

Calcium channels, external calcium concentration and cell proliferation.

Evidence for a role for calcium channel proteins in cell proliferation is numerous suggesting that calcium influx is essential in this physiological process. Several studies in the past thirty years have demonstrated that calcium channel expression levels are determinant in cell proliferation. Voltage-gated, store-operated, second messengers and receptor-operated calcium channels have been associated to cell proliferation. However, the relationship between calcium influx and cell proliferation can be uncoupled in transformed and cancer cells, resulting in an external calcium-independent proliferation. Thus, protein expression could be more important than channel function to trigger cell proliferation suggesting that additional channel functions may be responsible to reconcile calcium channel expression and cell proliferation. When needed, external calcium concentration is obviously important for calcium channel function but it also regulates calcium sensing receptor (CaSR) activity. CaSR can up- or down-regulate cell proliferation depending on physiological conditions. CaSR sensitivity to external calcium is within the 0.5 to 5 mM range and therefore, the role of these receptors in cell proliferation must be taken into account. We therefore suggest here that cell proliferation rates could depend on the relative balance between calcium influx and CaSR activation.

Are Orai1 and Orai3 channels more important than calcium influx for cell proliferation?

Transformed and tumoral cells share the characteristic of being able to proliferate even when external calcium concentration is very low. We have investigated whether Human Embryonic Kidney 293 cells, human hepatoma cell Huh-7 and HeLa cells were able to proliferate when kept 72h in complete culture medium without external calcium. Our data showed that cell proliferation rate was similar over a range of external calcium concentration (2µM to 1.8mM). Incubation in the absence of external calcium for 72h had no significant effect on endoplasmic reticulum (ER) Ca(2+) contents but resulted in a significant decrease in cytosolic free calcium concentration in all 3 cell types. Cell proliferation rates were dependent on Orai1 and Orai3 expression levels in HEK293 and HeLa cells. Silencing Orai1 or Orai3 resulted in a 50% reduction in cell proliferation rate. Flow cytometry analysis showed that Orai3 induced a small but significant increase in cell number in G2/M phase. RO-3306, a cdk-1 inhibitor, induced a 90% arrest in G2/M reversible in less than 15min. Our data showed that progression through G2/M phase after release from RO-3306-induced cell cycle arrest was slower in both Orai1 and Orai3 knock-downs. Overexpressing Orai1, Orai3 and the dominant negative non-permeant mutants E106Q-Orai1 and E81Q-Orai3 induced a 50% increase in cell proliferation rate in HEK293 cells. Our data clearly demonstrated that Orai1 and Orai3 proteins are more important than calcium influx to control cell proliferation in some cell lines and that this process is probably independent of ICRAC and Iarc.

Optimal differentiation of in vitro keratinocytes requires multifactorial external control.

For almost 30 years, keratinocyte differentiation has been studied in numerous cell models including keratinocyte primary culture with various supplemented culture media. In this respect, it has become quite difficult to draw comparisons between studies using such a variety of culture conditions. Serum-free condition with low calcium has been used to culture basal proliferating cells, though differentiation is induced by various procedures. These latter include the addition of calcium at mM concentration and a concomitant addition of serum and calcium. Lowering the incubation temperature of cells has also been reported to induce a premature differentiation of keratinocytes in organotypic skin culture. This effect of temperature on keratinocyte differentiation has been poorly depicted, although average human skin temperature has been shown to be about 32 °C. However, studying differentiation and quantifying shifts in the differentiation rate of a cell population implies to precisely know i) the proportion of differentiated cells in the whole population, and ii) to which extent and to which level of expression, the induction of a gene or a protein might be considered as a marker of differentiation. This lack has rarely been taken into consideration and has surely led to over-interpretations of single protein induction and to consequent extrapolations to real differentiation processes. By means of paralleled analyses with immunocytofluorescence, flow cytometry, and with multiple differentiation markers quantify by qPCR and western-blot, we studied the paradoxical connection between calcium, serum, multilayer culture and incubation temperature on the differentiation of in vitro keratinocytes. Conversely to previous reports, we have shown that calcium switch is indeed a potent model for inducing calcium-dependent genes, but is not an efficient procedure when one wishes to assess the keratinocyte differentiation rate. Moreover, we have demonstrated that a synergic stimulation by calcium, serum, confluence and lower incubation temperature amplified the differentiation rate.

