NALCN-mediated sodium influx confers metastatic prostate cancer cell invasiveness.

There is growing evidence that ion channels are critically involved in cancer cell invasiveness and metastasis. However, the molecular mechanisms of ion signaling promoting cancer behavior are poorly understood and the complexity of the underlying remodeling during metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques, we show that metastatic prostate cancer cells acquire a specific Na+ /Ca2+ signature required for persistent invasion. We identify the Na+ leak channel, NALCN, which is overexpressed in metastatic prostate cancer, as a major initiator and regulator of Ca2+ oscillations required for invadopodia formation. Indeed, NALCN-mediated Na+ influx into cancer cells maintains intracellular Ca2+ oscillations via a specific chain of ion transport proteins including plasmalemmal and mitochondrial Na+ /Ca2+ exchangers, SERCA and store-operated channels. This signaling cascade promotes activity of the NACLN-colocalized proto-oncogene Src kinase, actin remodeling and secretion of proteolytic enzymes, thus increasing cancer cell invasive potential and metastatic lesions in vivo. Overall, our findings provide new insights into an ion signaling pathway specific for metastatic cells where NALCN acts as persistent invasion controller.

TRPC3 shapes the ER-mitochondria Ca2+ transfer characterizing tumour-promoting senescence.

Cellular senescence is implicated in a great number of diseases including cancer. Although alterations in mitochondrial metabolism were reported as senescence drivers, the underlying mechanisms remain elusive. We report the mechanism altering mitochondrial function and OXPHOS in stress-induced senescent fibroblasts. We demonstrate that TRPC3 protein, acting as a controller of mitochondrial Ca2+ load via negative regulation of IP3 receptor-mediated Ca2+ release, is down regulated in senescence regardless of the type of senescence inducer. This remodelling promotes cytosolic/mitochondrial Ca2+ oscillations and elevates mitochondrial Ca2+ load, mitochondrial oxygen consumption rate and oxidative phosphorylation. Re-expression of TRPC3 in senescent cells diminishes mitochondrial Ca2+ load and promotes escape from OIS-induced senescence. Cellular senescence evoked by TRPC3 downregulation in stromal cells displays a proinflammatory and tumour-promoting secretome that encourages cancer epithelial cell proliferation and tumour growth in vivo. Altogether, our results unravel the mechanism contributing to pro-tumour behaviour of senescent cells.

Store operated calcium channels in cancer progression.

In recent decades cancer emerged as one of the leading causes of death in the developed countries, with some types of cancer contributing to the top 10 causes of death on the list of the World Health Organization. Carcinogenesis, a malignant transformation causing formation of tumors in normal tissues, is associated with changes in the cell cycle caused by suppression of signaling pathways leading to cell death and facilitation of those enhancing proliferation. Further progression of cancer, during which benign tumors acquire more aggressive phenotypes, is characterized by metastatic dissemination through the body driven by augmented motility and invasiveness of cancer cells. All these processes are associated with alterations in calcium homeostasis in cancer cells, which promote their proliferation, motility and invasion, and dissuade cell death or cell cycle arrest. Remodeling of store-operated calcium entry (SOCE), one of the major pathways regulating intracellular Ca2+ concentration ([Ca2+]i), manifests a key event in many of these processes. This review systematizes current knowledge on the mechanisms recruiting SOCE-related proteins in carcinogenesis and cancer progression.

Testosterone-androgen receptor: The steroid link inhibiting TRPM8-mediated cold sensitivity.

Recent studies have revealed gender differences in cold perception, and pointed to a possible direct action of testosterone (TST) on the cold-activated TRPM8 (Transient Receptor Potential Melastatin Member 8) channel. However, the mechanisms by which TST influences TRPM8-mediated sensory functions remain elusive. Here, we show that TST inhibits TRPM8-mediated mild-cold perception through the noncanonical engagement of the Androgen Receptor (AR). Castration of both male rats and mice increases sensitivity to mild cold, and this effect depends on the presence of intact TRPM8 and AR. TST in nanomolar concentrations suppresses whole-cell TRPM8-mediated currents and single-channel activity in native dorsal root ganglion (DRG) neurons and HEK293 cells co-expressing recombinant TRPM8 and AR, but not TRPM8 alone. AR cloned from rat DRGs shows no difference from standard AR. However, biochemical assays and confocal imaging reveal the presence of AR on the cell surface and its interaction with TRPM8 in response to TST, leading to an inhibition of channel activity.

