Ion channels in the regulation of apoptosis

AbstractApoptosis, a type of genetically controlled cell death, is a fundamental cellular mechanism utilized by multicellular organisms for disposal of cells that are no longer needed or potentially detrimental. Given the crucial role of apoptosis in physiology, deregulation of apoptotic machinery is associated with various diseases as well as abnormalities in development. Acquired resistance to apoptosis represents the common feature of most and perhaps all types of cancer. Therefore, repairing and reactivating apoptosis represents a promising strategy to fight cancer. Accumulated evidence identifies ion channels as essential regulators of apoptosis. However, the contribution of specific ion channels to apoptosis varies greatly depending on cell type, ion channel type and intracellular localization, pathology as well as intracellular signaling pathways involved. Here we discuss the involvement of major types of ion channels in apoptosis regulation. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers

Ca2+- and Volume-sensitive Chloride Currents Are Differentially Regulated by Agonists and Store-operated Ca2+ Entry

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca2+]i in a Ca2+-free medium followed by a secondary [Ca2+]i rise upon Ca2+ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca2+-activated (ICl(Ca)) and volume-regulated (ICl, swell) chloride currents. NPPB and DIDS more efficiently inhibited ICl(Ca) and ICl, swell, respectively. Cell swelling caused by hypotonic solution invariably activated ICl, swell while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling–induced ICl, swell, while its inactive analogue U-73343 had no effect. ICl(Ca) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca2+]i in the pipette solution at 500 nM. In contrast, ICl, swell could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP3 infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced ICl, swell in a nonadditive manner, suggesting their convergence on a common pathway. ICl, swell and ICl(Ca) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated ICl(Ca), but abolished ICl, swell, thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that ICl, swell can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a “selection” switch between closely localized channels

Functional implications of calcium permeability of the channel formed by pannexin 1

Although human pannexins (PanX) are homologous to gap junction molecules, their physiological function in vertebrates remains poorly understood. Our results demonstrate that overexpression of PanX1 results in the formation of Ca2+-permeable gap junction channels between adjacent cells, thus, allowing direct intercellular Ca2+ diffusion and facilitating intercellular Ca2+ wave propagation. More intriguingly, our results strongly suggest that PanX1 may also form Ca2+-permeable channels in the endoplasmic reticulum (ER). These channels contribute to the ER Ca2+ leak and thereby affect the ER Ca2+ load. Because leakage remains the most enigmatic of those processes involved in intracellular calcium homeostasis, and the molecular nature of the leak channels is as yet unknown, the results of this work provide new insight into calcium signaling mechanisms. These results imply that for vertebrates, a new protein family, referred to as pannexins, may not simply duplicate the connexin function but may also provide additional pathways for intra- and intercellular calcium signaling and homeostasis

TRPV6 Determines the Effect of Vitamin D3 on Prostate Cancer Cell Growth

Despite remarkable advances in the therapy and prevention of prostate cancer it is still the second cause of death from cancer in industrialized countries. Many therapies initially shown to be beneficial for the patients were abandoned due to the high drug resistance and the evolution rate of the tumors. One of the prospective therapeutical agents even used in the first stage clinical trials, 1,25-dihydroxyvitamin D3, was shown to be either unpredictable or inefficient in many cases. We have already shown that TRPV6 calcium channel, which is the direct target of 1,25-dihydroxyvitamin D3 receptor, positively controls prostate cancer proliferation and apoptosis resistance (Lehen'kyi et al., Oncogene, 2007). However, how the known 1,25-dihydroxyvitamin D3 antiproliferative effects may be compatible with the upregulation of pro-oncogenic TRPV6 channel remains a mystery. Here we demonstrate that in low steroid conditions 1,25-dihydroxyvitamin D3 upregulates the expression of TRPV6, enchances the proliferation by increasing the number of cells entering into S-phase. We show that these pro-proliferative effects of 1,25-dihydroxyvitamin D3 are directly mediated via the overexpression of TRPV6 channel which increases calcium uptake into LNCaP cells. The apoptosis resistance of androgen-dependent LNCaP cells conferred by TRPV6 channel is drastically inversed when 1,25-dihydroxyvitamin D3 effects were combined with the successful TRPV6 knockdown. In addition, the use of androgen-deficient DU-145 and androgen-insensitive LNCaP C4-2 cell lines allowed to suggest that the ability of 1,25-dihydroxyvitamin D3 to induce the expression of TRPV6 channel is a crucial determinant of the success or failure of 1,25-dihydroxyvitamin D3-based therapies