Regulation of activity of transient receptor potential melastatin 8 (TRPM8) channel by its short isoforms.

One important mechanism of the regulation of membrane ion channels involves their nonfunctional isoforms generated by alternative splicing. However, knowledge of such isoforms for the members of the transient receptor potential (TRP) superfamily of ion channels remains quite limited. This study focuses on the TRPM8, which functions as a cold receptor in sensory neurons but is also expressed in tissues not exposed to ambient temperatures, as well as in cancer tissues. We report the cloning from prostate cancer cells of new short splice variants of TRPM8, termed short TRPM8α and short TRPM8ß. Our results show that both variants are in a closed configuration with the C-terminal tail of the full-length TRPM8 channel, resulting in stabilization of its closed state and thus reducing both its cold sensitivity and activity. Our findings therefore uncover a new mode of regulation of the TRPM8 channel by its splice variants.

Regulation of IGF-1-dependent cyclin D1 and E expression by hEag1 channels in MCF-7 cells: the critical role of hEag1 channels in G1 phase progression.

Insulin-like Growth Factor-1 (IGF-1) plays a key role in breast cancer development and cell cycle regulation. It has been demonstrated that IGF-1 stimulates cyclin expression, thus regulating the G1 to S phase transition of the cell cycle. Potassium (K(+)) channels are involved in the G1 phase progression of the cell cycle induced by growth factors. However, mechanisms that allow growth factors to cooperate with K(+) channels in order to modulate the G1 phase progression and cyclin expression remain unknown. Here, we focused on hEag1 K(+) channels which are over-expressed in breast cancer and are involved in the G1 phase progression of breast cancer cells (MCF-7). As expected, IGF-1 increased cyclin D1 and E expression of MCF-7 cells in a cyclic manner, whereas the increase of CDK4 and 2 levels was sustained. IGF-1 stimulated p21(WAF1/Cip1) expression with a kinetic similar to that of cyclin D1, however p27(Kip1) expression was insensitive to IGF-1. Interestingly, astemizole, a blocker of hEag1 channels, but not E4031, a blocker of HERG channels, inhibited the expression of both cyclins after 6-8h of co-stimulation with IGF-1. However, astemizole failed to modulate CDK4, CDK2, p21(WAF1/Cip1) and p27(Kip1) expression. The down-regulation of hEag1 by siRNA provoked a decrease in cyclin expression. This study is the first to demonstrate that K(+) channels such as hEag1 are directly involved in the IGF-1-induced up-regulation of cyclin D1 and E expression in MCF-7 cells. By identifying more specifically the temporal position of the arrest site induced by the inhibition of hEag1 channels, we confirmed that hEag1 activity is predominantly upstream of the arrest site induced by serum-deprivation, prior to the up-regulation of both cyclins D1 and E. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Differential roles of STIM1, STIM2 and Orai1 in the control of cell proliferation and SOCE amplitude in HEK293 cells.

Orai1, together with STIM1 and STIM2, constitutes the molecular basis for store-operated calcium entry (SOCE) and we have investigated their role in cell proliferation and cell cycle progression in HEK293 cells. 48-h serum deprival, and a 24-h treatment with 1 mM hydroxyurea or with 10 microM RO-3306--a cyclin-dependent kinase 1 inhibitor--induced cell cycle block in G1, S and G2/M, respectively. SOCE amplitude, monitored in whole-cell voltage clamped cells, was markedly reduced (60-70%) in all conditions, with full reversibility within 4h. Silencing of Orai and STIM1 using siRNA resulted in a large inhibition of SOCE (70-80%) whereas siSTIM2 had a smaller but significant effect (30%). However, the cell population doubling time was not affected in siSTIM1 cells (18 h, the same as in control cells) but was increased in both siOrai1 cells (29 h) and in siSTIM2 (23 h) even when combined with siSTIM1. This suggests that STIM1 plays no role in cell proliferation in HEK293 cells while STIM2 is involved in both SOCE and cell proliferation in these cells. Finally, the cell cycle block induced SOCE inhibition was associated with reduced Orai1 expression with full recovery within 4h, whereas the expression of STIM1 and STIM2 remained unaltered. These observations reveal a tight relation between cell proliferation, calcium entry and Orai1 expression in HEK293 cells.

The transient receptor potential channel TRPM8 is inhibited via the alpha 2A adrenoreceptor signaling pathway.

