State-of-the-art and Aims - Following the “-omics” era (TASK 1), the goal is to model the activity and properties of ion channels both in vitro and in vivo, using preclinical models. The analysis performed at the cellular level requires the use of a wide variety of complementary approaches to efficiently link molecular information to complex phenotypes. A constant challenge of these studies is to use the appropriate cellular systems and the most accurate methodologies to probe the precise involvement of ion channels in a variety of physiological and pathological situations.
Aims - Most aspects of the molecular physiology of ion channels are explored in the ICST teams, including structure-function relationships, validation of partners and interacting networks, trafficking and quality control, electrophysiological and pharmacological properties, and phenotypic analysis. The ICST consortium aims to merge and share all the cellular systems and appropriate methodologies in a unique platform to facilitate the study of ion channel physiology at the molecular and cellular levels. This platform will provide access to all consortium members to one of the largest panels of validated cellular/tissue preparations and validated technical expertise to explore novel aspects of the channel regulation and the related phenotypes.
Established expertise available in the consortium - The ICST teams have set up and validated a unique set of model systems and complementary techniques to explore their favorite aspects of ion channel roles and activity. State-of-the-art analysis available within the consortium includes, exploration of the cardiac sinoatrial and atrioventricular nodes and conducting system properties (MM/SB), cardiac and brain ischemia models (MM/SB, CH), models of CNS and PNS phenotypes (EL, EB, MM, MdW, CH, PL), renal and vascular phenotypes (EH, JB), mechano-sensitive phenotypes (EH, EB), muscle and bone dysfunction (JB), nerve-skin sensory phenotypes (EL), proliferation/migration/apoptosis assays (NP), inflammatory assays (FR). This expertise will be available to all the ICST teams as a platform resource that will foster collaborative and innovative research programs on: 1) Structural analysis and molecular physiology studies - Heterologous expression systems (Xenopus oocytes, Sf9 insect cells, the mammalian HEK-293, COS-7, CHO cell lines) are used to perform structural analysis (MV) and to characterize various properties of their favorite ion channels, such as validation of partners or quality control mechanisms (FR, PL, EB, MdW, NP, EH, FL, MM, EL, CH). Electrophysiology is a dedicated technique to explore ion channel properties and all the ICST groups use the standard 2 electrodes voltage-clamp (oocytes) and/or the patch-clamp (isolated mammalian cells) techniques. Importantly, a platform for high throughput membrane protein crystallization, unique in France, is being finalized at IBS (MV), allowing structural studies of ion channels in the near future. 2) The use of specialized cellular models - Most ICST partners study their favorite channels in a native-like cellular environment. Many cell lines are used, including NG 108-15 (neuroblastoma), F11 (DRG-derived), LNCaP (prostate cancer), Jurkat, (T-lymphocytes), HL60 (monocytes), RBL-2H (mast cells), BV2 and N9 (microglia), RAW (macrophage). The ICST teams have also developed original cell lines, in which ion channels are overexpressed or silenced (NP), as well as cell lines derived from KO mice (EH). 3) The development of ex-vivo models - Several ICST groups have developed unique expertise for the preparation of primary cultures, including cardiac (MM/SB) and skeletal myocytes (JB, MdW), renal and vascular cells (EH), neurons (from DRG, hippocampus, cerebellum: EB, PL, EL, FL), prostate (NP), bone marrow-derived cells and microglia (FR). These labs have also established protocols to isolate and analyze freshly-dissociated cells from mice that available in the consortium for cardiac pacemaker cells, adrenocortical cells, a variety of primary neurons (DRG, hippocampal neurons) and astrocytes. Additional expertise also exists for the preparation and study of various freshly-cut brain slices (EL, MM), DRG and spinal cord slices (EB, EL), nerve-skin preparation (EL), adrenal slices (JB), vascular rings (EH). 4) The development of innovative methodologies to explore ion channel function - Several groups have developed ex-vivo electrophysiological recordings on brain slices (CH, EL, MM), nerve-skin preparation (EL), adrenal slices (JB). Innovative electrophysiological approaches include in situ patch-clamp and in vivo extracellular recordings of spinal dorsal horn neurons (EL). Fast perfusion- and fast temperature-clamp systems (EB, EL), light-activated and mechano-clamp techniques (EH) are complementary approaches available in the consortium to monitor and study non-voltage activated properties of channels. Imaging techniques enable complementary analysis of ion channel properties and most ICST teams are equipped with, or have access to, standard calcium imaging set-ups (Fura-2, Fluo4) and innovative imaging techniques, such as confocal, 2-photon and high-resolution OMX microscopy set-ups for fast imaging techniques (MM/SB). Gene silencing (RNA interference) and lentiviral-mediated overexpression or silencing techniques (shRNA) are used in several groups to probe the functional roles of ion channels in vivo (EH, PL). In addition, efforts are being made to perform pharmacological profiling and identify novel and selective compounds for some of ion channels studied in the consortium: automated patch-clamp set-ups are available for drug screening projects (IGF, NP) and a special interest resides in the search for spider and scorpion toxins targeting ion channels (EL, EB, MV, MdW).