Voltage- and cold-dependent gating of single TRPM8 ion channels

Transient receptor potential (TRP) channels play critical roles in cell signaling by coupling various environmental factors to changes in membrane potential that modulate calcium influx. TRP channels are typically activated in a polymodal manner, thus integrating multiple stimuli. Although much progress has been made, the underlying mechanisms of TRP channel activation are largely unknown. The TRPM8 cation channel has been extensively investigated as a major neuronal cold sensor but is also activated by voltage, calcium store depletion, and some lipids as well as by compounds that produce cooling sensations, such as menthol or icilin. Several models of TRPM8 activation have been proposed to explain the interaction between these diverse stimuli. However, a kinetic scheme is not yet available that can describe the detailed single-channel kinetics to gain further insight into the underlying gating mechanism. To work toward this goal, we investigated voltage-dependent single-channel gating in cell-attached patches at two different temperatures (20 and 30°C) using HEK293 cells stably expressing TRPM8. Both membrane depolarization and cooling increased channel open probability (Po) mainly by decreasing the duration of closed intervals, with a smaller increase in the duration of open intervals. Maximum likelihood analysis of dwell times at both temperatures indicated gating in a minimum of five closed and two open states, and global fitting over a wide range of voltages identified a seven-state model that described the voltage dependence of Po, the single-channel kinetics, and the response of whole-cell currents to voltage ramps and steps. The major action of depolarization and cooling was to accelerate forward transitions between the same two sets of adjacent closed states. The seven-state model provides a general mechanism to account for TRPM8 activation by membrane depolarization at two temperatures and can serve as a starting point for further investigations of multimodal TRP activation

Implication des canaux calciques de type TRP dans la croissance des kératinocytes humains

LILLE1-BU (590092102) / SudocSudocFranceF

Étude des mécanismes de modulation du canal cationique TRPM8 (implication dans la physiopathologie sensorielle et prostatique)

Le canal TRPM8 a été mis en évidence en tant que re cepteur au froid au sein des neurones sensoriels des ganglions rachidiens dorsaux (DRG) et trigéminaux. Il est activé par le froid (<28C) ainsi que par des molécules au pouvoir réfrigérant (menthol, iciline ou eucalyptol). Au niveau périphérique, ce canal est détecté, en outre, au niveau des cellules épithéliales prostatiques saines et cancéreuses. Si plusieurs travaux se sont attachés à déterminer ses modes d activation, au début de cette thèse, aucune équipe ne s était intéressée à rechercher des modulateurs physiologiques de ce canal, autres que le froid, aussi bien au niveau sensoriel que prostatique. Partant du constat que la thermosensation se voit altérée au cours de certaines situations physiologique (stress, traitements hormonaux, âge et sexe), nous avons étudié une possible régulation de ce récepteur au froid par les voies de signalisation empruntées par certains neuromodulateurs et hormones.Nos résultats mettent en évidence trois nouvelles voies de régulation de TRPM8 mettant en jeu les androgènes, les agonistes des récepteurs muscariniques et alpha2A adrénergiques. Nos données se confirment au sein des cellules épithéliales prostatiques et des neurones sensoriels. Ces travaux nous permettent d avancer une possible explication des variabilités inter-individuelles ainsi que des différences due l âge, au sexe ou face à des situations de stress vis-à-vis de la perception du froid. Enfin, nos résultats nous permettent également de proposer des pistes pour l établissement de nouvelles stratégies thérapeutiques pour des pathologies associant le canal TRPM8 telles que l allodynie au froid et le cancer de la prostate.TRPM8 is known as a cold receptor expressed in the subset of dorsal root ganglion (DRG) and trigeminal (TG) sensory neurons which are activated by cooling temperatures (<28C) or by chemical imitators of cooling sensation (menthol, icilin and eucalyptol). While screening for a new marker of prostate carcinoma, Larisa Tsavaler et al. detected TRPM8 channel expression in normal and cancer prostate epithelial cells. Expression of trpm8 gene is androgeno-dependent and change during cancer development. Even though many studies investigated the role of TRPM8 as a cold receptor and described its activation mechanisms, at the beginning of this work no research team had published any information about TRPM8 physiological modulators other than cold, be it in prostate or sensory neurons. Since several works reported modifications of thermosensation during physiological situations such as stress, hormonal therapy, age and gender, we investigated a possible regulation of the cold receptor by neuromodulators, hormones and their signalization pathways. Our results demonstrate three new regulatory mechanisms for TRPM8 involving respectively a new non genomic androgenic, a muscarinic or an alpha2A adrenergic receptors pathway.Our data are confirmed in two physiological models, epithelial prostate cells and dorsal root ganglia neurons. This work leads us to propose a possible explanation for variations which could be accounted for during stress, gender, as well as inter- and intra-individual disparity in thermosensation. Finally, our results could help establishing new therapeutic strategies for pathologies involving TRPM8 such as cold allodynia and prostate cancer.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Implication des canaux perméables au calcium dans l'homéostasie tissulaire de l'épiderme humain