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.

Ferroquine, the next generation antimalarial drug, has antitumor activity.

Despite the tremendous progress in medicine, cancer remains one of the most serious global health problems awaiting new effective therapies. Here we present ferroquine (FQ), the next generation antimalarial drug, as a promising candidate for repositioning as cancer therapeutics. We report that FQ potently inhibits autophagy, perturbs lysosomal function and impairs prostate tumor growth in vivo. We demonstrate that FQ negatively regulates Akt kinase and hypoxia-inducible factor-1α (HIF-1α) and is particularly effective in starved and hypoxic conditions frequently observed in advanced solid cancers. FQ enhances the anticancer activity of several chemotherapeutics suggesting its potential application as an adjuvant to existing anticancer therapy. Alike its parent compound chloroquine (CQ), FQ accumulates within and deacidifies lysosomes. Further, FQ induces lysosomal membrane permeabilization, mitochondrial depolarization and caspase-independent cancer cell death. Overall, our work identifies ferroquine as a promising new drug with a potent anticancer activity.

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.

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.

Molecular identification of P2X receptors in vascular smooth muscle cells from rat anterior, posterior, and basilar arteries.

BACKGROUND: Purinergic P2X receptors in vascular smooth muscle cells (VSMCs) play an important role in physiological stimulatory responses to the extracellularly released ATP. The aim of this work was to identify molecular P2X receptor subunits in VSMCs isolated from rat anterior, posterior and basilar arteries using a number of contemporary laboratory techniques. METHODS: P2X mediated ionic currents were recorded using amphotericin B perforated patch clamp method. Gene expression analysis was performed using RT-PCR in manually collected VSMCs. The expression of proteins was confirmed by fluorescent immunocytochemistry. RESULTS: Under voltage clamp conditions VSMCs stimulated by application of 10 µmol/l selective P2X receptor agonist αß-meATP, the biphasic currents consisting of rapidly rising rapidly desensitizing and slowly desensitizing components were observed in freshly isolated myocytes from all three arteries. Using RT-PCR, the expression of genes encoding only P2X1 and P2X4 receptor subunits was detected in preparations from all three arteries. The expression of corresponding P2X1 and P2X4 receptor subunit proteins was confirmed in isolated VSMCs. CONCLUSIONS: Our work therefore identified that in major arteries of rat cerebral circulation VSMCs express only P2X1 and P2X4 receptors subunits. We can propose that these P2X receptor subunits participate in functional P2X receptor structures mediating ATP-evoked stimulatory responses in cerebral vascular myocytes in vivo.

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.

TRP channel-associated factors are a novel protein family that regulates TRPM8 trafficking and activity.

TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor-activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named "TRP channel-associated factors" (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity.

Molecular mechanism for opioid dichotomy: bidirectional effect of µ-opioid receptors on P2X3 receptor currents in rat sensory neurones.

Here, we describe a molecular switch associated with opioid receptors-linked signalling cascades that provides a dual opioid control over P2X3 purinoceptor in sensory neurones. Leu-enkephalin inhibited P2X3-mediated currents with IC50 ~10 nM in ~25% of small nociceptive rat dorsal root ganglion (DRG) neurones. In contrast, in neurones pretreated with pertussis toxin leu-enkephalin produced stable and significant increase of P2X3 currents. All effects of opioid were abolished by selective µ-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), nonselective inhibitor naloxone, and by PLC inhibitor U73122. Thus, we discovered a dual link between purinoceptors and µ-opioid receptors: the latter exert both inhibitory (pertussis toxin-sensitive) and stimulatory (pertussis toxin-insensitive) actions on P2X3 receptors through phospholipase C (PLC)-dependent pathways. This dual opioid control of P2X3 receptors may provide a molecular explanation for dichotomy of opioid therapy. Pharmacological control of this newly identified facilitation/inhibition switch may open new perspectives for the adequate medical use of opioids, the most powerful pain-killing agents known today.

Impaired P2X signalling pathways in renal microvascular myocytes in genetic hypertension.

