Modulation of low-voltage-activated T-type Ca2+ channels

AbstractLow-voltage-activated T-type Ca2+ channels contribute to a wide variety of physiological functions, most predominantly in the nervous, cardiovascular and endocrine systems. Studies have documented the roles of T-type channels in sleep, neuropathic pain, absence epilepsy, cell proliferation and cardiovascular function. Importantly, novel aspects of the modulation of T-type channels have been identified over the last few years, providing new insights into their physiological and pathophysiological roles. Although there is substantial literature regarding modulation of native T-type channels, the underlying molecular mechanisms have only recently begun to be addressed. This review focuses on recent evidence that the Cav3 subunits of T-type channels, Cav3.1, Cav3.2 and Cav3.3, are differentially modulated by a multitude of endogenous ligands including anandamide, monocyte chemoattractant protein-1, endostatin, and redox and oxidizing agents. The review also provides an overview of recent knowledge gained concerning downstream pathways involving G-protein-coupled receptors. This article is part of a Special Issue entitled: Calcium channels

Voltage-Gated T-Type Calcium Channel Modulation by Kinases and Phosphatases: The Old Ones, the New Ones, and the Missing Ones

Calcium (Ca2+) can regulate a wide variety of cellular fates, such as proliferation, apoptosis, and autophagy. More importantly, changes in the intracellular Ca2+ level can modulate signaling pathways that control a broad range of physiological as well as pathological cellular events, including those important to cellular excitability, cell cycle, gene-transcription, contraction, cancer progression, etc. Not only intracellular Ca2+ level but the distribution of Ca2+ in the intracellular compartments is also a highly regulated process. For this Ca2+ homeostasis, numerous Ca2+ chelating, storage, and transport mechanisms are required. There are also specialized proteins that are responsible for buffering and transport of Ca2+. T-type Ca2+ channels (TTCCs) are one of those specialized proteins which play a key role in the signal transduction of many excitable and non-excitable cell types. TTCCs are low-voltage activated channels that belong to the family of voltage-gated Ca2+ channels. Over decades, multiple kinases and phosphatases have been shown to modulate the activity of TTCCs, thus playing an indirect role in maintaining cellular physiology. In this review, we provide information on the kinase and phosphatase modulation of TTCC isoforms Cav3.1, Cav3.2, and Cav3.3, which are mostly described for roles unrelated to cellular excitability. We also describe possible potential modulations that are yet to be explored. For example, both mitogen-activated protein kinase and citron kinase show affinity for different TTCC isoforms; however, the effect of such interaction on TTCC current/kinetics has not been studied yet

