Research team

Expertise

Electrophysiological analysis and Patch clamp.

Elucidating the pathogenicity of genetic variants of uncertain significance in Brugada syndrome patients by functional modelling in hiPSC-derived cardiomyocytes and zebrafish. 01/11/2020 - 31/10/2024

Abstract

Brugada syndrome (BrS) is an inherited arrhythmic disorder and is estimated to account for up to 12% of all sudden cardiac death cases, especially in the young (< 40 years old). Only in circa 30% of BrS patients the underlying genetic cause can be identified with current diagnostic arrhythmia gene panels. Moreover, the use of these panels result in detection of numerous genetic "Variants of Uncertain Significance" (so called VUS), but currently functional models to prove their causality are lacking. Therefore, in my project I will create two proof-of-concept models for a known pathogenic CACNA1C mutation associated with BrS: a cardiomyocyte cell model, created from human stem cells, and a novel transgenic zebrafish model with built-in fluorescent calcium and voltage indicators. By functionally characterising these models with innovative imaging and electrophysiological techniques, I will assess the mutation's effect on a cellular level and in the whole heart, proving its contribution to disease causation. After validating these models, I will apply this strategy to functionally assess the pathogenicity of two VUS identified in two BrS patients. Ultimately, by establishing the use of these state-of-the-art study models to predict the pathogenicity of BrS-related VUS, a more accurate risk stratification and proficient use of specialized prevention strategies can be implemented in the future, potentially also for other electrical disorders of the heart.

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Project type(s)

  • Research Project

Multi-well microelectrode array (MEA): a bridge to highthroughput electrophysiology. 01/05/2020 - 30/04/2024

Abstract

This project aims to upgrade the current electrophysiology technologies at UAntwerpen by acquiring a state-of-the-art MicroElectrode Array platform (MEA). To study the electrophysiological properties of excitable cells, currently patch-clamping is the gold standard. However, this is an extremely labour-intensive and invasive technique, limited to short-term measurements of individual cells at single time points. On the other hand, MEAs enable high-throughput non-invasive longitudinal real‐time measurements of functional cellular networks, without disrupting important cell-cell contacts, and thus provide a more physiologically relevant model. The multi-well format allows repeated recordings from cell cultures grown under various experimental conditions, including the opportunity to rapidly screen large drug libraries. Based on these advantages, multi-well MEAs are the most suitable instrument for functionally elucidating the pathomechanisms of neurological/cardiac disorders by performing (1) cardiac activity assays: measurement of field and action potentials from (iPSC-)cardiomyocytes to investigate wave-form, propagation and irregular beating; (2) neural activity assay based on three key measures: frequency of action potential firing, synchrony as measure for synaptic strength and oscillation as hallmark for neuronal organization in time; (3) (iPSC-)vascular smooth muscle contractility assay based on impedance alterations.

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  • Research Project

A zebrafish model system to assess pathogenicity of genetic variants in patients with cardiac arrhythmias. 01/01/2020 - 31/12/2023

Abstract

Inherited Cardiac Arrhythmia (ICA) refers to a group of genetic disorders in which patients present with abnormal and potentially harmful heart rhythm. These episodes often go unnoticed, but can lead to fainting and sudden cardiac death. At present, over 50 ICA genes have been identified. With the advent of next generation sequencing technology it is possible to test all of these genes simultaneously in multiple ICA patients with a single test. This method proficiently identifies clear disease causing genetic alterations. However, as the number of genes involved increases through better mechanistic insight into disease modifier genes and polymo hisms, we are confronted with a high number of genetic alterations for which causality is unsure. These pose a major challenge for the management of ICA patients. Therefore, the aim of this project is to develop a functional tool that will allow to test the functional impact of variants of unknown significance. We will develop a zebrafish assay in which the electrical dynamics of the heart are reported by fluorescent light signals. As zebrafish are translucent in early development, this model lends itself perfectly to visualize these signals 'in vivo' and at an exceptional resolution. After validating this tool with known pathogenic alterations, we will apply this method to evaluate variants of unknown significance. This innovative approach will allow the clinicians to deliver true personalized medicine.

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  • Research Project

Molecular elucidation of the unique gating behavior of cardiac ion channels thanks to their interaction with conopeptides. 01/01/2019 - 31/12/2022

Abstract

Venoms from cone snails (genus Conus) can be seen as an untapped cocktail of biologically active compounds, being increasingly recognized as new emerging source of peptide-based therapeutics. Cone snails are considered to be specialized predators that have evolved the most sophisticated peptide chemistry and neuropharmacology for their own biological purposes by producing venoms which contains a structural and functional diversity of neurotoxins. These neurotoxins or conopeptides are small cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion channels and receptors. The gamma- and omega-conopeptides their structurefunction activity relationships (QSARs) will be investigated by site-directed mutagenesis on both toxin and target in order to elucidate the molecular mechanisms involved.

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  • Research Project

New and safer tools to cure cardiac arrhythmias through genetic-manipulation-free photothermal stimulation and enhanced photothermal ablation of cardiomyocytes. 01/01/2019 - 31/10/2019

Abstract

Cardiac arrhythmias are the result of disorganised electrical signalling in the heart, affecting up to 33.5 million people worldwide and about 1-3% of the Belgian population. Moreover, as the prevalence of atrial fibrillation is expected to double the next 20 years, the clinical and economic impact of the disease is huge. The goal of the project is two-fold: (1) Establish a new and innovative tool for the synchronous stimulation of cardiomyocytes using light and circumventing the need for genetic manipulation and (2) Provide a proof of concept and initial protocol for a safer alternative for RF/cryo balloon cathether ablation. Both aims will be achieved by using a gold nanoparticle label, which specifically targets a cardiomyocyte surface marker. The gold nanoparticle will absorb LASER/LED light and produce heat. Depending on the rate of change in temperature, either photothermal stimulation or photothermal ablation is accomplished.

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    • Research Project

    Development of a functional assay to determine the pathogenicity of genetic variants with unknown significance identified in patients with cardiac arrhythmia. 01/10/2018 - 30/09/2022

    Abstract

    Inherited Cardiac Arrhythmia (ICA) refers to a group of genetic disorders in which patients present with abnormal and potentially harmful heart rhythm. These episodes often go unnoticed, but can lead to fainting and sudden cardiac death. At present, over 50 ICA genes have been identified. With the advent of next generation sequencing technology it is possible to test all of these genes simultaneously in multiple ICA patients with a single test. This method proficiently identifies clear disease causing genetic alterations. However, as the number of genes involved increases through better mechanistic insight into disease modifier genes and polymorphisms, we are confronted with a high number of genetic alterations for which causality is unsure. These pose a major challenge for the management of ICA patients. Therefore, the aim of this project is to develop a functional tool that will allow to test the functional impact of variants of unknown significance. We have developed a zebrafish assay in which the electrical dynamics of the heart are reported by fluorescent light signals. As zebrafish are translucent in early development, this model lends itself perfectly to visualize these signals 'in vivo' and at an exceptional resolution. After validating this tool with known pathogenic alterations, we will apply this method to evaluate variants of unknown significance and test the possible arrhythmogenic side effects of some drugs. This innovative approach will allow the clinicians to deliver true personalized medicine.

