WP1. Molecular architecture of K+ channels and their role in membrane excitability
Since the activity of K+ channels is determined by their molecular architecture which comprises
subunit tetramerization, co-assembly with
-subunits and secondary modifications, structure-function analysis will be performed in heterologous expression systems. We will use mutational analysis (P4) and probing with potent toxins (P2).
Functional studies (microelectrode and patch clamp recording) in isolated cells and multicellular preparations of native tissue will identify specific K+ channels that have a key role in cardiac repolarization (P1, EU1), in synaptic transmission and plasticity in the CNS (P3,6); molecular analysis (immunohistochemistry, RNAi) correlates functional observations and channel expression (P4).
WP2. Normal and abnormal pacemaker activity
This workpackage will focus on channels that are involved in spontaneous membrane depolarization, primarily HCN channels, and to a lesser extent also Nav and T-type Ca channels. Different isoforms are expressed in the brain and the heart under normal conditions, and the expression may change as part of a disease process. Central in the WP is the search for potent and specific ligands starting from the extensive existing library of natural venoms and venom-derived peptides (P2). Some specific aims are to compare the pharmacological profile of HCN1-4 channels expressed in Xenopus laevis oocytes versus mammalian cells (HEK, CHO) (P2, P4); to study in depth the biophysical properties of HCN1-4 (especially the gating behaviour) (P4); to make the toxins available to partners for functional studies in native tissues: dopaminergic neurons (P3); dorsal root ganglion (DRG) cells (P4), Glycine receptors (P6). In addition we will evaluate differential expression of HCN channels during remodeling (P1,P4).
WP3. Calcium homeostasis and feedback on membrane excitability
In this workpackage we analyze alterations in calcium homeostasis and the feedback on membrane excitability in processes of remodeling, with hypertrophy (P1, EU1). Specific aims are to examine alterations in Ca2+ release and feedback on membrane excitability in cardiac cells, in particular Ca2+-dependent modulation of Ca2+ channels, properties of the Na/Ca exchanger, inducibility of early and delayed afterdepolarizations and potential for pharmacological modulation. P5 will provide a fast 2D confocal imaging modality.
WP4. Cell-to-cell communication
In this WP we examine some less-studied and novel mechanisms of cell-to-cell communication in the CNS. We will examine how channels known to regulate neuronal excitability (like Ih and SK channels) seem to play a role in synaptic plasticity (Partner 3, Partner 2, 4). We examine communication between neurons and glial cells using a transmitter-receptor basis (Partner 6, Partner 2) and communication between glial cells and other non-neuronal cells via Ca2+ signals, focusing on the role of gap-junctions, as well as hemichannels (Partner 5). The aim of this WP is to assess the role of these mechanisms in basic synaptic communication as well as in synaptic plasticity.
WP5. Ion channel remodeling, plasticity and membrane excitability
This WP will integrate data on channel function, expression of different subunits and phosphorylation intrinsic to long term alterations in synaptic plasticity and in cardiac remodeling. Specific aims are to establish the basal spatial profile of ion channel expression and distribution in dopaminergic neurons (P4, EU2), to evaluate the plasticity of the ion channel profile in DA neurons (P3, P4), and of the Ca2+ channel in chronic ischemic heart disease (P1, P4), to evaluate the role of NO-dependent mechanisms compared to changes in expression of different subunits in the modulation of the Ca2+ channel in chronic atrial fibrillation, to examine plasticity of T-tubules as a mechanism for modulation of Ca2+ channel activity (P1, P4).