Role of voltage-gated T-type calcium channels in the viability of human melanoma

En aquest treball de tesi hem estudiat per primera vegada l’expressió funcional dels canals de calci dependents de voltatge (CCDV) en melanòcits humans i un ampli rang de línies cel.lulars i biòpsies de melanoma humà, mitjançant tècniques de biologia molecular i d’imatge. Els nostres resultats demostren que els melanòcits control i les cèl.lules de melanoma expréssen isoformes de les famílies de gens Cav1 i Cav2. De forma destacable, l’expressió d’isoformes de la família Cav3 (canals de tipus T) es troba restringida a les cèl.lules de melanoma, en les que promouen la progressió del cicle cel.lular. Aquests resultats motiven l’anàlisi dels CCDV-T com a dianes terapèutiques contra la tumorigènesi i/o progressió tumoral del melanoma. En aquesta línea, hem trobat que mibefradil i pimozida, dos bloquejants de CCDV-T d’us clínic, inhibeixen el creixement de les cèl.lules de melanoma in vitro, i que aquest efecte és degut tant a una reducció de la proliferació cel.lular, com a una inducció de la mort dependent de caspases. Hem explorat les vies moleculars implicades en el procés apoptòtic i hem trobat que ambdues drogues indueixen estrés de reticle endoplasmàtic (RE) i la inhibició subsequent de l’autofàgia basal constitutiva present a les cèl.lules de melanoma. Finalment, hem demostrat, a través del seu silenciament gènic, que la isoforma Cav3.2 és la diana molecular dels bloquejants de CCDV-T, en el que respecta als seus efectes sobre l’estrés de RE i l’autofàgia. Conjuntament, els resultats obtinguts en el decurs d’aquesta tesi apunten als canals de tipus T como a possibles marcadors de pronòstic i/o dianes terapèutiques contra la metastasi del melanoma.Hemos estudiado por primera vez la expresión funcional de los canales de calcio voltaje-dependientes (CCDV) en melanocitos humanos y un amplio rango de líneas celulares y biopsias de melanoma humano, mediante técnicas de biología molecular y de imagen. Nuestros resultados demuestran que los melanocitos control y las células de melanoma expresan isoformas pertenecientes a las famílias de genes Cav1 y Cav2. De forma destacable, la expresión de isoformas de la família Cav3 (canales de tipo T) se encuentra restringida a las células de melanoma, en las que promueven la progresión del ciclo celular. Estos resultados motivan el análisis de los CCDV-T como dianas terapéuticas contra la tumorigénesis y/o progresión tumoral del melanoma. En esta línea, hemos encontrado que mibefradil y pimozida, dos bloqueantes de CCDV-T de uso clínico, inhiben el crecimiento de las células de melanoma in vitro, y que este efecto es debido tanto a una reducción de la proliferación celular como a una inducción de la muerte dependiente de caspasas. Hemos explorado las vías moleculares implicadas en el proceso apoptótico y hemos hallado que ambas drogas inducen estrés de retículo endoplasmático (RE) y la inhibición subsiguiente de la autofagia basal constitutiva presente en las células de melanoma. Finalmente, hemos demostrado, a través de su silenciamiento génico, que la isoforma Cav3.2 es la diana molecular de los bloqueantes de CCDV-T en lo concerniente a sus efectos sobre el estrés de retículo endoplasmático y la autofagia. Conjuntamente, los resultados obtenidos en el curso de esta tesis apuntan a los canales de tipo T como posibles marcadores de pronóstico y/o dianas terapéuticas contra la metástasis del melanoma.We have addressed for the first time the functional expression of voltage-gated calcium channels (VGCCs) in human melanocytes and a range of melanoma cell lines and biopsies, by molecular biology and imaging techniques. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Cav1 and Cav2 gene families. Importantly, the expression of isoforms of Cav3 (T-type) channels is restricted to melanoma cells, in which they promote cell cycle progression. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and ⁄or tumour progression. In this regard, we have found that mibefradil and pimozide, two clinically-used T-type Ca2+ channel blockers, inhibit the in vitro growth of melanoma cells, and that this effect is due to both a reduction in the cell proliferation rate and an induction of caspase-dependent cell death. We have further explored the molecular pathways leading to T-type channels blockers-mediated apoptosis, and found that both drugs induce endoplasmic reticulum (ER) stress and a subsequent inhibition of the basal autophagy present in melanoma cells. Finally, we have demonstrated by a gene silencing approach that the Cav3.2 isoform is the molecular target of T-type channel blocker mediated effects on ER-stress and autophagy. Altogether, the results attained in this thesis point to T-type Ca2+ channels as putative prognosis markers and/or therapeutic targets to tackle melanoma metastasis