    Researcher(s)

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    • Research Project

    Inherited cardiac arrhythmias: identification of novel genes and development of a new diagnostic tool for translating genetic diagnosis into precision medicine. 01/01/2017 - 31/12/2020

    Abstract

    Inherited cardiac arrhythmias (ICA) are a group of predominantly autosomal dominant disorders characterized by a disturbed cardiac action potential that can lead to sudden cardiac death at a young age. Although currently more than 50 genes have been associated with ICA, in roughly 70% of the patients the precise genetic cause is still unknown. Moreover, this group of diseases is genetically and phenotypically heterogeneous and in a molecular diagnostic setting many variants of unknown pathogenic significance are detected, hampering proper risk stratification and efficient patient management. In a unique interfaculty collaboration between the Centre of Medical Genetics, the Cardiology department, the Laboratory of Experimental Hematology and Laboratory for Molecular Biophysics, Physiology and Pharmacology, we envision to address these needs in a project with two major aims: the identification of novel genes implicated in ICA and the development of a new diagnostic tool that allows functional phenotypic evaluation of the effect of genetic variants detected in ICA patients and family members. The first aim will be achieved using linkage analysis and state-of-the-art whole-genome sequencing in phenotypically well-characterized but genetically unresolved families, followed by functional characterization of the identified candidate variants. The second aim will be accomplished by the construction and electrophysiological characterization of patient-specific induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs). Focusing on the Brugada syndrome (BrS) as a proof-of-principle, iPSC-CMs will be created from fibroblasts of family members carrying an identical BrS-causing mutation but with different phenotypic expression of disease severity, and of BrS patients with a variant of unknown significance in the SCN5A gen. These powerful approaches in combination with the existing expertise in the different collaborating teams, will definitely allow accomplishing the envisioned ultimate goals of the project. As a result, a genetic diagnosis in a larger proportion of ICA families will be reached and can be translated into a personalized functional interpretation of the genetic result in patients and relatives. This will introduce the concept of precision medicine, tailoring proper risk stratification and efficient use of preventive and therapeutic measures for the individual patient.

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      • Research Project

      The pharmacological profile of homomtetrameric Kv2.1 and heterotetrameric Kv2.1/Kv6.4 channels depends on the channel's inactivation gating. 01/10/2015 - 30/01/2017

      Abstract

      Abstract (English version) Voltage−gated K+ (Kv) channels are K+ selective membrane proteins that open, close and/or inactivate in response to changes in the membrane potential. Kv channels set the resting membrane potential and are the major contributors of the repolarizing phase of the AP. Kv channels are targeted by a wide variety of pharmacological compounds that are capable of modulating Kv channel function through several mechanisms, including modifications of the gating machinery. Although the interaction of drug compounds with the closed and open state of the channel have been extensively investigated, the contribution of inactivation gating in Kv channel pharmacology remains largely unknown. In the current project we will investigate whether the inactivation process is capable of interfering with Kv channel pharmacology.

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        • Research Project

        "Silent" voltage-gated K+ channel subunits – no simple silent bystanders: analysis of their pharmacological profile, physiological role and gating mechanism. 01/10/2015 - 31/05/2016

        Abstract

        Voltage-gated K+ (Kv) channels play an important role in neuronal excitability which is reflected by several severe diseases caused by Kv channel dysfunction such as epilepsy. The Kv2 subfamily contains two members that display similar properties. The diversity is increased by assembly with members of the so-called silent Kv (KvS) subfamilies (Kv5-Kv6 and Kv8-Kv9). KvS subunits do not form functional homotetramers on their own but assemble into functional Kv2/KvS heterotetramers that display modified biophysical properties compared to Kv2.1 homotetramers. Further, diversity is increased by interactions with auxiliary ß-subunits. In addition, KvS subunits display a more tissue-specific expression compared to the ubiquitously expressed Kv2.1 creating tissue-specific functions for Kv2/KvS channels. Therefore, Kv2/KvS channels are more desirable pharmacological and therapeutic targets than Kv2 homotetramers. To fully understand Kv2.1/KvS channel complexes' pharmacological relevance as well as their role in neuronal physiology and diseases, it is important to unravel the molecular architecture of KvS containing channel complexes, to elucidate their role in neural excitability, to define how KvS subunits exert their unique effects and to determine the molecular determinants of channel opening and closing of these KvS containing channels. These are the topics of this "postdoctoral fellow renewal" application.

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          • Research Project

          Molecular architecture and assembly of heteromeric membrane proteins. 01/01/2015 - 31/12/2017

          Abstract

          The surface expression of plasma membrane proteins will be assessed using HA-tags on their extracellular segments, and the total amount using GFP-tagged versions. Heteromeric co-assembly will be tested using FRET between CFP and YFP tagged proteins. Internalization and endosomal recycling will be assessed using photoactivatable and pH sensitive fluorophores.

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            • Research Project

            Gating modulating peptide toxins of Shaker-type Kv1 channels derived from the sea anemone Bunodosoma cangicum and the sea snail Conus longurionis. 01/10/2013 - 30/09/2015

            Abstract

            The project focuses on the identification and biophysical characterization of gating modifier toxins of Kv1 channels. Thus far, only pore blocking toxins of the Kv1 subfamily have been described which implies that gating modification is a new method of modulating these channels. This gating modification mechanism is coupled to an unknown binding site on Kv1 channels. Based on the literature this binding site will most likely be present in the S3-S4 linker of the channel as previously shown for other gating modifiers such as hanatoxin, which acts on Kv2 channels [11].

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              • Research Project

              Research in the domain of molecular biophysics. 01/03/2013 - 28/02/2014

              Abstract

              This project represents a formal research agreement between UA and on the other hand Instituut Born Bunge. UA provides Instituut Born Bunge research results mentioned in the title of the project under the conditions as stipulated in this contract.

