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ION CHANNEL TRANSCRIPTOME  - Coordinator Sophie Demolombe

State-of-the-art - Turning on or turning off a gene at a different time or in a different place is an adaptive and coordinated response of the organism to novel physiological or pathological environments. Mammalian genomes contain more than 350 genes encoding ion channel subunits. Generalizing high-throughput methods (microarrays, sequencing, real-time PCR, etc) for ion channels will provide important information on how their transcripts are regionally expressed, and on how they are regulated by drugs or diseases in the context of a well-established cellular function.

Aims - Our investigations will explore how the ion channel gene network is dynamically regulated in clusters and how it links transcript expression with function. Ultimately, expression genomics of ion channels should be integrated into mathematical models of cellular electrical activity. Our research program will benefit from the broad experience of the IPMC genomic platform that belongs to the biotechnological infrastructure “France-Génomique” identified by the “Commissariat aux Grands Investissements” during its first call. This infrastructure is a network of 8 platforms providing cutting-edge expertise in genetics, genomics and associated bioinformatics. It has been created to provide rapid and cost effective genomics services.

Previous results: The team of S. Demolombe, who recently joined the EH team, addressed ion channel expression in the non-diseased human heart and in mouse pacemaker tissues with a genomic approach and highlighted significant differences, with potentially important implications for understanding regional electrophysiology, arrhythmia mechanisms and responses to ion channel blocking drugs (4,5). Concordance with previous functional studies suggests that regional regulation of cardiac ionic current expression may be primarily transcriptional. This project was conducted in collaboration with MM/SBL team. The EB team explored the changes of ion channel expression in sensory neurons in the context of chemotherapy induced neuropathy and demonstrated that the expression of ion channel genes is important for the detection of cold and mechanical perception and is altered by oxaliplatin (6).

Projects: 1) Large scale transcriptomic analysis such as those previously described will be used not only to analyze the impact of a given pain model on the expression of ion channel genes (EB, EL and FR teams), but also to study the consequences of calcium channel gene deletion (Cav3.2 conditional KO in sensory neurons in EB team) to the expression of other ion channel genes. Calcium channels are good candidates since they are strongly linked to excitation-transcription coupling, and high-throughput techniques will be key instrumental aspects to decipher these mechanisms. 2) In the FR team, the ion channel transcriptome will be used to evaluate the contribution of P2X4 channels to cell specific remodeling during inflammatory response, focusing on microglial cells, which are the resident macrophages of the central nervous system and are known to be central to inflammatory response within the brain. 3) The EH and MM/SBL teams will collaborate to study remodeling in expression and regulation of ion channels involved in cardiac pacemaking in mouse models of cardiac automaticity dysfunction. This approach will unravel novel pacemaker mechanisms, which may lead to new pharmacological targets for heart rate control in cardiac diseases.

Added value of the LabEX - Most previous studies have quantified the expression of a limited number (generally 5 or less) of ion channel genes and/or proteins in specific tissues or cells, excluding subunits that may have important but unsuspected variability. While, the large-scale studies of gene expression have used pan-genomic arrays, which are limited in their ability to detect differences in low-abundance gene products like those of most ion channel subunits. In our network, we will use high-throughput quantitative RT-PCR and sequencing for a comprehensive panel of ion channel and transporter subunit genes. This method has the advantages of being highly sensitive, specific, quantitative, and of generating the maximum amount of information from limited samples, like those available from human samples. Sharing the same experimental procedures from the handling of the biological samples to the data storage and analysis will be a major benefit of the ICST consortium, allowing the comparison between the models insight the network, to highlight their similarities and potentially discover new interactions between the ion channels of interest. 


State of the art - For years, pore-forming subunits of ion channels have been the main targets for the development of drugs. However, it is now well established that ion channels are multiple subunit assemblies belonging to larger signaling complexes regulating channel expression and function. These signaling complexes are cell-specific, opening new avenues for the development of cell (and tissue) specific ion channel modulators. Proteins interacting with ion channels are involved in trafficking and recycling, scaffolding and activity regulation of these ion channels. Another level of complexity comes from the dynamics of such protein networks as well as their post-translational modifications, both processes being poorly understood. Precisely deciphering the composition of ion channel complexes, dynamics and modifications as well as their tissue specificity represents the first step toward the identification of new targets for the development of novel families of ion channel modulators.

