Research project P7/10 (Research action P7)
Background and general objectives
Advances in treatment, improved living conditions, lifestyle and preventive measures, have led to a dramatic increase in life expectancy in affluent countries. In Europe, the challenges of an ageing population thus dominate the health agenda. In numbers of afflicted persons and impact on health, cardiovascular diseases lead the statistics for mortality, while disorders of the central nervous system (CNS) are among the leading causes of disability in the world (Anderson & Chu, NEJM 2007). To address these challenges, a broader multidisciplinary approach is required, between clinical and more basic research, and across traditional boundaries of topics. The current proposal brings together teams with different expertise and perspective but with a shared focus on electrical signaling and excitability, from basic mechanisms to clinical translation.
Electrical signaling through action potentials is the hallmark of excitable tissue, i.e. the central nervous system (CNS), the heart, skeletal muscle and some endocrine organs like the pancreas. Action potentials trigger complex biological signals that include short term responses, such as excitation-contraction coupling in cardiac myocytes and long term responses such as plasticity and memory in the CNS. Action potentials are generated through a concerted activity of ion channels, on a background of a negative membrane potential in the cell at rest. Ion channels form a large and complex superfamily of transmembrane spanning proteins that are shared across tissues. Tissue and cell specificity are obtained by differences in levels of expression, isoform switches and organization in macro-molecular complexes.
The overall objective of the project is to gain further understanding of specific mechanisms in electrical excitability in the heart and CNS focusing on complex molecular mechanisms, examining the associated cell physiology, as well as more integrative physiology and pathophysiology in vivo, complemented by in silico modeling. The participating labs have in-depth expertise in specific aspects of excitability, ion channels and ion transporters in normal and diseased tissue: structure-function relations of channels and their complexes (P3, P5), cardiac excitability and remodeling with disease (P1), modulation of ion channel activity through specific ligands (P2), physiology and pharmacology of CNS ion channels (P3), intercellular communication and hemichannels (P4), inhibitory ionotropic receptors and neuroglia channels (P5), synaptic plasticity and CNS network activity (P6), integrative modeling of excitability and cardiac arrhythmias (A. Panfilov, P4). The inclusion of European labs adds expertise in arrhythmias at a more integrated level (INT1), as well as ischemic heart disease (INT3) and synaptic transmission in the CNS (INT2).
At different levels, we initiate translational approaches exploring diagnostic potential and therapeutic targets. The span across CNS and cardiac fields has already shown specific added value: since the start of our network 4 years ago we have initiated joint studies on candidate ion channels across different tissues, such as the connexin hemichannels (P4, P1) and the SK channels (P3, INT2, P5), and partners in the network bring input at a more molecular level spanning across tissues (P2, P4). Exchange of junior scientists across the network generates a broad training platform while joint activities enhance visibility of the multidisciplinary approach. We have expanded the earlier consortium with additional Belgian partners to strengthen the network with transgenic (P6) and modeling expertise (A. Panfilov at P4), as well as additional foreign partners. The network objectives remain centered on joint research programs, exchange of expertise, shared tools, training of junior scientists and enhanced external communication and impact. The overall research objective is translated in 5 work packages.
Research plan
WP1. Molecular architecture of ion channels, macrocomplexes and multi-channel interactions
In this WP we address molecular properties, including the more recently established level of macromolecular units and specific membrane compartments, in two programs, one focusing on channel modulators and probing ligand interactions, and a second focusing on macrocomplexes of different subunits and multiprotein assemblies.
K+ channels are studied for subunit tetramerization, co-assembly with β-subunits and secondary modifications. Structure-function analysis includes probing with potent toxins (P2, collab D. Snyders, UA) and advanced molecular imaging (FRET, image correlation spectroscopy, single particle tracking, P5, collab D. Snyders) together with functional studies (microelectrode and patch clamp recording). Focus is on specific K+ channels in pacemaking (P2, INT2), in synaptic transmission and plasticity in the CNS (P3,6).
Connexin hemichannels are studied to identify the regions that confer voltage-dependence and Ca2+ regulation of gating through functional characterization (P4).
The Ca2+ release channel or ryanodine receptor, RyR, activity occurs in a multi-protein macrocomplex, further determined by colocalization with other ion channels. Structure and organization of this complex in normal and diseased cells is studied using advanced molecular imaging, including STED (P1) and probing of the interacting channels and proteins in molecular assays.
