Combining mass spectrometry and functional analyses for a comprehensive study of the phosphorylation of the Cav3.2 T-type calcium channel – Phospho-Cav

Le projet Phospho-Cav s’appuie sur le savoir-faire de la plateforme de protéomique de Montpellier (PPM), ainsi que sur l’expertise en électrophysiologie des canaux calciques de l’équipe du coordinateur, pour l’analyse moléculaire de la phosphorylation des canaux calciques de type T.

Ce projet de recherche fondamentale en cours de développement. Aucun résultat ne peut actuellement faire l’objet d’une communication vers le grand public.

Phospho-Cav est un projet pionnier concernant l’étude globale de la phosphorylation des canaux calciques de type T. Une perspective majeure de ce programme est une meilleure compréhension des mécanismes responsables de maladies neurologiques associées à ces canaux, en particulier l’épilepsie et la douleur.

En préparation

Phosphorylation is probably the most important post-translational regulatory mechanism and contributes to the regulation of the voltage-gated calcium channels (Cav channels) that are involved in a wide variety of physiological “excitable” functions and disease states. Cav3/T-type channels, especially Cav3.2 channels that play important role in cell differentiation, neuronal excitability, slow wave sleep, hormone secretion and sensory processing, are also regulated by phosphorylation. Understanding how these channels are regulated is critical for the interpretation of their roles in physiology and pathophysiology (absence epilepsy, neuropathic pain and cancer). Several recent studies have highlighted that Cav3 channels, especially the Cav3.2 subtype, are modulated by a wide variety of hormones, neurotransmitters and serine/threonine protein kinase pathways. These recent studies open new avenues for the study of phosphorylation of Cav3.2 channels, especially the search for phosphorylated residues and the functional consequences. This motivates the present project.

The Cav3.2 channels, that are studied in partner 1 lab for several aspects (search for domains involved in trafficking, impact of epilepsy absence mutations, search for partners…) can be directly phosphorylated and functionally modulated by several protein kinases (PKA, PKC,…) in heterologous expression systems. Classical biochemical approaches appear to be poorly reliable for the identification of the phosphorylated residues in Cav channels. Nowadays, because of the recent development in phosphoproteomic approaches, especially in partner 2's lab, a comprehensive study of the Cav3.2 channel phosphorylation can be performed, as recently reported for other ionic channels (Kv).

The main objectives of the “Phospho-Cav” project are listed below. (1) We will set-up a mass spectrometry approach (immuno-purification and phosphopeptide analysis) in order to provide a phospho-map of the residues expected to be important for Cav3.2 regulation. This part of the project will be achieved using a hemagglutinin (HA) tagged Cav3.2 channel. (2) A SILAC (stable isotope with amino acids in cell culture) procedure will be employed to estimate if, at some sites, phosphorylation is linked to specific regulatory pathways. (3) A functional analysis of the identified Cav3.2 phospho-sites will be performed using site-directed mutagenesis followed by heterologous expression in mammalian cells (electrophysiological properties, trafficking…). (4) The efficiency of antibodies for Cav3.2 immuno-purification will be characterized in order to purify native Cav3.2 channels from i) the neuroblastoma cell line NG108-15, in which Cav3.2 channels are involved in differentiation ; ii) mouse hippocampus in a temporal lobe epilepsy model, in which Cav3.2 activity is enhanced. (5) In these latter conditions, the mass spectrometry approach will allow identification of the phosphorylation status in a physiological context (differentiation) and in a disease state (epilepsy). (6) These novel active phosphorylation sites, possibly novel splice variants, will then be studied in heterologous expression as described above (3).

Completion of this project, especially the analysis of phospho-status of Cav3.2 channels, should provide the first global analysis of the active phospho-sites of Cav3.2 channels. Studies of functional consequences of native Cav3.2 channel phosphorylation will offer the first possibility to probe how signalling pathways may be involved in the regulation of Cav3.2 protein in normal and disease states. Using this global approach, we can expect to provide a comprehensive view of the important residues involved in PKA/PKC modulation of Cav3.2 channels, as well as other PK pathways. Ultimately, such an approach should allow a better understanding of the Cav3.2 channel regulation in a variety of physiological and pathophysiological situations.