Added value of the LabEx - The ICST consortium brings together complementary and established expertises in the field of ion channels. Merging these resources and know-how will allow carrying out one of the largest panels of functional assays on ex-vivo models. Importantly, all these functional assays - some of them being unique worldwide - have already been validated for one or more ion channel models and therefore will provide state-of the-art phenotypic analysis of the potential implication of other ion channel species. These ex-vivo approaches will ideally complement all the in vivo studies described in the next paragraphes. Finally, it is important to mention that several groups have now established collaborations with clinicians and access to human samples, e.g., cancer biopsies, myoblasts, enteric neurons, to initiate translational studies.
State-of-the-art – Ion channel dysfunction can induce multi-organ phenotypes or diseases. During the last years, ICST consortium teams have developed several mouse models in which ion channels have been inactivated or modified. Exploration of these mouse lines contributed to the understanding of ion channel role in important physiological functions. The consortium harbours cutting-edge technical platforms for exploration of mouse phenotypes that will be rendered available to all ICST teams to investigate unexplored functions of ion channels in multiple organs, as well as in normal and pain behaviour. We will also investigate the role of new channel isoforms and variants discovered by the consortium during the course of the project.
Aims -The ICST consortium will focus on the impact of specific ion channels in human diseases such as depression, epilepsy, pain sensation, cerebral and cardiac ischemia, cardiovascular pathology, kidney function and tumor genesis. Specific questions on the role of ion channels in inherited and acquired retinal pathologies will also be addressed. IPMC will centralize shared resources for analysis of the impact of ion channels on depression (18), anxiety, recovery from cerebral ischemia (19,20) and functional exploration of the retina (21-24). Two highly complementary platforms at the IPMC (EL) and at the IGF (EB) will study the functional role of ion channels in inflammatory, post-operative and neuropathic pain. Cardiovascular function will be investigated at the IGF (MM/SBL) and at the IPMC (EH). The platforms allow evaluating the effects of the inactivation or modification of ion channels on heart rate, cardiac automaticity, atrial and ventricular arrhythmias, as well as the impact of autonomic nervous system regulation on heart rhythm and vascular dynamics. Proper expertise for inducing cardiac infarct by coronary ligation and evaluation of infarct size and cellular apoptosis is also available.
Channels and behaviour: previous results and ongoing projects – CH team highlighted the role of TREK1 channels in depression(18). It has recently been demonstrated that TASK3, which has 50% of homology with TASK1 channels acts as a therapeutic target for antidepressant action (25). We will search new antidepressant targets by performing antidepressant tests on TASK1 KO mice and double KO mice TASK1/TREK1. The role of TASK1 in depression is still unexplored and can lead to validation of this channel as a pharmacological target. This will be important for searching for analogs of the specific blocker of TREK1 spadin (26) with a better stability/biodisponibility and without TREK1-related side effects. In the search for new neuroprotective molecules against cardiac arrest and stroke, the consortium will test antiapoptotic peptides previously characterized in MM/SBL team (15). EB and EL groups described important roles of Cav and ASIC channels in neuropathic and inflammatory pain (27-30). These groups will coordinate the in vivo investigation of the role of selected ion channels in pain sensation.
