Innovative approaches in ion channel biology

Michel De Waard, Jérôme Montnach, Gildas Loussouarn, Michel Ronjat


a) Molecular screening using exogenous chemical libraries
The molecular screening axis that we initiated two years ago will be reinforced further. We will continue to use the libraries of peptides from venoms, but we will also seek diversification by implementing libraries from marine unicellular algua or from plant extracts. We should be able to exploit the first set of screening campaigns launched during the preceding research period. Additional efforts will be devoted to enlarge our portfolio of stable cell lines expressing cardiac ion channels that will be used for the screening campaigns. A wider reflection will be led to identify new phenotypic assays compatible with medium throughput screening efforts. Here the objective is to obtain pharmacological drugs able to produce arrhythmias by themselves (pharmacological models of arrhythmias) or to correct for arrhythmias produced by channelopathies. Both activators and inhibitors will be sought after.

(b) Pharmacology and technological engineering for cardiac ion channels
Our screening campaigns are partly performed with natural peptide libraries, which allows for engineering during chemical syntheses. Our aim is of course to diversify the pharmacological arsenal available to us to control channel activity, both in terms of activation and in terms of inhibition. However, these tools may lack some interesting chemical properties that we could be engineered at our level once they have been discovered. Here are some of the engineering we aim to develop with biologically active peptides. Fluorescent peptides: one of the issues with ion channels is that the quality of antibodies to study them is most of the time particularly bad. We have done the proof of concept that high affinity (<50 nM) and slow Koff peptides (> 10 min), engineered to harbor a fluorescent tag can be used to specifically label their ion channel targets. We have successfully done that for maurocalcin, a peptide that labels RyR2 (manuscript submitted), and protoxin II, for labeling Nav1.7 a pain target in dorsal root ganglions (British Journal of Pharmacology, in revision). We will continue to diversify our portfolio of fluorescent peptides to replace advantageously antibodies. Photoactive peptides: here the aim is to photocontrol the activity of a pharmacological tool in order to possess temporal and spatial control over channel activation or inhibition. While there are biophysical approaches for the control of arrhythmias (radiofrequency), there are no valid spatially defined pharmacological approaches to control the heart rate and arrhythmias. In this part of the project, we aim to develop photo-activated or photo-inhibited peptides using photolabile protecting groups on important amino acids of the pharmacophore or azobenzene-modified peptides using a click chemistry approach. These peptides will be validated in vitro on appropriate cell lines with the automated patch clamp system and on cardiomyocytes derived from iPS on the CardioExcite or the MEA system. The next stage will be to demonstrate their efficacy on isolated working hearts. Photolabeling peptides: peptides control channel activity by binding through their pharmacophore to binding site strategically located to influence the channel activity (pore blockers or gating modifiers). The knowledge of these binding sites is instrumental if one wishes to design smaller compounds with better pharmacokinetics and availability by oral administration (peptides are poorly-performing compounds for this route of administration). Our objective is to engineer natural peptide ligands containing a photoaffinity-labelling moiety that allows for irreversible covalent coupling of the ligand to the ion-channel-binding site. Using this strategy along with biochemical purification and MS/MS analyses of the channel/peptide complex we should be able to define with precision the binding site of our newly discovered peptide ligands. Needless to say, these pharmacological innovative tools can also be used to irreversibly block ion channels in a spatially defined approach on cardiac tissue and assess the importance of channel distribution in cardiac excitability.

CARDIAG: Building of a diagnostic/prognostic database by high-throughput multiplexed assays for human ERG variant effects, funded by the FFC (2019-2022) and ANR (2022-2025)

Channelopathies induce severe heart rhythm or conduction disorders. Mutations of the KCNH2 gene, that encodes the human (h)ERG channel, is responsible for 30% of all cases of long QT syndrome. Besides, hERG is frequently responsible for off-target effects of several pharmacological agents. With the advent of Next Generation Sequencing, hundreds of new KCNH2 variants are accumulating in various databases, many being of unknown significance to clinicians which hampers the value of their diagnosis and the quality of patient management. Therefore, there is an urgent need to functionally characterize a large fraction of KCNH2 variants and provide access of this information to hospital clinicians. We assembled a consortium of clinicians, geneticists, biophysicists and computer bioscientists to build the largest web-accessible diagnostic/prognostic database of hERG-related channelopathies. To this end, we will take advantage of high-throughput techniques of channel variant phenotyping.

CoV2-E-TARGET: Screening for inhibitors of SARS‐CoV-2 E protein, funded by Région Pays de la Loire (2020-2022)

Many viruses have genes coding for proteins with ion channel activity. Among the proteins encoded by the SARS-CoV and SARS-CoV-2 viruses responsible for the SARS epidemics of 2002 and 2019-2022, the E protein (for envelope) has such a function. Interestingly, it has been shown in several coronaviruses, including SARS-CoV, that channel activity is correlated with the replication power of the virus. In this project, using a high-throughput electrophysiology system, we aim to identify candidates for the inhibition of channel activity of E protein using our large collection of venoms. Our experience shows that each venom contains several dozens of channel inhibitors and activators. To identify venoms containing inhibitors of CoV-2-SARS E protein, we will establish a cell line stably expressing CoV-2-SARS E protein. The high-throughput patch-clamp will allow us to rapidly implement the screening. Once the active venoms fractions identified, we will isolate the peptides responsible for the inhibition. Finally, in collaboration with the Virology Department of the Institut Pasteur, we will test the effect of these peptides on the replication power of SARS-CoV-2.


Fluorescent- and tagged-protoxin II peptides: potent markers of the Nav 1.7 channel pain target. Montnach J, De Waard S, Nicolas S, Burel S, Osorio N, Zoukimian C, Mantegazza M, Boukaiba R, Béroud R, Partiseti M, Delmas P, Marionneau C, De Waard M. Br J Pharmacol. 2021;178:2632-2650. doi: 10.1111/bph.15453.

A standardised hERG phenotyping pipeline to evaluate KCNH2 genetic variant pathogenicity. Oliveira-Mendes B, Feliciangeli S, Ménard M, Chatelain F, Alameh M, Montnach J, Nicolas S, Ollivier B, Barc J, Baró I, Schott JJ, Probst V, Kyndt F, Denjoy I, Lesage F, Loussouarn G, De Waard M. Clin Transl Med. 2021 Nov;11(11):e609. doi: 10.1002/ctm2.609. PMID: 34841674

Modelling sudden cardiac death risks factors in patients with coronavirus disease of 2019: the hydroxychloroquine and azithromycin case. Montnach J, Baró I, Charpentier F, De Waard M, Loussouarn G. Europace. 2021 Jul 18;23(7):1124-1133. doi: 10.1093/europace/euab043. PMID: 34009333

Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp. Montnach J, Lorenzini M, Lesage A, Simon I, Nicolas S, Moreau E, Marionneau C, Baró I, De Waard M, Loussouarn G. Sci Rep. 2021 Feb 8;11(1):3282. doi: 10.1038/s41598-021-82077-8. PMID: 33558601 Free PMC article.

Functional Impact of BeKm-1, a High-Affinity hERG Blocker, on Cardiomyocytes Derived from Human-Induced Pluripotent Stem Cells. De Waard S, Montnach J, Ribeiro B, Nicolas S, Forest V, Charpentier F, Mangoni ME, Gaborit N, Ronjat M, Loussouarn G, Lemarchand P, De Waard M. Int J Mol Sci. 2020;21:7167. doi: 10.3390/ijms21197167.


  • AFM
  • Agence National pour la Recherche
  • Région Pays de la Loire
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Mis à jour le 26 June 2023.