Troubles du rythme cardiaque dans les modèles murins transgéniques

Thèse en cotutelle avec Université de Nantes - Pays de La Loire - France (2005-2010)Les maladies cardio-vasculaires sont la première cause de mortalité dans le monde. L’hypertrophie cardiaque est un processus de remodelage provoqué par une surcharge de travail du muscle cardiaque afin de mieux répondre à la demande de l’organisme. Bien que bénéfique à court terme, une hypertrophie trop accentuée conduira à long terme, à une insuffisance cardiaque. L’hypertrophie est associée à un remodelage électrique qui conduit généralement à un allongement du potentiel d’action, une des causes des arythmies ventriculaires et de la mort subite. Généralement, le mécanisme causal est la fibrillation ventriculaire, un trouble du rythme irréversible dont les mécanismes sont complexes et méconnus. Si les conséquences fonctionnelles in vitro des mutations génétiques ou du remodelage ionique sont relativement simples à étudier ou à prévoir, leur rôle dans les mécanismes des troubles du rythme in vivo sont plus difficiles à appréhender. Parmi les nombreux modèles animaux développés pour la recherche sur les troubles du rythme, la souris est de plus en plus utilisée en raison de notre capacité à muter, invalider ou sur-exprimer les gènes d'intérêt chez ces animaux. L'objectif de mon travail de thèse était de mieux comprendre le rôle des canaux ioniques en physiopathologie cardiaque, en particulier dans la survenue des troubles du rythme in vivo. Ces travaux ont permis d'améliorer notre connaissance du rôle des anomalies génétiques impliquant des canaux ioniques et du remodelage ionique dans la physiopathologie des troubles du rythme et pourrait ainsi ouvrir de nouvelles perspectives thérapeutiques dans le traitement anti-remodelage cardiaque et la prévention de la mort subite.Cardiovascular disease is the leading cause of death in the world each year. If no action is taken to improve cardiovascular health and current trends continue, WHO estimates that 25% more healthy life years will be lost to cardiovascular disease globally by 2020. Cardiac hypertrophy is the consequence of an excessive workload of the heart muscle leading to cardiac remodeling process. As the workload increases, the ventricular walls grow thicker, lose elasticity and eventually may fail to pump with as much force as a healthy heart. Furthermore, hypertrophied myocardium is not physiologically normal and may confer a predisposition to potentially fatal arrhythmias. Generally, the causal mechanism is ventricular fibrillation, a cardiac rhythm disorder which is irreversible but the pathophysiological mechanisms are complex and poorly understood. The functional consequences of mutations or ionic remodeling are relatively simple to study in vitro, but their role in the pathophysiology of arrhythmias in vivo is more difficult to grasp. Among the different animal models developed in cardiac arrhythmias research, the mouse is increasingly used because of our ability to mutate, knock-out or over-express genes of interest. The objective of my thesis was to study the role of ion channels in physiology as well as cardiac pathophysiology, particularly in the involvement of the occurrence of cardiac arrhythmias in vivo. This thesis will improve our understanding of the role of genetic abnormalities involving ionic remodeling in the pathogenesis of the heart and may also open new therapeutic perspectives in the treatment of cardiac remodeling as well as sudden cardiac death

Molekulární fyziologie kanálů T-typu aktivovaných nízkým napětím u neuropatických bolestí

Nízkonapěťově aktivované iontové kanály se významně podílí na přenosu signálu ve vzestupné dráze bolesti. Jejich elektrofyziologické a biochemické vlastnosti jim umožňují regulovat neuronální excitabilitu a uvolňování neurotransmiterů. Změny v elektrických proudech spojovaných s nervovými poruchami včetně neuropatické bolesti a epilepsie, jsou spojovány s tímto subtypem vápenných kanálů, naznačujíc tak jejich zásadní roli v modulaci neuronální odpovědi na různé obtěžující stimuly. Povícero nemocí, jako třeba cukrovka, rakovina nebo chronický úraz nervu, jsou doprovázeny bolestivými neuropatickými stavy. Specifické inhibitory T-typu kanálů vykazují zlehčující účinky na neuropatickou bolest na zvířecích modelech, ukazujíce tak jejich potenciál k vývoji nových typů léků možná učinnějších než tradiční analgetika, která vykazují malý efekt při léčbě neuropatické bolesti.Low-voltage activated T-type channels contribute significantly to signal transmission in ascending pain pathway. Their electrophysiological and biochemical properties allow them to modulate neuronal excitability and neurotransmitter release. Alterations of electric currents associated with a number of neuronal disorders, including neuropathic pain and epilepsy, have been linked to this subtype of calcium channel, suggesting its prominent role in modulation of neuronal response to various noxious stimuli. Multiple diseases, such as diabetes, cancer or chronic nerve injury, are accompanied by painful neuropathic conditions. Specific inhibitors of T-type channels have been demonstrated to alleviate symptoms of neuropathic pain in mouse models, showing their potential for development of novel type of drugs possibly more effective than traditional analgesics, which exhibit minor effect in neuropathic pain treatment.Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologiePřírodovědecká fakultaFaculty of Scienc

Activity-Dependent Regulation of Neuronal Ion Channel Expression During Epileptogenesis