Cardiovascular disease and ion channels: previous results and on-going projects – The MM/SBL team has described the role of Cav1.3, Cav3.1 and HCN channels in heart automaticity (see (31) for review). The ICST now aims to explore the link between ion channel dysfunction, neurological disease and the risk of sudden cardiac death. For instance, we will explore the role of TREK1 in sudden cardiac death associated with epilepsy by simultaneous ECG/EEG recording in TREK1 KO mice. EH team provided outstanding studies to uncover the role of TRPP channels in the regulation of pressure sensing (7,32). This new knowledge will be important to investigate the role of TRPP channels in heart rate regulation. This point is of special interest, since, while it is well known that mechanical stretch and pressure sensing regulate heart performance, the molecular basis of this regulation is completely unknown. The expertise of EL team will be important to study the role of ASIC channels in cardiac ischemia, using mice in which one or more ASIC isoforms have been inactivated.
Functional role of ion channels in body homeostasis – JB team has contributed to uncover the specific physiological functions of channel isoforms of the K2P family (33,34). This expertise is pivotal within the consortium for investigating the role of ion channels in the kidney and ionic homeostasis. The consortium is particularly interested in two important functions: the roles of TASK channels in the adrenal gland in respect to its functional differentiation and the control of the aldosterone secretion, as well as the roles of TASK channels in cardiorespiratory function in respect to breathing and vascular adaptation to O2 and CO2/pH changes.
Ion channels in retinal disease - Among the organs, the eye concentrates the largest number of listed genetic diseases. It is also the organ most collectively affected in systemic diseases of genetic origin. The IPMC platform of functional exploration of the retina offers the possibility to assess retinal function by non-invasive retinogram in rodent models of photodegeneration and retinal ischemia (21-24). The consortium will investigate the role of ASIC channels in retinal ischemia. Knowledge acquired on protective strategies against global or partial cerebral and cardiac ischemia will also be tested in retinal ischemia.
Ion channels and in vivo models of tumor genesis – NP group has developed state-of-the-art techniques to investigate ion channel-mediated tumor genesis using prostate cancer (PCa) as a model. MdW team develops models of glioma for screening toxins targeting ion channels able to inhibit progression of tumorigenesis. An objective of the consortium is to identify a set of ion channels with pro- or anti-oncogenic action that may be used for the selection of therapeutical molecules against PCa and glioma in humans. Candidate ion channels will be tested by siRNA-based approach to study their role in tumour growth and metastasis development. Ion channel targets of selected toxins will be identified by proteomic approaches. Selected channels will be validated for their oncogenic activity in in vivo xenograft mouse models. The anti-tumor potential of the selected toxins will be increased by chemically grafting anti-tumor agents with known organ toxicity. The functional exploration platform at the PhyCel and the INPA platform (Imagerie Nucléaire du Petit Animal) at the GIN will combine their expertise to perform non-invasive whole-body bioluminescence imaging of tumour growth, analysis of individual tissues, pharmacological tracking of toxins and assessing the bio availability of active anti-oncogenic peptides. The role of Cav channels will be studied in collaboration with teams at the IGF, while the expertise of the IPMC will be precious to study other interesting candidates such as TRPM8 or TRPV6 channels.
Added value to the LabEx - Functional exploration of animal models of genetic and acquired disease is of paramount importance for understanding the physiopathological and molecular bases of human illness. Furthermore, testing of candidate molecules, peptides and toxins with potential therapeutic impact constitutes a fundamental step to provide proof-of-concepts in preclinical studies. For ion channels inducing multi-organ phenotype, or for new ion channels of unknown physiological role, it is crucial to provide phenotype exploration at the multi-organ level, a need that is rarely met within a single Institute. Sharing technical expertise and platforms within the ICST consortium will open the way to better understand ion channel function and their pharmacological interest in the whole organism with an unprecedented degree of integration, an approach that would not be feasible outside the consortium. For instance, exploring the link between ion channel dysfunction, neurological disease and the risk of sudden cardiac death is an ambitious task that can be accomplished only by the ICST consortium. Similarly, exploring the interest of cardioprotective peptides against brain ischemia has a major medical interest, but development of such a research program requires a large collaborative effort.