La structure de l épiderme est le résultat de la régulation de l équilibre entre la prolifération, la migration et la différenciation des kératinocytes. Le calcium est un second messager contrôlant ces mécanismes. Parmi les différents acteurs de l homéostasie calcique, les canaux ioniques jouent un rôle majeur. Ainsi, l identification de ces canaux et des mécanismes par lesquels ils régulent la différenciation, la prolifération et la migration des kératinocytes est primordiale pour une meilleure compréhension de la physiologie de la peau humaine. Mes travaux se sont donc focalisés sur l implication de ces canaux calciques dans les mécanismes impliqués dans le développement de l épiderme. Les résultats ainsi obtenus ont pu mettre en évidence que la présence du canal TRPV6 à la membrane des kératinocytes est importante pour leur différenciation et qu elle pouvait être régulée par un mécanisme de translocation à la membrane plasmique. Ces travaux montrent également, pour la première fois, que le canal Orai1 est exprimé dans les kératinocytes de l épiderme humain et qu il participe au courant calcique de type SOC. Le canal Orai1 n est pas nécessaire à l induction de la différenciation mais il est principalement impliqué dans la prolifération et la migration des kératinocytes des cellules de la couche basale de l épiderme. L implication d Orai1 dans la migration des kératinocytes est liée à son rôle dans la dynamique des adhésions focales passant par l activation de la protéine kinase FAK.Structure of epidermis is the result of a fine regulated balance between proliferation, differentiation and migration of keratinocytes. Calcium is a second messenger controlling these mechanisms. Among the different players in calcium homeostasis, ionic channels play a major role. The study of these channels and the mechanisms by which they regulate the differentiation, the proliferation and the migration of keratinocytes is therefore crucial in the understanding of skin physiology. This study is focused on these different Ca2+ channels and their implication in the development of the epidermis. The results obtained have shown that TRPV6 plasma membrane expression in keratinocytes is important for differentiation and that it could be regulated by its translocation to the plasma membrane. These results also showed, for the first time, that Orai1 channels are expressed in human epidermis keratinocytes and are involved in SOC current. Also, Orai1 is not necessary for the induction of differentiation, but is mostly implicated in proliferation and migration of non-differentiated keratinocytes, like the cells in the basal layer of epidermis. Orai1 is, in fact, involved in the dynamic of the formation and the turnover of focal adhesions through activation of the protein kinase FAK. Finally, preliminary results have shown that Ca2+-induced differentiation of keratinocytes induces several phases of cytosolic Ca2+ concentration variations involving TRPV6 and Orai1 channels in different ways.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

TRP channels in cell survival and cell death in normal and transformed cells

International audienceTRP channels form a superfamily of channel proteins exhibiting versatile regulatory characteristics with many channels participating in the regulation of Ca(2+) homeostasis and influencing the cell fate. Multitude of evidence is emerging that the colocalization of TRP channels with Ca(2+)-sensing elements of specific regulatory pathways leading to either proliferation or apoptosis is what makes these channels participate in cell fate regulation and, in turn, determines the final effect of Ca(2+) entry via the particular channel. This review focuses on the aspects of TRP channel localization and function that affect the balance between cell survival and death and how various dysregulations of these channels may lead to perturbed balance and onset of cancer