Caractérisation biophysique et moléculaire du complexe récepteurs P2X7 et canaux TMEM16F découverte de nouvelles cibles immunorégulatrices

Purinergic P2X7 receptors (P2X7R) are ligand-gated non-selective cation channels involved in several pathophysiological conditions. P2X7R activation is triggered following elevated concentrations of extracellular ATP, similarly to those observed in tissues injury, chronic inflammation, and in the scrambling of phospholipids leading to membrane blebbing and apoptosis, occurring during elevated entry of Ca2+ ions. Very recently a major family of regulated scramblase and calcium-activated chloride channels, known as anoctamins/TMEM16s were proposed as promising interacting partner to P2X7 receptors. In this research thesis we demonstrated, for the first time, a physical coupling between these two proteins and identified regulatory components/regions critical to complex stability and interaction, contributing, as such to the biophysical and molecular characterisation of the complex also exploring its implications in immune responses. Thus, providing insights into novel potential pharmacological targets.Les récepteurs purinergiques P2X7 (P2X7R) sont des canaux cationiques non sélectifs dépendants d'un ligand, impliqués dans plusieurs conditions pathophysiologiques. L'activation des P2X7R est déclenchée par des concentrations élevées d'ATP extracellulaire, similaires à celles observées dans les lésions tissulaires, les inflammations chroniques, et dans le brouillage des phospholipides conduisant à l'hémorragie membranaire et à l'apoptose, survenant lors de l'entrée élevée d'ions Ca2+. Très récemment, une famille importante de scramblases régulées et de canaux chlorure activés par le calcium, connue sous le nom d'anoctamines/TMEM16s, a été proposée comme partenaire d'interaction prometteur des récepteurs P2X7. Dans cette thèse de recherche, nous avons démontré, pour la première fois, un couplage physique entre ces deux protéines et identifié des composants/régions régulateurs critiques pour la stabilité et l'interaction du complexe, contribuant ainsi à la caractérisation biophysique et moléculaire du complexe et explorant également ses implications dans les réponses immunitaires. Ainsi, nous avons pu découvrir de nouvelles cibles pharmacologiques potentielles

Caractérisation biophysique et moléculaire du complexe récepteurs P2X7 et canaux TMEM16F découverte de nouvelles cibles immunorégulatrices

Les récepteurs purinergiques P2X7 (P2X7R) sont des canaux cationiques non sélectifs dépendants d'un ligand, impliqués dans plusieurs conditions pathophysiologiques. L'activation des P2X7R est déclenchée par des concentrations élevées d'ATP extracellulaire, similaires à celles observées dans les lésions tissulaires, les inflammations chroniques, et dans le brouillage des phospholipides conduisant à l'hémorragie membranaire et à l'apoptose, survenant lors de l'entrée élevée d'ions Ca2+. Très récemment, une famille importante de scramblases régulées et de canaux chlorure activés par le calcium, connue sous le nom d'anoctamines/TMEM16s, a été proposée comme partenaire d'interaction prometteur des récepteurs P2X7. Dans cette thèse de recherche, nous avons démontré, pour la première fois, un couplage physique entre ces deux protéines et identifié des composants/régions régulateurs critiques pour la stabilité et l'interaction du complexe, contribuant ainsi à la caractérisation biophysique et moléculaire du complexe et explorant également ses implications dans les réponses immunitaires. Ainsi, nous avons pu découvrir de nouvelles cibles pharmacologiques potentielles.Purinergic P2X7 receptors (P2X7R) are ligand-gated non-selective cation channels involved in several pathophysiological conditions. P2X7R activation is triggered following elevated concentrations of extracellular ATP, similarly to those observed in tissues injury, chronic inflammation, and in the scrambling of phospholipids leading to membrane blebbing and apoptosis, occurring during elevated entry of Ca2+ ions. Very recently a major family of regulated scramblase and calcium-activated chloride channels, known as anoctamins/TMEM16s were proposed as promising interacting partner to P2X7 receptors. In this research thesis we demonstrated, for the first time, a physical coupling between these two proteins and identified regulatory components/regions critical to complex stability and interaction, contributing, as such to the biophysical and molecular characterisation of the complex also exploring its implications in immune responses. Thus, providing insights into novel potential pharmacological targets

Untangling macropore formation and current facilitation in P2X7

This article belongs to the Special Issue The Role of P2X7 Receptor in Human Health and Diseases.Macropore formation and current facilitation are intriguing phenomena associated with ATP-gated P2X7 receptors (P2X7). Macropores are large pores formed in the cell membrane that allow the passage of large molecules. The precise mechanisms underlying macropore formation remain poorly understood, but recent evidence suggests two alternative pathways: a direct entry through the P2X7 pore itself, and an indirect pathway triggered by P2X7 activation involving additional proteins, such as TMEM16F channel/scramblase. On the other hand, current facilitation refers to the progressive increase in current amplitude and activation kinetics observed with prolonged or repetitive exposure to ATP. Various mechanisms, including the activation of chloride channels and intrinsic properties of P2X7, have been proposed to explain this phenomenon. In this comprehensive review, we present an in-depth overview of P2X7 current facilitation and macropore formation, highlighting new findings and proposing mechanistic models that may offer fresh insights into these untangled processes.This work described in this review was supported by grants from the French National Research Agency ANR (ANR-20-CE14-0016-02), the Fondation Jean-Marie Lehn (Labex CSC-TGR-18), the Région Grand Est and the École Universitaire de Recherche Euridol (Programme d’Investissement d’Avenir, ANR-17-EURE-0022).Peer reviewe

P2X7 Receptors and TMEM16 Channels Are Functionally Coupled with Implications for Macropore Formation and Current Facilitation

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as “macropore” formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities