Research project P6/31 (Research action P6)
Electrical excitability is the hallmark of the primary vital organs, the brain and the heart. Transient changes in the electrical potential across the cell membrane are the result of a highly coordinated activity of ion channels and transporters. These electrical signals convey information over long distances and initiate complex events such as memory and learning and cardiac muscle contraction; they are also the basis of the spontaneous rhythmic activity in the brain and in the heart. Dysfunction of ion channels is responsible for several major congenital and acquired diseases, such as epilepsy, deafness and sudden cardiac death. For congenital diseases resulting from specific mutations in ion channel subunits, the term ‘channelopathies’ is often used.
The large diversity in electrical signals within the CNS and also in the different regions of the heart is the result of the specific expression of a collection of ion channels, including ionotropic receptors, and transporters in each cell. Individual ion channels are transmembrane proteins that consist of several subunits; regulatory proteins such as kinases and phosphatases often associate with these channels to from large protein complexes. Within the membrane, ion channel complexes can furthermore be organized in specific subdomains such as caveolae and lipid rafts; this organization further modulates their function.
Insight into the process of normal excitability in the heart and CNS requires an understanding of the basic properties of ion channels, their structure-function relation, co-assembly, trafficking and insertion into the membrane and turnover. Unraveling mechanisms of disease with abnormal excitability relies on the knowledge of basic properties and the availability of tools to probe and define the specific alterations in channel function. Taking this another step forward, the insight gained from basic properties and mechanisms of dysfunction provides a rational basis for therapeutic development.
Against this background, the consortium presents a joint program to investigate at the tissue, cellular and molecular level the expression, function and regulation of a number of ion channels, transporters and ionotropic receptors underlying normal and abnormal excitability in the heart and CNS. A number of shared and conserved properties of these molecules across different types of excitable cells justify and facilitate the use of common platforms and knowledge basis, whereas the unique function in different tissues enriches the exchange and collaboration between the different partners.
The participating labs have established expertise in specific aspects of excitability, ion channels and ion transporters in normal and diseased tissue: structure-function relations of channels and their complexes (UA, D. Snyders), cardiac excitability and remodeling with disease (KUL, K. Sipido), modulation of ion channel activity through specific ligands (KUL, J. Tytgat), physiology and pharmacology of CNS ion channels and synaptic plasticity (ULg, V. Seutin), intercellular communication in the CNS (UGent, L. Leybaert), inhibitory ionotropic receptors (UHasselt, J.-M. Rigo). So far, collaboration between partners has been sporadic and not structured. Both from a scientific point of view as well as of an organizational point of view within the Belgian research area, this consortium therefore provides a major added value. The inclusion of two expert European labs (UUtrecht, M. Vos, cardiac electrophysiology; University of Bristol, N. Marrion, synaptic transmission in the CNS) further increases the potential of this network.
Through the IAP the consortium will address questions that surpass the capabilities of individual labs; they are identified as specific research goals in the next section. The project relies on collaboration and exchange to enhance scientific knowledge, but also to improve efficiency and return on available means.
Specific objectives for the collaboration are
• Setting up common technology platforms
Common use of major equipment (sequencer - confocal imaging, including live cell imaging – brain slice recording) and molecular tools (cloning - mutagenesis - in vitro expression in a variety of cell types); to develop specific tools for application within the network.
• Combining multiple level investigations
From in vivo electrophysiology, to tissue electrophysiology, cellular recordings and molecular analysis; complementary studies and exchange of data and material; normal and abnormal function in animal models (transgenic mice, large animal models for heart disease).
• Exchange of expertise and knowledge.
To be able to address larger research questions; exchange of material, methods for study; exchange of postdoctoral researchers; programs across the network for PhD students.
The research activities are organized in work packages (WPs) focusing on major mechanistic aspects of excitability. Each WP addresses specific research goals:
• to define the molecular architecture and pharmacology of K+ channels and their role in excitability
• to study and compare mechanisms of normal and abnormal pacemaking
• to characterize the feedback on membrane excitability of secondary changes in [Ca2+]i
• to study properties of ion channels and ionotropic receptors in cell-to-cell communication
• to examine the plasticity of ion channel expression and function in normal and pathological conditions
Research plan
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 β-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).
