Final Report Summary - CHANNEL TARGETING (Identification of the dynamic mechanisms regulating the targeting and clustering of sodium and potassium channels in neurons) Electrical excitability is a fundamental property of neurons. Diversity in intrinsic neuronal excitability and function is generated by the variable expression, subcellular localization, and activity of a complex repertoire of neuronal ion channels. Dynamic regulation of intrinsic excitability can further alter the behavior of neurons and confer plasticity to neuronal signaling. Moreover, aberrant expression, localization, and function of ion channels can result in channel-based pathophysiologies. Thus, an understanding of how neurons regulate the expression and localization of ion channels is critical to understanding the complexity of normal neuronal function, its dynamic modulation to achieve plasticity, and defects that lead to neuronal dysfunction. The Axon Initial Segment (AIS) and the Nodes of Ranvier are key sub-compartments that generate and maintain signal conduction along the axon. This is where voltage-dependent sodium (Nav) and potassium (Kv) channels are critically concentrated to ensure proper axon potential propagation. At the molecular level, Nav and Kv channels are held in place by an intricate assembly of adhesion molecules, and cytoskeletal scaffold proteins. One cytoskeletal scaffold proteins, ankyrinG (ankG), has been identified as a “master organizer” of both AIS and Nodes of Ranvier, and is essential for the clustering of Nav1 channels. Despite the crucial role of Kv1 and Nav1 channels in neuronal physiology and of ankG in axonal organization, the molecular and cellular determinants governing their appropriate targeting and assembly are just beginning to emerge. We recently identified two new processes based on the phosphorylation Nav and Kv complexes by two kinases: casein kinase 2 (CK2) and cyclin-dependant kinase (Cdks). The dynamic phosphorylation/dephosphorylation modifications of these channels indeed modify their localization at the AIS. In this project, there are three specific aims that I intended to accomplish with a multidisciplinary approach during the four-year funding period: (i) Aim 1: Molecular dissection of the CK2 pathway in regulating AIS component clustering; (ii) Aim 2: Role of Cdk signaling in Kv1 localization and clustering at the AIS; (iii) Aim 3: Characterization of the mechanisms regulating ion channel targeting and clustering at the Node of Ranvier. At the end of the funding period, we showed for the first time, that Nav1 channels are phosphorylated in vivo in their ankG-binding motif using a phosphospecific antibody that we developed against serine 1112 of this motif. This phosphorylation follows Nav1 expression levels during neuronal development. We also demonstrated that CK2 accumulation at the AIS depends on Nav1 channels, with which they form tight complexes. The existence of such complexes likely leads to Nav1-phosphorylation during neuronal development and, consequently, Nav1–membrane anchoring by enhanced binding to ankG (article currently being written). By accomplishing aim 2, we revealed a new regulatory mechanism for the targeting of Kv1 complexes to the axonal membrane through the reversible CDK phosphorylation-dependent binding of Kvβ2 auxiliary subunits to the microtubule plus-end tracking protein (+TIP) EB1. These results were published in the Journal of Cellular Biology in 2011. Finally, we characterized an exciting and novel mechanism regulating the axonal targeting of ankG and, consequently, Nav1 axonal localization/clustering. We showed that ankG targeting to the axon of hippocampal neurons is regulated by the kinase CDK5 and by its serine rich domain.The completion of this project greatly favored progress towards understanding basic processes that determine Nav1 and Kv1 channel organization/clustering at distinct sub-domains of the plasma membrane of excitable cells. Understanding these mechanisms is one of the most basic and essential questions in biology. Moreover, recent studies are beginning to give us new views concerning the structure of axonal compartments, which are not as static and uniform as once thought. They are actually diverse and dynamic in order to produce a fine-tuning of neuronal excitability in normal and pathological conditions. It is likely that this plasticity depends upon modifications of local ion channel densities and/or distributions. Therefore, the identification of our new molecular mechanisms will also help to understand neuronal physiology in normal and pathological contexts.