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                • Research Project

                "Silent" voltage-gated K+ channel subunits: analysis of the molecular determinants of modulatory properties and electrically silent behavior and elucidation of the physiological role. 01/10/2012 - 30/09/2015

                Abstract

                The overall goal of this project is to obtain novel insights in the molecular aspects of the interaction within KvS and the association between KvS and Kv2.1 subunits, using several complementary techniques to study protein-protein interaction, subcellular distribution and biophysical channel properties. Furthermore, we aim to determine the physiological role of Kv2/KvS heterotetrameric channels, using a transgenic model system. Because Kv6.4 induces quite marked shifts in the biophysical parameters and all constructs as well as the transgenic model system (targeted deletion of Kv6.4) for Kv6.4 are present in the lab, Kv6.4 will be used as a primary test model. Three specific aims are proposed: 1) Determine the molecular mechanism by which Kv6.4 influences the Kv2.1 gating in heterotetrameric Kv2.1/Kv6.4 channels. 2) Identify the molecular determinants in Kv6.4 responsible for its silent behavior. 3) Transgenic analysis of the physiological role of Kv6.4.

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                  • Research Project

                  Molecular mechanisms of the interaction between neurotoxins and potassium and sodium channels. 01/01/2012 - 31/12/2015

                  Abstract

                  This project focuses on the novel lipid exposed toxin/drug binding site in Kv channels that we recently characterized using the marine neurotoxin gambierol. This binding site is most likely the molecular equivalent of Nav channel 'site 5'. Site 5 (and site 2) toxins disrupt fast inactivation and cause a negative shift in the activation leading to persistent Na-current (i.e. potentiation). However, in Kv channels the toxin binding had an opposite effect and resulted in channel block. The overall goal is to expand our knowledge on this novel type of binding site(s) in Kv channels and unravel the molecular mechanism by which these toxins potentiate Nav channels but block Kv channels.

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                    • Research Project

                    Molecular mechanism and function of modulatory subunits of Kv channels. 01/01/2011 - 31/12/2014

                    Abstract

                    The overall goal of this project is to obtain novel insights in the molecular aspects of the association and interaction of KvS and KCNE subunits with the Kv2 and KCNQ1 subunits, respectively, using several complementary techniques to study protein-protein interaction, subcellular distribution and biophysical channel properties. Furthermore, we aim to determine the physiological role of Kv2/KvS heterotetrameric channels, using a transgenic model system (targeted deletion of Kv6.4).

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                      • Research Project

                      Molecular mechanism of modulation of Kv channels by PIP2 and small peptide modulators. 01/01/2010 - 31/12/2011

                      Abstract

                      This project represents a formal research agreement between UA and on the other hand the Flemish Public Service. UA provides the Flemish Public Service research results mentioned in the title of the project under the conditions as stipulated in this contract.

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                        • Research Project

                        Characterization of biological toxins on tension-sensitive K+ canals. 01/01/2010 - 31/12/2011

                        Abstract

                        The focus of this project (in collaboration with Prof. Jan Tytgat, KULeuven) is to expand the knowledge of the mechanism of action of biological toxins. . Using these toxins, we will also explore in more detail the structure, function and pharmacology of potassium (K+) channels. The goal is to find toxins or mechanisms of action that have important therapeutic or physiological implications. The first step is to screen the crude venoms electrophysiologically for toxins that have an effect on K+ channels. These venoms will be tested on all K+ channels available and those displaying an effect will be purified and characterized (1). Toxins with an interesting/remarkable effect that may be innovative or therapeutically beneficial will be investigated further (2). 1) The effect of purified toxins will be investigated using the voltage-clamp technique. By analyzing the evoked K+ currents (originating from K+ ions flowing trough the channel pore) we will determine the working mechanism (permeation block or gating-modifier) and blocking parameters like affinity, kinetics and voltage-dependency. 2) An in-depth structure-function analysis of interesting toxins starts with the characterization of the toxin binding site on the channel (receptor) by site-directed mutagenesis and investigating the effect of the substitution on the blocking parameters. Once the binding site on the channel has been characterized, the active site of the toxin will be determined by mutating/altering the toxin sequence/structure. Knowing both the binding-site on the channel and the active site of the toxin, we can elucidate the mechanism of action of the toxin and the coupling energy of the interaction through "mutant cycle analysis" and computational docking.

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                          • Research Project

                          Molecular structure-function analysis of a novel class of K-channel blocking scorpion toxins. 01/01/2008 - 31/12/2011

                          Abstract

                          The general aim of this project is the identification of the ion channels that are the targets of these novel toxins. Secondly, the specificity of the interaction will be used to perform structure-function analysis on these channels and toxins. Specific aims; 1. The biological target of k-Hefutoxin1 2A. Structure-function analysis of channels with a high binding affinity for k-Hefutoxin1 2B. Structure-function analysis of the k-Hefutoxin1 3. Biological targets and structure-function analysis of novel toxins.

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                            • Research Project

                            Molecular mechanisms of voltage-dependent gating in potassium channels. 01/01/2008 - 31/12/2011

                            Abstract

                            Specific aims: 1. Elucidate the dynamic behavior of channel opening with FRET during voltage clamp. 2. Do the S1 and S5 domains of Kv2.1 interact with each other? 3. Which residues in the S6 C-terminus in KCNQ1 are relevant for channel gating? 4. The KIKER sequence modulates gating through charge interactions.

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                              • Research Project

                              Molecular coupling between voltage sensing and gating in Kv-channels. 01/01/2008 - 31/12/2011

                              Abstract

                              This proposal aims to expand our insight in the molecular mechanism of K-channel gating, using several Kv subunits: Kv1.5 (forms homotetrameric channels like Shaker), Kv2.1 (forms heterotetrameric channels with the 'silent' subunits) en Kv6.3 (as a prototype for the 'silent' subunits because Kv2.1-Kv6.3 channels differ conspicously from Kv2.1 homomeric channels). In addition to standard state-of-the-art techniques, we will use a combination of electrical and optical techniques (dynamic fluorescence spectroscopy during gating).

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                                • Research Project

                                Interactions of silent Kv subunits and their role in DRG neurons. 01/01/2008 - 31/12/2009