The transient receptor potential channel melastatin member 8 (TRPM8) is expressed in sensory neurons, where it constitutes the main receptor of environmental innocuous cold (10-25 degrees C). Among several types of G protein-coupled receptors expressed in sensory neurons, G(i)-coupled alpha 2A-adrenoreceptor (alpha 2A-AR), is known to be involved in thermoregulation; however, the underlying molecular mechanisms remain poorly understood. Here we demonstrated that stimulation of alpha 2A-AR inhibited TRPM8 in sensory neurons from rat dorsal root ganglia (DRG). In addition, using specific pharmacological and molecular tools combined with patch-clamp current recordings, we found that in heterologously expressed HEK-293 (human embryonic kidney) cells, TRPM8 channel is inhibited by the G(i) protein/adenylate cyclase (AC)/cAMP/protein kinase A (PKA) signaling cascade. We further identified the TRPM8 S9 and T17 as two key PKA phosphorylation sites regulating TRPM8 channel activity. We therefore propose that inhibition of TRPM8 through the alpha 2A-AR signaling cascade could constitute a new mechanism of modulation of thermosensation in both physiological and pathological conditions.

IGF-1 activates hEAG K(+) channels through an Akt-dependent signaling pathway in breast cancer cells: role in cell proliferation.

Previous work from our laboratory has shown that human ether à go-go (hEAG) K(+) channels are crucial for breast cancer cell proliferation and cell cycle progression. In this study, we investigated the regulation of hEAG channels by an insulin-like growth factor-1 (IGF-1), which is known to stimulate cell proliferation. Acute applications of IGF-1 increased K(+) current-density and hyperpolarized MCF-7 cells. The effects of IGF-1 were inhibited by hEAG inhibitors. Moreover, IGF-1 increased mRNA expression of hEAG in a time-dependent manner in parallel with an enhancement of cell proliferation. The MCF-7 cell proliferation induced by IGF-1 is inhibited pharmacologically by Astemizole or Quinidine or more specifically using siRNA against hEAG channel. Either mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K) are known to mediate IGF-1 cell proliferative signals through the activation of extracellular signal-regulated kinase 1/2 (Erk 1/2) and Akt, respectively. In MCF-7 cells, IGF-1 rapidly stimulated Akt phosphorylation, whereas IGF-1 had little stimulating effect on Erk 1/2 which seems to be constitutively activated. The application of wortmannin was found to block the effects of IGF-1 on K(+) current. Moreover, the inhibition of Akt phosphorylation by the application of wortmannin or by a specific reduction of Akt kinase activity reduced the hEAG mRNA levels. Taken together, our results show, for the first time, that IGF-1 increases both the activity and the expression of hEAG channels through an Akt-dependent pathway. Since a hEAG channel is necessary for cell proliferation, its regulation by IGF-1 may thus play an important role in IGF-1 signaling to promote a mitogenic effect in breast cancer cells.

The antiestrogen tamoxifen activates BK channels and stimulates proliferation of MCF-7 breast cancer cells.

In the present study, we investigated the effect of the antiestrogen compound tamoxifen on BK channels by the use of the patch-clamp technique. The perfusion of 10 nM tamoxifen significantly increased the magnitude of a voltage-dependent K+ current by 22.6 +/- 10.6% (n = 23). The effect of tamoxifen was always obtained in the first minute, peaked at 5.9 +/- 2.2 min (n = 23), and was abolished by the perfusion of tetraethylammonium (0.5 mM), charybdotoxin (50 nM), or iberiotoxin (100 nM). The stimulatory effect of 10 nM tamoxifen was the same at low (50 nM) and high (700 nM) internal calcium concentration and was not additive to that of 17-beta-estradiol (E2) or its membrane-impermeant form, beta-estradiol 6-(O-carboxymethyl)oxime:bovine serum albumin. Furthermore, the effect of tamoxifen was still recorded in the presence of the selective estrogen receptor antagonist faslodex (ICI-182,780; 1 microM). At the single-channel level, tamoxifen significantly increased the open probability of the BK channel by 46.2 +/- 10.1% (n = 4) without changing its unitary conductance. Moreover, we show here that the stimulation of BK channel activity by tamoxifen is involved in MCF-7 cell proliferation. Taken together, these results permitted us to identify the BK channel as the molecular target of tamoxifen that probably acts at the same extracellular molecular level as E2. The site of action of tamoxifen is probably the channel itself or the auxiliary beta subunits.