Two-pore domain potassium channels (K2P) play a central role in the control of cellular excitability and the regulation of the cell's electrical membrane potential. K2Ps have been widely conserved throughout evolution. They are polymodal ion channels that are subjected to extensive regulation by a diverse set of physical (pH, temperature, mechanical force) and biological signals (lipids, G-protein coupled receptor pathways). They are broadly expressed in excitable and non-excitable cells, and have in turn been implicated in a large spectrum of physiopathological processes, ranging from the regulation of neuronal excitability, respiratory and cardiac function to the control of cell volume, hormone secretion and cell proliferation. Recently, loss- and gain-of-function mutations in K2P channels have been directly linked to human pathologies (Birk Barel syndrome, familial migraine with aura, cardiac conduction disorder, FHEIG neuro-developmental disorder).
In contrast to many other ion channel families, comparatively little is known about the molecular and cellular processes that regulate different aspects of the cell biology of K2P channels. For instance we know only of very few factors that specifically regulate the expression, the activity and the localisation of K2P channels at the cell surface. Therefore the central question addressed by our team is: How is the number of active two-pore domain potassium channels present at the cell surface controlled in vivo?
To identify novel genes and conserved cellular processes that regulate the biology of K2P channels in vivo we take advantage of the powerful genetic tools available in the model nematode Caenorhabditis elegans. We use the full array of techniques available in C. elegans including genetics, live imaging, electrophysiology and state-of-the-art CRISPR/Cas9 genome engineering and next-generation DNA sequencing. These studies will provide new leads to understand the cellular pathways that control K2P function in other organisms.
The starting point for our 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 recent 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 et al., Nature Communications 2019). This has allowed us to rationally design novel 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.
Many different mechanisms could control the number, the activity and the localisation of K2P channels in vivo. From the control of channel expression, biosynthesis and trafficking, to the regulation of membrane insertion, recycling and channel activity at the cell surface (figure). Very few experimental strategies can identify, at once, genes acting in such diverse aspects of cellular physiology. We propose that genetic strategies could address many of these levels of regulation.
Genetic approaches have been vastly underused by past research focusing on K2P channels. Yet, genetic screens in model organisms such as flies and worms are ideally suited to dissect complex cellular processes and gene regulatory networks in vivo. C. elegans has proven a powerful, integrated model system for the study of synaptic function, development, aging, cell death or gene regulation by microRNAs and RNA interference. Due to its short life cycle (< 3 days), C. elegans is well suited for large-scale genetic screens. In addition, most mutants of neuronal genes are viable, and even paralyzed worms are able to develop and reproduce.
Recent estimates show that 40% to 75% of human disease genes have homologues in C. elegans. Conversely, about 38 % of the ~21,000 predicted genes of C. elegans have clear homologues in humans (Culetto and Sattelle, 2000; Silverman et al., 2009; Shaye and Greenwald, 2011). Because of this evolutionary conservation, the genes or pathways that regulate K2P function in C. elegans should enable us to identify genes or mechanisms that are also relevant to the regulation of vertebrate K2P channels in excitable and non excitable cells.
Questions that we would like to solve
- What are the cellular mechanisms that control the distribution of K2P channels in vivo in C. elegans?
- Are these pathways conserved in other organisms, including vertebrates?
- How are the number and the activity of K2P channels at the cell surface controlled?
- Are there specific factors that differentially regulate K2P channels in distinct tissues or cell types?
- Do regulators of K2P channels also control the biology of other ion channel families?
Localisation of K2P channels in C. elegans
Using CRISPR/Cas9 genome engineering, we have generated the first knock-in of a K2P channel. This fluorescently-labeled ion channel is fully functional and visible in vulval muscles and VC motor neurons associated with the vulval muscles (A).>
In a mutant strain isolated in our genetic screens, K2P channel expression is lost specifically in vulval muscles but not in neurons (B). This suggests that K2P channel expression may be differentially regulated in neurons and muscle.
Direct visualisation of a K2P channel in vivo and at physiological expression levels opens the way to further analyse the cellular and molecular pathways that regulate the biology of these important ion channels.