Aims - The ISCT consortium will pursue its efforts to characterize native ion channel complexes using state-of-the-art approaches. These include adapting protocols to improve specificity in the identification of protein partners, developing new approaches to reach a cell specific ion channel proteome, and generating proof-of-concept data establishing protein-protein interactions as potential therapeutic targets. This program will benefit from the expertise of the IGF proteomic facility that is specialized in membrane protein complex analysis as well as in phosphoproteomics.

Previous results: The ICST partners have a strong history in ion channel proteomics, as well as in targeting protein-protein interactions by mimetic peptides or small molecules. Interacting proteins identified by the partners of the consortium are related to different levels of regulation: 1) Sensor proteins: Filamin A has been identified as a partner of TRPP2, the polycystin-2 involved in polycystic kidney disease. Filamin A is necessary for transducing pressure sensing to the channel (7). Vilip-1, a neuronal calcium sensor interacts with the P2X2 channel. This interaction is dynamically regulated by calcium influx mediated by P2X2 activation (8). 2) Scaffolding proteins: PICK-1 and NHERF-1, two scaffolding proteins have been identified as interacting with PDZ domain of ASIC channels. These proteins play a crucial role in the modulation of ASIC channel function through PKC-mediated phosphorylation (9,10). AKAP150 interacts with TREK channels converting their activity from low to fully active at rest, and regulating PKC- and PKA-dependent phosphorylation of the channels (11). 3) Trafficking: Different proteins involved in the regulation of ion channel trafficking have been discovered. These include the association between TREK channels and MAP2 (12,13), TRPM8 and the new proteins TCAF1 and 2 (in press). 4) Signaling proteins: Cav channels interact directly with signaling molecules such as GPCR (14) or nNOS (unpusblished results). Members of the consortium are also actively involved in developing new tools to modulate protein-protein interactions. The rationale for this approach is that one way to regulate ion channel function with higher specificity is through specific interference with known interacting proteins. The proof-of-concept of this approach has been given by SBL who succeeded in inhibiting protein-protein interaction through systemic administration of a small vectorized peptide (patent pending) (15). The MdW team who has a great expertise in cell-penetrating peptides develops similar approaches (16,17).

Projects: The ICST partners are actively engaged in proteomic analysis of ion channels in different systems. Without being exhaustive, one can mention the identification of interacting proteins regulating the trafficking of channels to the plasma membrane, which represents a new strategy in the development of molecules controlling their function. The FL team is involved in the analysis of TWIK1 channel trafficking between recycling endosomes and the plasma membrane. A similar approach is developed by FR team to understand the rapid recycling of P2X4 channels in macrophages. The MdW team is characterizing trafficking of Cavß subunits between the plasma membrane and the cell nucleus. Other proteomic investigation of ion channel regulation and functions are: TRPP2 and the intracellular calcium homeostasis (EH), identification of Cav3.2 channels partners through GFP trap (EB), P2X receptors and activation of inflammasome though TAP-tag proteomics (FR), phosphosproteomic of Cav channels (PL) and the mechanisms of the functional crosstalk between Cav1.3 and ryanodine receptors in the generation of cardiac pacemaking (EH, MM/SBL).

Added value of the LabEX - Proteomic methods are constantly improving, and it is now conceivable to bring identification of interacting proteins to a medium throughput level. Furthermore, approaches are being developed to analyze posttranslational modification of proteins. These technological advances can only be handled by specialized facilities. The consortium will benefit from the expertise and equipment of FPP, the Montpellier Proteomic facility (www.fpp.cnrs). The platform is equipped with the latest technology for mass spectrometry (LTQ Orbitrap XL-ETD and VELOS), allowing for high sensitivity and high-resolution analysis. FPP also takes advantage of a wide array of new and efficient technologies to develop differential proteomics studies. These include quantitative mass spectrometry with or without labeling (e.g label-free, SILAC metabolic labeling), TMT or dimethylation (chemical labeling). FPP participates in a variety of programs, including the characterization of multiple protein complexes involved in cell signaling, the dynamic analysis of post-translational modifications (phosphorylation, sumoylation, etc). The ICST consortium will set optimized protocols for membrane protein isolation and solubilization, tandem affinity purification, GFP-trap and mass spectrometry analysis, allowing efficient exploration of proteins associated with ion channels.