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Molecular mechanisms of ion channels voltage-dependency

Final Report Summary - MECAVDEP (Molecular mechanisms of ion channels voltage-dependency)

BACKGROUND

Voltage-gated ion channels are ubiquitously expressed in human tissues where they play diverse physiological functions such as the generation and modulation of the electrical activity in excitable cells (neurons, myocytes) and the modulation of neurotransmitter and hormone release. Mutations and/or altered regulation of these channels are associated to many pathologies such as neuronal, cardiac and muscular diseases. As a result, structural and functional characterization of those channels will certainly lead to new pharmacological, genetic and cellular therapies. Crystallization of voltage-gated potassium (Kv) and sodium (Nav) channels provided a lot of information on their structure, and represented a new template for investigations on key molecular characteristics. This allowed the team of Gildas Loussouarn to shed light on the molecular mechanism of a cardiac Kv and a skeletal muscle Nav channel voltage-dependence. They showed that the channel protein contains an intramolecular ligand (the linker between the fourth and the fifth transmembrane domain, S4-S5) that binds to the channel gate (S6) and locks it in a closed state, in a voltage-dependent fashion. This channel property allowed them constructing inhibitory S4-S5 peptides and activatory S6 peptides. Their final goal is to test if the channels regulation by S4-S5 and S6 peptides may be of therapeutic interest, in the context of cardiac and skeletal muscle pathologies.

PERIOD I

During his visit to the laboratory of Daniel Minor at UCSF, Gildas Loussouarn has confirmed that the molecular model of voltage-dependence implicating S4-S5 and S6 interaction also applies to a bacterial channel: NavSp1. This introduces bacterial channels with known structure as a powerful tool to decipher the molecular details of voltage dependence.

Second Gildas Loussouarn has collaborated with the team of Daniel Minor to get structural information on NavSp1 channel. The structural information is still in use to optimize the S4-S5 and S6 peptides effects. A collaborator, Mounir Tarek, is building a dynamic model of Navsp1 channel voltage-gating. This model should allow (i) better understanding the interactions between S4-S5 and S6 at work for channel function, and (ii) designing S4-S5 and S6 peptides with enhanced potency, as a new step toward therapeutic tools.

All the results obtained during the one-year visit to the laboratory of Daniel Minor supported and consolidated the mechanistic model of voltage dependence in which S4-S5 binds to the channel gate (S6) and locks it in a closed state, in a voltage-dependent fashion.

Impact for Gildas Loussouarn and his team:

This year allowed initiating a strong scientific collaboration with the laboratory of Daniel Minor, and sharing expertise, in electrophysiology on one hand, structural biology on the other hand.


PERIOD II

Back to Nantes, Gildas Loussouarn and his team confirmed the same mechanistic model also applies to a sodium channel and another potassium channel implicated in several channelopathies.

Fayal Abderemane-Ali (Post-doctorate) and Yue Wei (Master 2 student) studied the Nav channel. In more than 500 cells expressing this channel, they looked at the effect of 27 peptides, corresponding to the S4-S5 and S6 of domain I to IV, on the biophysical characteristics of the Nav channel. The results obtained show that S4-S5 and S6 interactions of domains I, II and III control the activation gate, and S4-S5 and S6 interactions of domains IV control the inactivation gate. Furthermore, the results suggest that S4-S5 peptides have an expected inhibiting effect on the activation gate when they are individually expressed. On the contrary two S6 peptides need to be co-expressed to show the expected activating effect, suggesting that one S4-S5 binds to two S6 to exert its control of the activation gate. This is especially interesting because Nav and Kv channels show a major difference: Kv channels are tetramers of identical subunits whereas Nav channels are made of four distinct domains, consistent with the requirement of two distinct S6 peptides, issued from two distinct domains.

In parallel, Zeineb Es-Salah Lamoureux (Post-doctorate) and Olfat Malak (Master 2 student) studied the Kv channel (different than Kv7.1) and obtained similar results: one inhibiting S4-S5 peptide and one activating S6 peptide.

At last, Fabien Coyan (PhD student) and Fayal Abderemane-Ali (Post-doctorate) initially observed that concerning Kv7.1 an activating S6 peptide is able to correct, in COS-7 cells, the effect of a loss of function mutation responsible for the Long QT syndrome: R555C. They planned to test peptides on other mutations in Kv7.1 expressed in COS-7. However some results obtained this year on a Kv7.1 mutation that is at first very similar to R555C made them think differently. This mutation, R539W, not only modifies the channel biophysical characteristics, but it also suppresses its sensitivity to a variation in membrane PIP2, through a de novo interaction with cholesterol. This defect will not be corrected by the peptides that can only act on the channel biophysical characteristics. A mutation such as R555C is more simple and a better candidate to evaluate the potency of the peptides. Also this urges the team to use more pathophysiological models such as iPS-derived cardiomyocytes issued from the Long QT patient, now available in the laboratory.

Impact for Gildas Loussouarn and his team:
This year allowed maintaining the strong scientific collaboration with the laboratory of Daniel Minor, leading to an exchange of student: Fayal Abderemane Ali, will work in the laboratory of Daniel Minor for two years, as a post-doc.

As scheduled, Gildas Loussouarn followed a training on “team management” provided by the CNRS.

To summarize, there is no doubt that the Marie Curie experience helped Gildas Loussouarn in consolidating his team which counted 7 members, including two post-docs, two PhD students and a part time technician.