                                Abstract

                                Voltage-gated K+ channels are widely expressed in native tissues. Four ¿-subunits tetramerize in a fully assembled K+ channel. Based on homology, voltage-gated K+ channels are subdivided into several (sub)families. De Kv family consist of 11 subfamilies. Members of the Kv1-Kv4 subfamilies produce a outward K+ current. Through assembly into heteromultimeric channels, the heterogenity of these subfamilies increased. Only members of the same subfamily can form heterotetramers. The Kv2 subfamily only consists of two members, Kv2.1 and Kv2.2. Both channels and their heteromeric complexes display similar currents resulting in a small functional diversity of the subfamily. The heterogenity of Kv2.x channels is however increased through tetramerization with subunits of the Kv5-Kv11 subfamilies. The latter are "silent" Kv subunits as these can not produce outward current in a homotetrameric conformation. At present, every "silent" Kv subunit is able to tetramerize with the Kv2.x subunits into a functional heterotetrameric channel resulting in biophysical properties that are altered compared homotetrameric Kv2 channels. In a yeast-two-hybrid assay also the association of the silent Kv6.3, Kv10.1 and Kv11.1 subunits with Kv3.1 and Kv5.1 was demonstrated. By means of electrophysiological experiments we will test if these associations occur in vivo. Morever, the interaction will be confirmed on the molecular level with FRET and co-immunoprecipitation experiments. Kv2 subunits are widely expressed in neuronal tissue while "silent" subunits show a more specific expression pattern. With RT-PCR de expression of "silent" Kv subunits wille be investigated in DRG neurons. The functional effects of the identified "silent" subunits on the electrical properties of the DRG neurons will be determined with the patch-clamp technique. The biophysical parameters such as kinetics and voltage dependence of activation will be analyzed of native delayed rectifier channels. In "current-clamp" mode the role of these "silent" Kv subunits in the action potential and the firing frequence of the DRG neurons will be investigated. The functional effects of the "silent" Kv subunits will be confirmed through overexpression and RNAi silencing. The function of Kv6.3 will also be determined in a complementary study by behavioural analysis of Kv6.3 overexpression mice. The mice DRG neurons will be isolated and similar analysis as with wild-type DRG neurons, would be performed. This project allows to aquire a better knowledge of the association, distribution and physiological role of "silent" Kv subunits.

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                                  • Research Project

                                  Characterization of biological toxins on tension-sensitive K+ canals. 01/01/2008 - 31/12/2009

                                  Abstract

                                  The focus of this project (in collaboration with Prof. Jan Tytgat, KULeuven) is to expand the knowledge of the mechanism of action of biological toxins. . Using these toxins, we will also explore in more detail the structure, function and pharmacology of potassium (K+) channels. The goal is to find toxins or mechanisms of action that have important therapeutic or physiological implications. The first step is to screen the crude venoms electrophysiologically for toxins that have an effect on K+ channels. These venoms will be tested on all K+ channels available and those displaying an effect will be purified and characterized (1). Toxins with an interesting/remarkable effect that may be innovative or therapeutically beneficial will be investigated further (2). 1) The effect of purified toxins will be investigated using the voltage-clamp technique. By analyzing the evoked K+ currents (originating from K+ ions flowing trough the channel pore) we will determine the working mechanism (permeation block or gating-modifier) and blocking parameters like affinity, kinetics and voltage-dependency. 2) An in-depth structure-function analysis of interesting toxins starts with the characterization of the toxin binding site on the channel (receptor) by site-directed mutagenesis and investigating the effect of the substitution on the blocking parameters. Once the binding site on the channel has been characterized, the active site of the toxin will be determined by mutating/altering the toxin sequence/structure. Knowing both the binding-site on the channel and the active site of the toxin, we can elucidate the mechanism of action of the toxin and the coupling energy of the interaction through "mutant cycle analysis" and computational docking.

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                                    • Research Project

                                    Molecular and cellular mechanisms of electrical excitability. 01/01/2007 - 31/12/2011

                                    Abstract

                                    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. The IAP consortium consists of 7 research teams form Belgian universities (Leuven, Antwerpen, Gent, Hasselt, Liège, Mons) and 2 European partners (Utrecht, Bristol). The project encompasses a joint program to investigate - from the molecular level to the in vivo situation - the expression, function and regulation of specific ion channels, transporters and ionotropic receptors underlying normal and abnormal excitability in the heart and CNS. The research activities are organized into 5 work packages (WPs) focusing on major mechanistic aspects of excitability: WP1: Molecular architecture of K+ channels and their role in excitability WP2: Normal and abnormal pacemaker activity WP3: Calcium handling and feedback on membrane excitability WP4: Cell-to-cell communication WP5: Ion channel remodeling, plasticity and membrane excitability The UA partner is involved with WP1, 2 and 5 and participates in all network-wide activities (e.g. common technology platforms, training and exchange programs)

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                                      • Research Project

                                      Characterization of the function of biological toxins on K+ canals. 01/01/2007 - 31/12/2007

                                      Abstract

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                                        • Research Project

                                        Study of the role of electrically silent voltage-gated potassium channels in the insulin secretion of the pancreas. 01/10/2006 - 30/09/2009

                                        Abstract

                                        The insulin secretion in the pancreas is highly regulated by ion channels. When the plasma glucose concentration increases, the membrane of the ¿-cell depolarizes, which leads to insulin secretion. The repolarization of the ¿-cell is caused by a slowly inactivating, delayed rectifier current, IDR, resulting in an inhibition of insulin secretion. It was shown by a dominant-negative knockout strategy that Kv2 subunits contribute approximately 60-70% of the IDR in insulin secreting cells and that inhibition of Kv2.1 enhances the glucose-dependent insulin secretion. Kv2 channels are known to form heterotetrameric channels with the members of the Kv5-Kv11 subfamilies. The latter subunits are not able to generate current by themselves and are therefore often referred to as "electrically silent". Co-expression with Kv2.1 results in heterotetrameric channels with clearly distinguishable properties from homotetrameric Kv2.1 currents. We and other have shown that Kv6.1, Kv6.2, Kv9.2, Kv9.3, Kv10.1 and Kv11.1 are expressed in the pancreas as a whole. In this project we study the role of silent Kv subunits in the regulation of insulin secretion using insulin secreting cell lines and isolated ß-cells. Using RT-PCR we will investigate which "silent" subunits are expressed. Overexpression and knock-down of these subunits will allow us to study the effect on the insulin secretion and on the electrophysiological properties of the cell. We will generate antibodies against the "silent" subunits to determine their exact localization in the pancreas using immunohistochemistry and to confirm their interaction with Kv2.1 in vivo. Finally, transgenic mice (overexpression and knock-out) will be generated and the function of the ß¿cells in these mice will be studied.

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                                          • Research Project

                                          Transgenic Service Facility: support central facilities. 01/05/2006 - 31/12/2006

                                          Abstract

                                          The Transgenic mice Service Facility is centered on several aspects of Mouse transgenesis and is operational in the Specific Pathogen Free facility of the Mouse housing facility of the University of Antwerp ¿ CDE: production of genetically modified mice via pronuclear injections, production of knock-out ands knock-in clones via embryonic stem cells, re-derivation of non-SPF mice and cryopreservation van important mouse lines. At the end of 2007 the TgSF activities were stopped.

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                                            • Research Project

                                            Replacement of CO2 incubator and the repair of a gel documentation system. 11/01/2006 - 31/12/2006

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                                              • Research Project

                                              Role of modulation subunits of K-channels in the heart. 01/01/2006 - 31/12/2009

                                              Abstract

                                              Specific aims of the project : 1) Detection of silent Kv subunits in the heart and their role in the slowly inactivating delayed rectifier current. 2) Transgenic analysis of the physiological role of silent subunits. 3) Do KChIP and KVI.5 interact stably and what are the functional consequences ? 4) What are the molecular determinants of the KChIP interaction with Kv1.5 subunits?

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                                                • Research Project

                                                Role of silent Kv subunits in DRG neurons. 01/01/2006 - 31/12/2007

                                                Abstract

                                                Voltage-gated K+ channels are widely expressed in native tissues. Four ¿-subunits tetramerize in a fully assembled K+ channel. Based on homology, voltage-gated K+ channels are subdivided into several (sub)families. De Kv family consist of 11 subfamilies. Members of the Kv1-Kv4 subfamilies produce a outward K+ current. Through assembly into heteromultimeric channels, the heterogenity of these subfamilies increased. Only members of the same subfamily can form heterotetramers. The Kv2 subfamily only consists of two members, Kv2.1 and Kv2.2. Both channels and their heteromeric complexes display similar currents resulting in a small functional diversity of the subfamily. The heterogenity of Kv2.x channels is however increased through tetramerization with subunits of the Kv5-Kv11 subfamilies. The latter are "silent" Kv subunits as these can not produce outward current in a homotetrameric conformation. At present, every "silent" Kv subunit is able to tetramerize with the Kv2.x subunits into a functional heterotetrameric channel resulting in biophysical properties that are altered compared homotetrameric Kv2 channels. In a yeast-two-hybrid assay also the association of the silent Kv6.3, Kv10.1 and Kv11.1 subunits with Kv3.1 and Kv5.1 was demonstrated. By means of electrophysiological experiments we will test if these associations occur in vivo. Morever, the interaction will be confirmed on the molecular level with FRET and co-immunoprecipitation experiments. Kv2 subunits are widely expressed in neuronal tissue while "silent" subunits show a more specific expression pattern. With RT-PCR de expression of "silent" Kv subunits wille be investigated in DRG neurons. The functional effects of the identified "silent" subunits on the electrical properties of the DRG neurons will be determined with the patch-clamp technique. The biophysical parameters such as kinetics and voltage dependence of activation will be analyzed of native delayed rectifier channels. In "current-clamp" mode the role of these "silent" Kv subunits in the action potential and the firing frequence of the DRG neurons will be investigated. The functional effects of the "silent" Kv subunits will be confirmed through overexpression and RNAi silencing. The function of Kv6.3 will also be determined in a complementary study by behavioural analysis of Kv6.3 overexpression mice. The mice DRG neurons will be isolated and similar analysis as with wild-type DRG neurons, would be performed. This project allows to aquire a better knowledge of the association, distribution and physiological role of "silent" Kv subunits.

                                                Researcher(s)

                                                Research team(s)

                                                  Project type(s)

                                                  • Research Project

                                                  Do heterotetrameric K+-channels contribute to the diversity of the native K+-currents in vivo? 01/01/2006 - 31/12/2006

                                                  Abstract

                                                  Researcher(s)

                                                  Research team(s)

                                                    Project type(s)

                                                    • Research Project

                                                    Structure analysis of proteins by means of spin labeling and EPR spectroscopy. 01/10/2005 - 30/09/2009

                                                    Abstract

                                                    This project focusses on the structure and structure-function determination of (i) cardial hERG PAS domain, (ii) the KChip subunits that influence ion channels and (iii) globin-coupled sensors that have been related to oxygen-sensing. The analyses will be done using advanced electron paramagnetic resonance techniques.

                                                    Researcher(s)

                                                    Research team(s)

                                                    Project type(s)

                                                    • Research Project

                                                    Analysis of the channel and gating mechanism of Shaker-type Kv channels. 01/10/2005 - 30/09/2008

                                                    Abstract

                                                    Specific aims: 1)Which channel regions make up the S4-gate link? 2)Is the single channel conductance determined by the channel gate ? 3)Clarifying the mechanism of channel gate opening and closure.

                                                    Researcher(s)

                                                    Research team(s)

                                                      Project type(s)

                                                      • Research Project

                                                      Functional analysis of potassium channel mutations underlying the long QT syndrome. 01/10/2005 - 30/09/2007

                                                      Abstract

                                                      Torsade de pointes (Tdp ), one of the most feared cardiac arrythmias is associated with QT prolongation on surface Ecg. This disease already known for some years as long QT syndrome (LQTS) is caused by underlying acquired or congenital disturbances in myocardial transmembrane ion channels causing action potential duration (APD) prolongation. At least six loci (LQTl-6) have already been identified causing congenital LQTS. Although a widespread variety of drugs have QT prolonging properties, many underlying causes for acquired LQTS still have to be identified. Moreover at the level of the cardiac ion channels many biophysical, cell biological and pharmacological mechanisms underlying QT prolongation stili remain unclear. Therefor, the purpose of this research is to study the electrophysiological characteristics of LQT-related cardiac ion channels and the cell biological processes necessary for functional expression to identify the pathogenesis of both acquired and congenital LQTS. The studies are conducted through Whole cell Patch Clamp techniques and if necessary during pharmacological incubation. Confocal microscopy is used to study possible trafficking deficiencies in LQTS.

                                                      Researcher(s)

                                                      Research team(s)

                                                        Project type(s)

                                                        • Research Project

                                                        Analysis of the structure of the hERG PAS domain and of KChIP y EPR spectroscopy. 01/01/2005 - 31/12/2008

                                                        Abstract

                                                        The N-terminal part of the cardiac K+-channel hERG contains a sequence (AA 1-90) belonging to the family of the PAS domains (characterized by 3D homology). Mutations identified in this domain have been shown to cause the LQT syndrome, in some cases by changing biophysical properties while in other cases by changing the trafficking and thus impeding the transport out of the ER to the plasmamembrane. The low expression of hERG-1b lacking this domain is consistent with the hypothesis that the PAS domain is important for trafficking. We and others have shown that mutations in' this domain are associated with the LOT syndrome. Some of these mutations display temperature dependence suggesting that these mutations change the folding of these domains. The KChlP subunjts were i.dentified as cytoplasmic 13-subunits 15. This group has expanded rapidly and we recently identified a new splice variant of KCh1P116. These subunits associate with the Kv4 subfamily increasing the expression at the level of the plasmamembrane bya factor 5-10. These results indicate that the association of KChlP with a o- subunit alters the trafficking of ion channels and thereby also determines in some extent the expression level. Furthermore, the subcellular localization (axon, dendrite, cell body) might be altered by the presence of KChIP. Recent results indicate that KChlP also associates with Kv1.5 indicating a more general role for these subunits.

