Dynamic ion selectivity of K+ channels in cell excitability and development – Dynaselect

We have identified residues located in the ionic pore that are involved in the dynamic selectivity of TWIK1 We have identified channels having residues compatible with a dynamic selectivity. We will clone and characterize these channels. We have also obtained mutant versions of TWIK1 that are locked in the selective state or in the unselective state. We'll prepare Knock In ( KI ) mice expressing these two different forms and compare them with wild type mice expressing the normal version of TWIK1, and to KO mice which do not express TWIK1 . On the other hand , and because there is no selective pharmacology TWIK1 , we will create a photoswitchable TWIK1 channel. With this channel, we will study how TWIK1 activity dynamically affects the electrical membrane potential

In the frame of this project we have shown that the inactivation of TWIK1 causes heart defects and arrhythmias in zebrafish. Normal function is restored by expressing the human channel in this tissue. This shows that TWIK1 is involved in the cardiac function and that the fish is a good study model (which is not the case of the mouse that unlike humans and zebrafish does not express TWIK1 in the heart). An article is in press in Journal of Molecular and Cellular Cardiology. We built and tested different TWIK1 channels sensitive to light. These channels will allow us to study the mechanisms that control the selectivity changes. We also demonstrated that TWIK1 does not assemble with TREK1, unlike to what was suggested by a recent article in Cell. Heteromerization is limited to the members of the same subfamily TREK1, TREK2 and TRAAK. Two articles are in press in the Proceedings of the National Academy of Sciences, USA.

The ultimate goal is to improve our knowledge of the mechanisms that regulate cellular excitability . There are many applications, including the identification of new targets for the development of therapeutic agents of interest in epilepsy , cardiac arrhythmias , diabetes , cancer ...

Three original articles were published (one in Journal of Molecular and Cellular Cardiology , two in the Proceedings of the National Academy of Sciences, USA)

K+ channels play a key role in regulating cellular excitability. It was thought that the strong K+-selectivity of these channels was static, only altered by genetic mutations in their selectivity filter, which cause severe disorders. Recently, we have challenged this dogma by showing that selectivity of K+ channels can also exhibit dynamic changes in physiological conditions. Under acidic conditions or in low extracellular K+ concentrations, the two-pore domain K+ channel (K2P) TWIK1 becomes permeable to Na+, shifting from an inhibitory role to an excitatory role. This phenomenon is responsible for the paradoxical depolarization of human cardiomyocytes in pathological hypokalaemia, and therefore may contribute to cardiac arrhythmias. In other cell types including kidney and pancreatic cells, TWIK1 produces depolarizing leak currents under physiological conditions. Sequence comparisons suggest that dynamic ion selectivity occurs in other mammalian K2P channels, as well as in the nematode Caenorhabditis elegans. Our research proposal aims to better understand K2P channel dynamic selectivity. In particular we will determine the molecular basis, of dynamic selectivity and its biological role in the many tissues and cell types that express TWIK1. We will also evaluate the conservation of dynamic selectivity during evolution. Together, the results obtained should confirm the physiological relevance of this novel regulatory mechanism of the cellular excitability.

The specific objectives are to identify the conformational rearrangements associated with dynamic ion selectivity by obtaining crystal structures of TWIK1 in K+-selective and Na+-permeable states, and to evaluate the impact of TWIK1 and dynamic selectivity on endosomal function and membrane protein recycling, and on cell excitability and development of the brain, kidney, pancreas and adrenal glands. For these purposes we will develop cell and animals models including knock-out and knock-in mice as well as tools for live cell imaging of TWIK1-containing endosomes. The techniques that we plan to use will range from cell biology, electrophysiology and proteomics to animal behaviour. Two additional specific objectives are to evaluate the potential implication of TWIK1 in human atrial fibrillations and aldosterone-producing adenomas by analysing mutations from patients, and finally to clone and characterize other human and nematode ion channels with permanently or dynamically-altered ionic selectivity.

The research project relies on an established collaboration between 3 partners with different areas of interest and complementary expertise (pharmacology, molecular biology, biochemistry, cell biology and physiology). The 3 partners are founding members of the Laboratoire of Excellence - Ion Channel Science and Therapeutics (LabEx ICST), a national consortium dedicated to the identification of novel targets for the next generation of medicines. Roughly 15% of the current pharmaceutical drugs have their primary action on ion channels. These modulators include anaesthetics, and drugs for treating diabetes, hypertension, cardiac arrhythmia, pain, insomnia, anxiety and epilepsy. Despite being a promising source of new targets for a number of other pathologies, ion channels have proven to be particularly resistant to molecular-driven approaches to drug discovery. Their biogenesis, addressing, recycling and degradation require cellular factors that are only poorly understood. Identification of novel targets requires a thorough understanding of the processes driving channel diversity and regulation. Additionally, validation of potential targets requires testing the physiological relevance of the identified mechanisms by functional exploration in cell and animal models where ion channels and regulatory cellular factors can be handled. Our work on TWIK1 and dynamic ionic selectivity is necessary to estimate their potential as a drug targets.