Les canaux ioniques sont des déterminants majeurs de la forme et de la durée du potentiel d'action (PA) cardiaque. Leur expression fonctionnelle à la membrane plasmique résulte d'une balance entre les voies antérograde et rétrograde du trafic intracellulaire, ainsi que de leur prise en charge par des compartiments endosomaux afin d'être recyclés ou dégradés. Le canal KV1.5 porte le courant principal de repolarisation atriale chez l'homme, IKur, et est impliqué dans la physiopathologie de la fibrillation atriale (FA). La FA est caractérisée par un raccourcissement de la durée du PA lié à un courant IKur augmenté et un courant ICaL diminué et est favorisée par l'augmentation des contraintes mécaniques. Ainsi, le canal KV1.5 constitue une cible majeure pour le développement d'anti-arythmiques spécifiques de l'oreillette. Ce projet avait pour but de mieux comprendre comment est régulée l'expression fonctionnelle des canaux KV1.5 dans les myocytes atriaux. Dans un premier temps, nous avons montré que le shear stress entraîne une augmentation du courant IKur impliquant la voie de mécanotransduction intégrine?1/FAK et l'endosome de recyclage lent. Dans les cellules hypertrophiées, cette voie de mécanotransduction est hyperactivée et le courant IKur est ainsi augmenté. Dans un second temps, nous avons montré que la voie d'endocytose des canaux KV1.5 est dépendante de la clathrine et que les microtubules sont principalement impliqués dans l'internalisation et la dynamique du canal à la surface des cellules. Ainsi, ce travail a permis de mieux caractériser les acteurs du trafic impliqués dans la régulation de l'expression fonctionnelle du canal KV1.5 dans les cardiomyocytes atriaux. / Ion channels are major determinants of shape and duration of the cardiac action potential (AP). Their functional expression at the sarcolemma is a dynamic process resulting from a balance between anterograde (exocytosis) and retrograde (endocytosis) pathways, and the involvement of the endosomal compartments which direct ion channels towards recycling or degradation. KV1.5 channel carries IKur current which constitutes the main atrial repolarizing current in human and which is involved in atrial fibrillation (AF). Mechanical forces and shortening of the AP duration are linked to an increased in IKur current and decreased ICaL current. Therefore, KV1.5 channel constitutes a major target for the development of atria-selective antiarrhythmic drugs. The aim on this project was to better understand how functional expression of KV1.5 channels in atrial myocytes is regulated. Firstly, we showed that shear stress triggers an increase in IKur current implying the integrinβ1/FAK mecanotransduction pathway. This process requires an intact microtubule network and involves the Rab11-associated recycling endosome. In hypertrophied cells, the mecanotransduction pathway is overactivated. Consequently, IKur is increased. Secondly, we demonstrated that KV1.5 channel endocytosis is mediated by the clathrin pathway. We showed that microtubules are involved in the internalization and dynamics of KV1.5 channel in the membrane. Therefore, this work provides a better understanding of the different players involved in the trafficking of KV1.5 channel and shed new lights on the functional regulation of this atria-specific channel.

Over the last few decades, computer simulation techniques have been established as an essential tool for understanding biochemical processes. This thesis deals mainly with the application of free energy calculations to ribosomal complexes and a cardiac ion channel. The linear interaction energy (LIE) method is used to explore the energetic properties of the essential process of codon–anticodon recognition on the ribosome. The calculations show the structural and energetic consequences and effects of first, second, and third position mismatches in the ribosomal decoding center. Recognition of stop codons by ribosomal termination complexes is fundamentally different from sense codon recognition. Free energy perturbation simulations are used to study the detailed energetics of stop codon recognition by the bacterial ribosomal release factors RF1 and RF2. The calculations explain the vastly different responses to third codon position A to G substitutions by RF1 and RF2. Also, previously unknown highly specific water interactions are identified. The GGQ loop of ribosomal RFs is essential for its hydrolytic activity and contains a universally methylated glutamine residue. The structural effect of this methylation is investigated. The results strongly suggest that the methylation has no effect on the intrinsic conformation of the GGQ loop, and, thus, that its sole purpose is to enhance interactions in the ribosomal termination complex. A first microscopic, atomic level, analysis of blocker binding to the pharmaceutically interesting potassium ion channel Kv1.5 is presented. A previously unknown uniform binding mode is identified, and experimental binding data is accurately reproduced. Furthermore, problems associated with pharmacophore models based on minimized gas phase ligand conformations are highlighted. Generalized Born and Poisson–Boltzmann continuum models are incorporated into the LIE method to enable implicit treatment of solvent, in an effort to improve speed and convergence. The methods are evaluated and validated using a set of plasmepsin II inhibitors.

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