                                                        Researcher(s)

                                                        Research team(s)

                                                        Project type(s)

                                                        • Research Project

                                                        Molecular architecture of ion channels and electrical remodelling. 01/10/2004 - 30/09/2006

                                                        Abstract

                                                        Researcher(s)

                                                        Research team(s)

                                                          Project type(s)

                                                          • Research Project

                                                          Fluorescent live cell imaging and CFP/YFP based FRET analysis of the interaction and tetramerization of the subunits of 'silent' ion channels. 01/01/2004 - 31/12/2007

                                                          Abstract

                                                          Researcher(s)

                                                          Research team(s)

                                                            Project type(s)

                                                            • Research Project

                                                            Excitability and connectivity of neural circuits. 01/01/2004 - 31/12/2007

                                                            Abstract

                                                            This project combines the technical expertise of the 3 research groups to study 3 topics: the cerebellum, enteric nervous system and potassium channels. Specifically we will study: classification of neurons in the granular layer, potassium channels in Purkinje cells and their inhibition of the deep nuclei; expression of potassium channels in viscerosensitive neurons and changes due to inflammatory mediators released by mast cells; and expression and function in the brain of 5 new potassium channel subunits.

                                                            Researcher(s)

                                                            Research team(s)

                                                              Project type(s)

                                                              • Research Project

                                                              Molecular and functional analysis of the interaction between voltage gated K+ channels and beta-subunits. 01/01/2004 - 31/12/2005

                                                              Abstract

                                                              Ion channels form the molecular basis of electrical excitability. These channels are integral membrane proteins that regulate membrane permeability and control several physiological functions such as neuronal signaling, muscle contraction, hormone secretion, cell volume and ionic homeostasis. Different components are critical for the realization of these diverse functions. The heterogeneity derives in part of the large group of a-subunits with their distinct characteristics and is further increased by heterotetramerization of a-subunits and/or association with accessory ß-subunits. The properties of these heteromeric channels can be clearly distinct from homotetrameric channels, thus increasing the in vivo diversity. The function altering properties of ß-subunits can be impressive e.g. KvLQT/MinK. Whereas heterologous expression of the KvLQT a-subunit alone results in a rapidly activating current with no relation to a known cardiac current, coexpression of KvLQT and MinK results in a slowly activating current similar to the native IK,s. Mutations in either KvLQT or MinK disrupt cardiac repolarization causing the Long QT syndrome with potentially lethal arrhythmia. As a detailed knowledge of ß-subunits and their function is essential to assess the in vivo situation, further investigations will be directed towards the discovery of new ß-subunits and the determination of their function. For the detection of new ß-subunits the MAPPIT system will be used with appropiate baits. Additionally new splice variants will be uncovered by using conserved regions of ß-subunits as probe in the Cloncapture technique (Clontech). In this search we will focus on the KChIP and the MinK related family. The interaction partners of the new ß-subunits or splice variants will be determined using the Cytotrap system (Stratagene) or MAPPIT system (RUG). Based upon bait-prey interaction nearby the membrane, these systems are better suited for the detection of protein-protein interactions for proteins that are targeted to the membrane. Interaction will be confirmed by co-immuno-precipitation. The tissue expression pattern of the interacting subunits will be compared using the MTC and MTE panels (Clontech) to determine the in vivo relevance. As the interaction between a- and ß-subunits often modifies the subcellular distribution, the membrane expression level of GFP or DsRed tagged interaction partners will be investigated by confocal microscopy. The functional effects of ß-subunits on the biophysical properties of the interaction partners will be investigated using the patch-clamp technique. Finally interacting and/or modulating domains will be determined using chimeric constructs and mutation analysis.

                                                              Researcher(s)

                                                              • Promoter: Snyders Dirk
                                                              • Co-promoter: Raes Adam
                                                              • Fellow: Van Hoorick Diane

                                                              Research team(s)

                                                                Project type(s)

                                                                • Research Project

                                                                Functional analysis of potassium channel mutations underlying the long QT syndrome. 01/10/2003 - 30/09/2005

                                                                Abstract

                                                                Torsade de pointes (Tdp ), one of the most feared cardiac arrythmias is associated with QT prolongation on surface Ecg. This disease already known for some years as long QT syndrome (LQTS) is caused by underlying acquired or congenital disturbances in myocardial transmembrane ion channels causing action potential duration (APD) prolongation. At least six loci (LQTl-6) have already been identified causing congenital LQTS. Although a widespread variety of drugs have QT prolonging properties, many underlying causes for acquired LQTS still have to be identified. Moreover at the level of the cardiac ion channels many biophysical, cell biological and pharmacological mechanisms underlying QT prolongation stili remain unclear. Therefor, the purpose of this research is to study the electrophysiological characteristics of LQT-related cardiac ion channels and the cell biological processes necessary for functional expression to identify the pathogenesis of both acquired and congenital LQTS. The studies are conducted through Whole cell Patch Clamp techniques and if necessary during pharmacological incubation. Confocal microscopy is used to study possible trafficking deficiencies in LQTS.

                                                                Researcher(s)

                                                                Research team(s)

                                                                  Project type(s)

                                                                  • Research Project

                                                                  Molecular architecture of ion channels and electrical remodelling. 01/04/2003 - 30/09/2004

                                                                  Abstract

                                                                  Researcher(s)

                                                                  Research team(s)

                                                                    Project type(s)

                                                                    • Research Project

                                                                    Molecular mechanism for the linkage between S4 movement and channel opening in voltage-gated potassium channels 01/01/2003 - 31/12/2004

                                                                    Abstract

                                                                    Ion channels are transmembrane proteins that allow selective ion permeation through a central pore which opens and closes depending on an external stimulus. In the case of voltage dependent ion channels it is the movement of S4 ' the voltage sensor ' that opens such gate in the channel pore. The heterogeneity in the voltage dependence of current activation reflects differences in coupling of S4 movement to channel opening. Shaker K channels and hyperpolarization activated (HCN) channels display a similar channel structure but activate with opposite voltage-dependence. This difference will be exploited to study the molecular nature of the mechanism that links S4 activation to channel opening. In both channels the area from S4 to S6 will be studied a candidate domain for the linkage-mechanism. This will be studied using biophysical analysis of ion channel gating using patch clamp techniques in combination with molecular manipulation of the channel structure such as channel chimeras, site-directed mutagenesis, cysteine scanning (SCAM) and site directed fluorescence. The specific clones employed in this study will be hKv1.5 as Shaker type and HCN1 as HCN type.

                                                                    Researcher(s)

                                                                    Research team(s)

                                                                      Project type(s)

                                                                      • Research Project

                                                                      Molecular genetics and cell biology. 01/01/2002 - 31/12/2006

                                                                      Abstract

                                                                      This network proposal has as major theme the molecular genetics and cell biology of human inherited disorders. This network groups 11 excellent research laboratories at the University of Antwerp active in molecular genetics of diseases such as Alzheimer dementia, psychiatric disorders, mental retardation, peripheral neuropathies, hearing impairment and bone disorders. The availability of the human genome sequence will not only provide the molecular geneticists new tools to accelerate their research topics (such as bioinformatics, SNPs, etc.), but also lead to functional studies of the respective disease causing genes. The post-genome era will therefore need the integration of high-throughput techniques and bio-informatics, but also collaboration with excellent cell biology laboratories in the network located at other Belgian universities.