AIMS: P2X receptors (P2XRs) mediate sympathetic control and autoregulation of renal circulation triggering preglomerular vasoconstriction, which protects glomeruli from elevated pressures. Although previous studies established a casual link between glomerular susceptibility to hypertensive injury and decreased preglomerular vascular reactivity to P2XR activation, the mechanisms of attenuation of the P2XR signalling in hypertension remained unknown. We aimed to analyse molecular mechanisms of the impairment of P2XR signalling in renal vascular smooth muscle cells (RVSMCs) in genetic hypertension. METHODS AND RESULTS: We compared the expression of pertinent genes and P2XR-linked Ca(2+) entry and Ca(2+) release mechanisms in RVSMCs of spontaneously hypertensive rats (SHRs) and their normotensive controls, Wistar Kyoto (WKY) rats. We found that, in SHR RVSMCs, P2XR-linked Ca(2+) entry and Ca(2+) release from the sarcoplasmic reticulum (SR) are both significantly reduced. The former is due to down-regulation of the P2X1 subunit. The latter is caused by a decrease of the SR Ca(2+) load. The SR Ca(2+) load reduction is caused by attenuated Ca(2+) uptake via down-regulated sarco-/endoplasmic reticulum Ca(2+)-ATPase 2b and elevated Ca(2+) leak from the SR via ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors. Spontaneous activity of these Ca(2+)-release channels is augmented due to up-regulation of RyR type 2 and elevated IP3 production by up-regulated phospholipase C-ß1. CONCLUSIONS: Our study unravels the cellular and molecular mechanisms of attenuation of P2XR-mediated preglomerular vasoconstriction that elevates glomerular susceptibility to harmful hypertensive pressures. This provides an important impetus towards understanding of the pathology of hypertensive renal injury.

Mechanisms of the sarcoplasmic reticulum Ca2+ release induced by P2X receptor activation in mesenteric artery myocytes.

BACKGROUND: ATP is one of the principal sympathetic neurotransmitters which contracts vascular smooth muscle cells (SMCs) via activation of ionotropic P2X receptors (P2XRs). We have recently demonstrated that contraction of the guinea pig small mesenteric arteries evoked by stimulation of P2XRs is sensitive to inhibitors of IP3 receptors (IP3Rs). Here we analyzed contribution of IP3Rs and ryanodine receptors (RyRs) to [Ca(2+)]i transients induced by P2XR agonist αß-meATP (10 µM) in single SMCs from these vessels. METHODS: The effects of inhibition of L-type Ca(2+) channels (VGCCs), RyRs and IP3Rs (5 µM nicardipine, 100 µM tetracaine and 30 µM 2-APB, respectively) on αß-meATP-induced [Ca(2+)]i transients were analyzed using fast x-y confocal Ca(2+) imaging. RESULTS: The effect of IP3R inhibition on the [Ca(2+)]i transient was significantly stronger (67 ± 7%) than that of RyR inhibition (40 ± 5%) and was attenuated by block of VGCCs. The latter indicates that activation of VGCCs is linked to IP3R-mediated Ca(2+) release. Immunostaining of RyRs and IP3Rs revealed that RyRs are located mainly in deeper sarcoplasmic reticulum (SR) while sub-plasma membrane (PM) SR elements are enriched with type 1 IP3Rs. This structural peculiarity makes IP3Rs more accessible to Ca(2+) entering the cell via VGCCs. Thus, IP3Rs may serve as an "intermediate amplifier" between voltage-gated Ca(2+) entry and RyR-mediated Ca(2+) release. CONCLUSIONS: P2X receptor activation in mesenteric artery SMCs recruits IP3Rs-mediated Ca(2+) release from sub-PM SR, which is facilitated by activation of VGCCs. Sensitivity of IP3R-mediated release to VGCC antagonists in vascular SMCs makes this mechanism of special therapeutic significance.

ORAI1 calcium channel orchestrates skin homeostasis.

To achieve and maintain skin architecture and homeostasis, keratinocytes must intricately balance growth, differentiation, and polarized motility known to be governed by calcium. Orai1 is a pore subunit of a store-operated Ca(2+) channel that is a major molecular counterpart for Ca(2+) influx in nonexcitable cells. To elucidate the physiological significance of Orai1 in skin, we studied its functions in epidermis of mice, with targeted disruption of the orai1 gene, human skin sections, and primary keratinocytes. We demonstrate that Orai1 protein is mainly confined to the basal layer of epidermis where it plays a critical role to control keratinocyte proliferation and polarized motility. Orai1 loss of function alters keratinocyte differentiation both in vitro and in vivo. Exploring underlying mechanisms, we show that the activation of Orai1-mediated calcium entry leads to enhancing focal adhesion turnover via a PKCß-Calpain-focal adhesion kinase pathway. Our findings provide insight into the functions of the Orai1 channel in the maintenance of skin homeostasis.

Opiates modulate thermosensation by internalizing cold receptor TRPM8.

Stimulation of µ-opioid receptors (OPRMs) brings powerful pain relief, but it also leads to the development of tolerance and addiction. Ensuing withdrawal in abstinent patients manifests itself with severe symptoms, including cold hyperalgesia, often preventing addicted patients from successfully completing the rehabilitation. Unsurprisingly, OPRMs have been a central point of many studies. Nonetheless, a satisfactory understanding of the pathways leading to distorted sensory responses during opiate administration and abstinence is far from complete. Here, we present a mechanism that leads to modulation by OPRMs of one of the sensory responses, thermosensation. Activation of OPRM1 leads to internalization of a cold-sensor TRPM8, which can be reversed by a follow-up treatment with the inverse OPRM agonist naloxone. Knockout of TRPM8 protein leads to a decrease in morphine-induced cold analgesia. The proposed pathway represents a universal mechanism that is probably shared by regulatory pathways modulating general pain sensation in response to opioid treatment.

Mechanisms of [Ca2+]i elevation following P2X receptor activation in the guinea-pig small mesenteric artery myocytes.

BACKGROUND: There is growing evidence suggesting involvement of L-type voltage-gated Ca2+ channels (VGCCs) in purinergic signaling mechanisms. However, detailed interplay between VGCCs and P2X receptors in intracellular Ca2+ mobilization is not well understood. This study examined relative contribution of the Ca2+ entry mechanisms and induced by this entry Ca2+ release from the intracellular stores engaged by activation of P2X receptors in smooth muscle cells (SMCs) from the guinea-pig small mesenteric arteries. METHODS: P2X receptors were stimulated by the brief local application of αß-meATP and changes in [Ca2+]i were monitored in fluo-3 loaded SMCs using fast x-y confocal Ca2+ imaging. The effects of the block of L-type VGCCs and/or depletion of the intracellular Ca2+ stores on αß-meATP-induced [Ca2+]i transients were analyzed. RESULTS: Our analysis revealed that Ca2+ entry via L-type VGCCs is augmented by the Ca2+-induced Ca2+ release significantly more than Ca2+ entry via P2X receptors, even though net Ca2+ influxes provided by the two mechanisms are not significantly different. CONCLUSIONS: Thus, arterial SMCs upon P2X receptor activation employ an effective mechanism of the Ca2+ signal amplification, the major component of which is the Ca2+ release from the SR activated by Ca2+ influx via L-type VGCCs. This signaling pathway is engaged by depolarization of the myocyte membrane resulting from activation of P2X receptors, which, being Ca2+ permeable, per se form less effective Ca2+ signaling pathway. This study, therefore, rescales potential targets for therapeutic intervention in purinergic control of vascular tone.

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.

Identifying an uptake mechanism for the antiepileptic and bipolar disorder treatment valproic acid using the simple biomedical model Dictyostelium.

Valproic acid (VPA) is the most highly prescribed epilepsy treatment worldwide and is also used to prevent bipolar disorder and migraine. Surprisingly, very little is known about its mechanisms of cellular uptake. Here, we employ a range of cellular, molecular and genetic approaches to characterize VPA uptake using a simple biomedical model, Dictyostelium discoideum. We show that VPA is taken up against an electrochemical gradient in a dose-dependent manner. Transport is protein-mediated, dependent on pH and the proton gradient and shows strong substrate structure specificity. Using a genetic screen, we identified a protein homologous to a mammalian solute carrier family 4 (SLC4) bicarbonate transporter that we show is involved in VPA uptake. Pharmacological and genetic ablation of this protein reduces the uptake of VPA and partially protects against VPA-dependent developmental effects, and extracellular bicarbonate competes for VPA uptake in Dictyostelium. We further show that this uptake mechanism is likely to be conserved in both zebrafish (Danio rerio) and Xenopus laevis model systems. These results implicate, for the first time, an uptake mechanism for VPA through SLC4-catalysed activity.