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Genetics and cell biology of K2P channels

Final Report Summary - KELEGANS (Genetics and cell biology of K2P channels)

Two-pore domain potassium channels form a large family of well-conserved ion channels that play a central role in the establishment and maintenance of the resting membrane potential of almost all animal cells. They regulate neuronal excitability, hormone secretion, respiratory and cardiac functions. Recently, mutations in K2P channels have been implicated in rare human diseases affecting the function of the heart and the nervous system (Birk Barel mental retardation with dysmorphism syndrome, severe cardiac conduction disorder, FHEIG neuro-developmental disorder). Their role as modulators of cellular excitability make them interesting entry points to affect neuronal function.
In contrast to many other ion channel families, comparatively little is known about factors that specifically regulate the expression, the activity and the localization of K2P channels at the cell surface. Therefore, the central question addressed by our project was: how is the number of active K2P potassium channels present at the cell surface controlled in vivo?
To address this basic question, we have used forward genetic screens in the model nematode C. elegans to identify factors that regulate the trafficking, expression, and subcellular localization of K2P channels. The starting point for these genetic screens are gain-of-function mutants of K2P channels that cause strong and easily identifiable behavioral phenotypes. This approach has become possible because of a crucial discovery of our group. By combining electrophysiology in Xenopus oocyte and CRISPR/Cas9-based gene editing in C. elegans, we have shown that a single conserved residue controls the activity of all K2P channels from vertebrates and invertebrates (Ben Soussia, Nature Communications 2019). This has allowed us to rationally design gain-of-function mutants for C. elegans K2P channels and to identify genes that are specifically required to control their expression, biogenesis, trafficking, and subcellular distribution.
Using this genetic approach, we have identified previously unknown functional links between K2P channels and major regulators of cellular function such as Ankyrin, Notch signaling, and Dystrophin. Furthermore, we have discovered that K2P channels are not randomly localized at the surface of excitable cells as previously thought. On the contrary, different channels are partitioned to distinct subcellular domains of the plasmamembrane. This has led us to identify a novel planar polarity paradigm in C. elegans which was entirely unsuspected, and fully revisit the function of Dystrophin and its associated proteins in C. elegans, resolving a long-lasting controversy in the field about the structure of the DAPC in worms.
By employing state-of-the-art gene editing techniques, we have generated fluorescently-labeled knockin lines for many of the 47 K2P channels present in the C. elegans genome. This systematic effort has revealed striking cell- and tissue-specific expression patterns that hint at a complex combinatorial code that could be at the basis of the precise control of cellular excitability in the nematodes nervous system. This could add a highly relevant layer of complexity to our understanding of this apparently simple and well-described model nervous system.
By combining a wide array of experimental strategies including genetics, gene editing, fluorescence imaging, and electrophysiology, the Kelegans project has produced a more complete picture of the conserved molecular players that regulate K2P channels in vivo and identified unexpected cellular processes that pattern the cell.