                                                                      Researcher(s)

                                                                      Research team(s)

                                                                        Project type(s)

                                                                        • Research Project

                                                                        Molecular and functional analysis of novel K+-channel subunits identified in the human genome. 01/01/2002 - 31/12/2005

                                                                        Abstract

                                                                        Based on the public human genome draft sequence data we have identified three novel K-channel sequences. Thry are located on chromosome 2, 9 and 16 and contain at least two exons. Using available EST's and PCR amplification from various libraries we assembled the full length cDNA for each clone. They display the typical voltage-gated K+ channel topology (S1-S6, P-loop with GYG -motif etc.), but their S6 sequences lack a few conserved residues from the `functional' Kv1-Kv4 subfamilies. Thus they resemble the Kv5-Kv9 subunits. Further sequence comparison revealed that the chromosome 16 sequence is a novel Kv6 sequence (Kv6.3, ~55% identity with Kv6.1 and Kv6.2) while the other two sequences have approximately 35% identity with Kv2, Kv8 and Kv9. Since this is insufficient to assign them to the existing subfamilies these novel sequences are tentatively designated Kv10.1 and Kv11.1. Heterologous expression of these subunits in Ltk- or HEK293 cells did not yield K-currents, as suspected from the sequence analysis. The overall goal of this proposal is to evaluate the molecular and functional properties of these novel subunits identified in the human genome. The overall strategy of this project combines a molecular approach to determine the gene expression profile and identify interacting partners with an electrophysiological and microscopical approach to determine structure-function relationships.

                                                                        Researcher(s)

                                                                        Research team(s)

                                                                          Project type(s)

                                                                          • Research Project

                                                                          Molecular and functional analysis of the interaction between voltage gated K+ channels and beta-subunits 01/01/2002 - 31/12/2003

                                                                          Abstract

                                                                          Ion channels form the molecular basis of electrical excitability. These channels are integral membrane proteins that regulate membrane permeability and control several physiological functions such as neuronal signaling, muscle contraction, hormone secretion, cell volume and ionic homeostasis. Different components are critical for the realization of these diverse functions. The heterogeneity derives in part of the large group of a-subunits with their distinct characteristics and is further increased by heterotetramerization of a-subunits and/or association with accessory ß-subunits. The properties of these heteromeric channels can be clearly distinct from homotetrameric channels, thus increasing the in vivo diversity. The function altering properties of ß-subunits can be impressive e.g. KvLQT/MinK. Whereas heterologous expression of the KvLQT a-subunit alone results in a rapidly activating current with no relation to a known cardiac current, coexpression of KvLQT and MinK results in a slowly activating current similar to the native IK,s. Mutations in either KvLQT or MinK disrupt cardiac repolarization causing the Long QT syndrome with potentially lethal arrhythmia. As a detailed knowledge of ß-subunits and their function is essential to assess the in vivo situation, further investigations will be directed towards the discovery of new ß-subunits and the determination of their function. For the detection of new ß-subunits the MAPPIT system will be used with appropiate baits. Additionally new splice variants will be uncovered by using conserved regions of ß-subunits as probe in the Cloncapture technique (Clontech). In this search we will focus on the KChIP and the MinK related family. The interaction partners of the new ß-subunits or splice variants will be determined using the Cytotrap system (Stratagene) or MAPPIT system (RUG). Based upon bait-prey interaction nearby the membrane, these systems are better suited for the detection of protein-protein interactions for proteins that are targeted to the membrane. Interaction will be confirmed by co-immuno-precipitation. The tissue expression pattern of the interacting subunits will be compared using the MTC and MTE panels (Clontech) to determine the in vivo relevance. As the interaction between a- and ß-subunits often modifies the subcellular distribution, the membrane expression level of GFP or DsRed tagged interaction partners will be investigated by confocal microscopy. The functional effects of ß-subunits on the biophysical properties of the interaction partners will be investigated using the patch-clamp technique. Finally interacting and/or modulating domains will be determined using chimeric constructs and mutation analysis.

                                                                          Researcher(s)

                                                                          • Promoter: Snyders Dirk
                                                                          • Co-promoter: Raes Adam
                                                                          • Fellow: Van Hoorick Diane

                                                                          Research team(s)

                                                                            Project type(s)

                                                                            • Research Project

                                                                            Allowance for research equipment : electrophysiological setup. 01/01/2002 - 31/12/2002

                                                                            Abstract

                                                                            The proposed setup is suited to measure ionic currents from cells expressing native or cloned channels. The capabilities include voltage clamp and current clamp to probe biophysical and molecular properties, down to the individual protein (single channel recordings). The fast solution switch allows for experiments to elucidate structure using accessiblity analysis by cysteine scanning and MTS reagents. The configuration is expandable for future FRET / voltage clamp analysis.

                                                                            Researcher(s)

                                                                            Research team(s)

                                                                              Project type(s)

                                                                              • Research Project

                                                                              Altered patterns of gene expression during electrical remodelling causing cardiac arrhythmias: a DNA microarray analysis. 01/09/2001 - 31/08/2003

                                                                              Abstract

                                                                              In this project an existing human DNA microarray (HuGem 1, VIB) will be used to de termin e the altered gene expression causing 'electrical remodelling' which underlies atrial fibrillation. Also a 'custom' DNA microarray will be constructed for investigating ion channel expression in human and dog. This strategy will also be used for a dog modelof atial fibrillation.

                                                                              Researcher(s)

                                                                              Research team(s)

                                                                                Project type(s)

                                                                                • Research Project

                                                                                Molecular mechanism for the linkage between S4 movement and channel opening in voltage-gated potassium channels 01/01/2001 - 31/12/2002

                                                                                Abstract

                                                                                Ion channels are transmembrane proteins that allow selective ion permeation through a central pore which opens and closes depending on an external stimulus. In the case of voltage dependent ion channels it is the movement of S4 ' the voltage sensor ' that opens such gate in the channel pore. The heterogeneity in the voltage dependence of current activation reflects differences in coupling of S4 movement to channel opening. Shaker K channels and hyperpolarization activated (HCN) channels display a similar channel structure but activate with opposite voltage-dependence. This difference will be exploited to study the molecular nature of the mechanism that links S4 activation to channel opening. In both channels the area from S4 to S6 will be studied a candidate domain for the linkage-mechanism. This will be studied using biophysical analysis of ion channel gating using patch clamp techniques in combination with molecular manipulation of the channel structure such as channel chimeras, site-directed mutagenesis, cysteine scanning (SCAM) and site directed fluorescence. The specific clones employed in this study will be hKv1.5 as Shaker type and HCN1 as HCN type.

