[
International Worm Meeting,
2003]
Sudden cardiac death is a major cause of mortality in the western world. Change to currents that direct the action potential of cardiac tissue is the single largest predisposing factor that triggers lethal arrhythmias and heart failure. Voltage-gated K+ channels are essential for controlling action potential repolarization and frequency in muscles, neurons and other excitable cells. Kv4.3 is the major transient outward K+ channel component in ventricle myocytes, making it a particular interesting therapeutic target. Drugs that would improve Kv4.3 mediated K+ current, for instance by blocking the channel in the open state, could be of therapeutic benefit for the treatment of arrhythmic heart failure and other diseases related to interruptions in smooth muscle action potential.Kv4.3 and other valuable molecular drug targets such ion channels, vesicular transporters and synaptic proteins require a complex physiological environment to function. For example, assessment of the gating properties of compounds on ion channels at high throughput remains an industry challenge, particularly for voltage gated ion channels. Some ion channel drugs interact only with a specific conformation of the protein molecule and block the channel either in the open or in the closed configuration. Such 'smart' drugs can only be identified in a functional screen. The pharynx of C. elegans is a validated model system for neuronal signalling and membrane excitability. For example, the voltage-gated K+ channels EXP-2 in C. elegans influences the shape and duration of the action potential of pharyngeal muscle cells. Loss-of-function mutations in this voltage-gated K+ channel lead to prolonged repolarization and reduced frequency of pharyngeal action potentials. The relevant physiology of C. elegans offers the opportunity to study ion channels and other tough transmembrane targets in an in vivo environment. Devgen has developed standardized C. elegans assays that enable these targets to be screened at relatively high throughput (up to 9K compounds/week) in homogeneous assays using 30 micromolar compound concentrations. The potential of this technology will be illustrated by presenting data generated during assay development and HTS of the human KV4.3 channel.
[
European Worm Meeting,
2006]
Impaired Processing of FLP and NLP Precursors in C. elegans Lacking Active Prohormone Convertase 2 (EGL-3) and Carboxypeptidase E (EGL-21): Mutant Analysis by Mass Spectrometry. Steven J. Husson, Elke Clynen, Geert Baggerman, Tom Janssen and Liliane Schoofs. Biologically active (neuro)peptides are synthesized as larger proprotein precursors which are further processed by the concerted action of a cascade of enzymes. Among these, proprotein convertase 2 (PC2), which cleave the precursors after dibasic residues, and carboxypeptidase E (CPE), which remove these basic amino acids from the carboxyterminals, play a pivotal role. We have examined the role of the C. elegans orthologues PC2/EGL-3 and CPE/EGL-21 in the processing of FMRFamide-like peptide (FLP) precursors and neuropeptide-like protein (NLP) precursors. Recently, we performed the peptidomic analysis of C. elegans by two dimensional nanoscale liquid chromatography - Quadrupole Time-Of-Flight tandem mass spectrometry (2D-nanoLC Q-TOF MS/MS). This setup yielded the identification of sixty endogenously present (neuro)peptides. We now expand this list of identified peptides by using an off-line approach in which HPLC fractions are analyzed by a Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometer (MALDI-TOF MS). This way we compared the peptide profile of different C. elegans strains, among which PC2/EGL-3 and CPE/EGL-21 mutants. This differential peptidomics approach now unambiguously proves the role of these enzymes in the processing of FLP and NLP precursors.