For glycine receptors, GlyR, clustering of channels and interactions with other (sub)membrane proteins determine their synaptic/extra-synaptic location and some of their dynamic behavior.
WP2. Mechanisms of normal and abnormal pacemaking
In the heart and in the CNS, specific cell types generate spontaneous electrical activity, so-called pacemaker cells. Several channels (HCN, Ca2+ and Na+ channels, Ca2+ transporters,..) conspire to pacemaking providing a safety factor but also providing cell specificity. In this WP we focus on pacemaking in the CNS, where evidence is growing for alterations in pacemaking as part of disease processes.
A first program focuses on mechanisms of so-called “slow” pacemakers (P3, P6). We will explore in functional and in silico experiments whether cooperation between L-type Ca2+ channels and Na+ channels, characterized by P3 in dopaminergic neurons, determines pacemaking similarly in all subpopulations of these neurons. Conditional gene inactivation of key channel proteins using Cre-specific mice (DAT-Cre or ChAT-Cre) and ShRNA approaches allow correlation to integrated function and behaviour.
A second program focuses on GABAergic fast spiking interneurons (FSI), which are under the regulation of these dopaminergic neurons and are spontaneously active neurons that exert a strong feed-forward inhibition on the striatal output. Our objective is to understand whether FSI activity is driven by network oscillations or is due to intrinsic pacemaking. This will be explored in functional studies (P6) using purified toxins (P2) or pharmacological tools (P3).
WP3. Linking Ca2+ homeostasis and electrical excitability
Many ion channels in excitable cells participate in regulation of [Ca2+]i but are also modulated by [Ca2+]i establishing a tight link between Ca2+ and excitability. In this WP we explore specific channels but also include a more integrative approach.
Connexin hemichannels form a non-selective transmembrane pathway, modulated by Ca2+, which also passes Ca2+ ions and can contribute to Ca2+ oscillations. Preliminary data (P4, P1) indicate their presence in the heart and decreased activation voltage under conditions of increased [Ca2+]i. We will further characterize the conditions for activation of these channels in cardiac myocytes. Functional studies of single channel properties, using specific peptides (P1, P4) will be complemented with in silico modeling (A. Panfilov, P4).
The role of RyR organization in [Ca2+]i and feedback on membrane excitability will be studied using confocal [Ca2+]i imaging and FRET (P1). We will further examine the role of Ca2+ in lability of repolarization in intact cells (P1) and the contribution to functional biomarkers for arrhythmia (P1, INT1). Large animal models of arrhythmia will be central to this task.
WP4. Ligand-gated ion channels (LGC) in plasticity of excitability
In this WP, we address the role of neurotransmitter ionotropic receptors in plastic changes that occur during CNS development, as well as in adulthood both in normal and in pathological conditions.
The role of inhibitory ligand-gated channels, with focus on the GlyR, in the proliferation and migration of cortical projection neurons and interneurons during development will be studied by means of dynamic calcium imaging (time-lapse), patch-clamp on slices, shRNA, migration 3D reconstructions (P5, P6).
The influence of the essential NMDA receptor subunit NR1 on intrinsic excitability and network activity - synaptic transmission and synaptic plasticity - in specific subsets of neurons in the basal ganglia system will be studied in conditional transgenic mice (P6, P3) and will be correlated with its influence on motor and motivational behavior in normal and pathological conditions (addiction and Parkinson’s disease, P6).
WP5. Exploring novel approaches and tools to gain further insight in the role of ion channels in normal and abnormal excitability
Partners within the network participate in development of specific modulators of ion channels, such as small synthetic peptides, venom-derived toxins, and organic compounds through library screening. Adult DRG neurons are used as a model to screen natural venom toxins for lead compounds for further development. Equivalent screening in Xenopus laevis oocytes (P2) and in transfected cell lines is operated in combination with the automated patch-clamp system acquired under the previous collaborative project (P5) to develop novel toxin-derived tools and evaluate the potential of Nav1.6 and P2X as therapeutic target.
Uncontrolled opening of connexin hemichannels occurs in response to ischemia, pro-inflammatory cytokines and elevation of [Ca2+]i. Novel probes are developed to investigate whether these channels have a role in arrhythmias (INT3, P1, P4). Central to these studies is the availability of small synthetic peptides that result in hemichannel inhibition without affecting the function of gap junctions (P4).
We explore the potential for integrating ionic channel remodeling and morphological data obtained in vivo in a sheep model of atrial fibrillation into a computational model to identify critical stages in disease progression (Willems P1, Panfilov P4).