                                                                                Researcher(s)

                                                                                Research team(s)

                                                                                  Project type(s)

                                                                                  • Research Project

                                                                                  Transgenesis for the functional analysis of eukaryotic genes 01/01/2000 - 31/12/2003

                                                                                  Abstract

                                                                                  The aim of the project is to establish a centralised facility for transgenesis within the University of Antwerp. This facility will be responsible for the transgenesis and gene targeting of ES cells, the generation of animal models and the in vitro culture work of genetically modified non-differentiated cells ( ES cells) and differentiated cells. The transgene technology will be applied in the functional studies of: Genes involved in osteo/chondrogenic differentiation (Ecm1,Itm2a); Genes involved in the pathogenesis of affective disorders; Genes coding for voltage-gated K+ channels .

                                                                                  Researcher(s)

                                                                                  Research team(s)

                                                                                    Project type(s)

                                                                                    • Research Project

                                                                                    Molecular and functional analysis of the genetic basis for pacemaking activity in the heart and the nervous system 01/01/2000 - 31/12/2003

                                                                                    Abstract

                                                                                    The rhythmic activity of the heart originates in the sino-atrial node. Pacemaking cells display a slow diastolic depolarization which is controlled to a large extent by a mixed Na/K current. In contrast to most voltage-gated channels, this current activates slowly upon hyperpolarization. Therefore the current was named If (f for funny) or Ih (h for hyperpolarization-activated). A total of four subunits have already been cloned for these hyperpolarization-activated & cyclic nucleotide gated (HCN) channels. Their molecular structure displays remarkable similarities with highly-selective K channels that open upon depolarization. The specific aims of this proposal are to: (1) Clone novel isoforms of the HCN family and test for the existence of alternatively spliced variants or beta-subunits; (2)Test whether the GYG motif in the pore segment is critical for the (cation) selectivity and permeation properties of HCN channels; (3) Test whether S4 moves outwardly as in Shaker channels but couples differently to the activation gate.

                                                                                    Researcher(s)

                                                                                    Research team(s)

                                                                                      Project type(s)

                                                                                      • Research Project

                                                                                      Molecular determinants for the difference in gating mechanism between hKv1.5 and HCN ion channels. 01/01/2000 - 30/09/2000

                                                                                      Abstract

                                                                                      Ion channels are transmembrane proteins that allow selective ion permeation through a central pore which opens and closes depending on an external stimulus. In the case of voltage dependent ion channels it is the movement of S4 ' the voltage sensor ' that opens such gate in the channel pore. The heterogeneity in the voltage dependence of current activation reflects differences in coupling of S4 movement to channel opening. Shaker K channels and hyperpolarization activated (HCN) channels display a similar channel structure but activate with opposite voltage-dependence. This difference will be exploited to study the molecular nature of the mechanism that links S4 activation to channel opening. In both channels the area from S4 to S6 will be studied a candidate domain for the linkage-mechanism. This will be studied using biophysical analysis of ion channel gating using patch clamp techniques in combination with molecular manipulation of the channel structure such as channel chimeras, site-directed mutagenesis, cysteine scanning (SCAM) and site directed fluorescence. The specific clones employed in this study will be hKv1.5 as Shaker type and HCN1 as HCN type.

                                                                                      Researcher(s)

                                                                                      Research team(s)

                                                                                        Project type(s)

                                                                                        • Research Project

                                                                                        Molecular analysis of function, pharmacology and architecture of voltage-gated ion channels. 01/04/1998 - 31/03/2003

                                                                                        Abstract

                                                                                        Ion channels are transmembrane proteins that allow selective ion permeation through a central pore which opens and closes depending on an external stimulus. In the case of voltage dependent ion channels it is the movement of S4 ' the voltage sensor ' that triggers the opening of the gate in the channel pore (mainly S6). Specific aims of this project are (1) to test that specific pore lining residues in the S6 segment are involved not only in drug binding but also in channel activation, (2) to distinguish between molecular determinants for channel block and agonist effects of fatty acids and antiarrhythmic drugs, and (3) to evaluate whether stable cell lines expressing specific channel subunits can be used in high throughput screening assays for antiarrhythmic drugs or as an in vitro system to assess the anti- or pro-arrhythmic potential of various drugs (antihistaminics, antibiotics). This will be studied using biophysical analysis of ion channel gating using patch clamp techniques in combination with molecular manipulation of the channel structure such as channel chimeras, site-directed mutagenesis, cysteine scanning and site directed fluorescence. The specific clones employed in this study will be hKv1.5, Kv4.2, HERG and KvLQT1 which are major components of repolarizing K currents in cardiac tissue.

                                                                                        Researcher(s)

                                                                                        Research team(s)

                                                                                          Project type(s)

                                                                                          • Research Project

                                                                                          Molecular determinants of K+-channel drug block. 01/01/1998 - 28/02/2005

                                                                                          Abstract

                                                                                          The long term goal of this research is to understand the molecular basis of cardiac excitability. In this proposal the investigators will study molecular determinants of voltage-gated K+ channel block by antiarrhythmic drugs and local anesthetics. Voltage-gated K+ channels play a crucial role in controlling cardiac excitability and repolarization and are the molecular target for many new antiarrhythmic agents. The investigators will utilize cloned subunits that are considered to contribute to native cardiac currents (Kv1.5, Kv4.2, HERG, KvLQT1). Increasing evidence indicates that the molecular architecture of the channel protein complex includes function-altering accessory subunits (? subunits, minK) which may impact on drug binding. The hypothesis to be tested include that specific pore residues in the S6 segment are involved in binding of open channel blocking drugs. Furthermore, the investigators will test whether hydrophobic interactions determine affinity and stereoselectivity of drug block. In addition, they will test whether amino acid differences in the pore lining segments explain isoform-specific affinities for antiarrhythmic drugs. They will address whether antiarrhythmic drugs utilize a conserved receptor site for inactivating ?-subunits by testing for mutual interactions in the inner mouth of the pore. Finally, they will test whether the intrinsic pharmacology of KvLQT1 is altered when it co-assembles with the minK subunit to reconstitute the native current IKs. In these studies they will use contemporary techniques of molecular biology to modify channel structure and a variety of patch clamp techniques to test for specific functional changes. The results will be interpreted in terms of mathematical and thermodynamic models for channel gating and drug action. These studies will address molecular determinants of drug block and drug interactions with activation and inactivation `gates'. The information gained from this project will expand our knowledge of the molecular pharmacology of these important channels, which may ultimately result in improved understanding of mechanisms of arrhythmias and the development of better antiarrhythmic treatments.

                                                                                          Researcher(s)

                                                                                          Research team(s)

                                                                                            Project type(s)

                                                                                            • Research Project