Status epilepticus (SE) is known to cause numerous structural and functional alterations in brain. In this study, expression of various voltage-gated ion channels in rat hippocampal CA1 neurons were investigated at different time points after an episode of SE induced by pilocarpine. First, with microarray, gene expression profiling data revealed expression alterations in several voltage-gated ion channel subfamilies. Then, with absolute real-time RT-PCR quantification, we found GAPDH were significantly increased in the SE experienced neurons, whereas the neuron-specific gene synaptophysin appeared stable. This was also proved by in situ hybridization. Using synaptophysin as reference gene, real-time RT-PCR relative quantification was applied to determine the expression changes of several groups of voltage-gated ion channels related to intrinsic excitability in CA1 neurons. They were voltage-gated Ca2+ and Na+ channels; KCNQ channels, A-type K+ channels; and HCN channels. The most important and interesting parts are below: (1) Cav3.2 mRNA that underlies the T-type Ca2+ currents was transiently increased at day 3 in SE experienced samples. (2) And the persistent down-regulation of KCNQ2 mRNA was also found after SE. This channel is responsible to the M-currents, one type of voltage-gated K+ currents, which play a critical roll in determining the sub-threshold electrical excitability of neurons. We thus suggest that intrinsic plasticity is a potent mechanism that, in conjunction with synaptic plasticity, can fundamentally alter the input-output properties of neuronal networks in the mammalian brain

The calcium-cancer signalling nexus

The calcium signal is a powerful and multifaceted tool by which cells can achieve specific outcomes. Cellular machinery important in tumour progression is often driven or influenced by changes in calcium ions; in some cases this regulation occurs within spatially defined regions. Over the past decade there has been a deeper understanding of how calcium signalling is remodelled in some cancers and the consequences of calcium signalling on key events such as proliferation, invasion and sensitivity to cell death. Specific calcium signalling pathways have also now been identified as playing important roles in the establishment and maintenance of multidrug resistance and the tumour microenvironment

Investigating Mechanisms in Nociceptors Driving Ongoing Activity and Ongoing Pain

Ongoing (apparently spontaneous) pain at rest is a major complaint of patients suffering from many forms of acute and chronic pain, including acute and persistent postsurgical pain. Accumulating evidence suggests ongoing activity in nociceptors is a major driver of ongoing pain. Ongoing activity can be generated in sensory neurons in the absence of sensory generator potentials if one or more of three neurophysiological alterations occur – prolonged depolarization of resting membrane potential (RMP), hyperpolarization of action potential (AP) threshold, and/or increased amplitude of depolarizing spontaneous fluctuations of membrane potential (DSFs) to bridge the gap between RMP and AP threshold. Cellular signaling pathways that increase DSF amplitude and promote ongoing activity acutely in nociceptors were unknown for any neuromodulator, as were the neurophysiological alterations that underly ongoing activity in nociceptors after deep tissue incision. The work presented in this dissertation sought to identify a cellular signaling pathway and injury- and inflammation-related neuromodulator that induces alterations that drive hyperexcitability, such as enhanced DSFs, and thereby potentiates ongoing activity in nociceptors, as well as to determine the alterations that contribute to nociceptor hyperactivity associated with postsurgical pain. A combination of whole-cell patch clamp electrophysiology, pharmacology, and high content microscopy was used to define the effects of low concentrations of the injury-related proinflammatory mediator serotonin and the major pathway by which it exerts its effects. This study shows that serotonin enhances DSFs, hyperpolarizes AP threshold, and thereby potentiates OA in isolated rat DRG neurons at a concentration of 100 nM, and serotonin exerts these effects on nonpeptidergic nociceptors via the 5-HT4 receptor and downstream cAMP signaling via PKA and EPAC. Furthermore, whole-cell patch clamp recordings of primary DRG neurons, analysis of spontaneous pain behavior, and RNA sequencing were used to reveal an unexpected persistence of hyperexcitability in isolated DRG neurons and potentially associated differential gene expression, both of which were found after the behavioral expression of postsurgical pain had resolved. In addition to elucidating cell signaling mechanisms that can contribute to acute ongoing activity in nociceptors, these studies set the stage for future investigations addressing important questions about nociceptor contributions to pain