Ontdekking van de fysiologie en functies van twee poriedomein kaliumkanalen in Caenorhabditis elegans

Abstract

Two-pore domain potassium (K2P) channels are important regulators of the resting membrane potential in various cell types. They are conserved across different phyla and are involved in a wide range of physiological processes and biological functions (Chapter 1). The K2P gene family is greatly diversified and expanded in nematodes, specifically in the model organism Caenorhabditis elegans (C. elegans). The C. elegans genome contains 47 different K2P-encoding genes, more than three times the number found in mammals (15).Biophysically, this large group of K2P channels in C. elegans are likely to contribute greatly to the maintenance of the negative resting membrane potential of cells. However, the biological significance of having such an expanded and diversified K2P gene family is unclear, given that only a few K2P channels have been functionally characterized, mostly in gain-of-function (GOF) mutations. In this PhD thesis, I investigated the possible physiological and biological functions in different aspects of several body wall muscle- or neuron-enriched K2P channels, in combination with loss-of-function (LOF) mutants and various behavioral assays. Chapter 2 focuses on examining the role of two K2P channels, TWK-26 and TWK-46, in egg-laying behaviors, due to their abundant transcriptomic levels in egg-laying-related neurons. Using the CRISPR/Cas9 gene-editing technique, two individual LOF mutants were generated. These two mutants were subsequently assessed using various egg-laying assays, including egg retention, quantification of egg-laying events on and off food, characterization of temporal egg-laying patterns, and responses to exogenous serotonin. However, no differences were observed in any of the assays between the LOF mutants and wild-type animals. Therefore, I concluded I was unable to identify roles for TWK-26 and TWK-46 in egg-laying behaviors. In Chapter 3, I focused on the body wall muscle-expressing K2P channel TWK-28, GOF mutants of which have been reported to exhibit severe locomotory defects. Using a LOF mutant, we found that TWK-28 exerts its regulatory effects in the body wall muscles to maintain body curvature during general locomotion on food. More importantly, loss of TWK-28 significantly increased the arousal speed in response to multiple aversive cues, such as mechanical tapping and hypoxia. Rescue experiments indicated that TWK-28 functions in the body wall muscles to temper the speed of animals aroused by unfavorable stimuli. In vitro electrophysiology and pilot in vivo calcium imaging experiments suggested that TWK-28 might affect the excitability of body wall muscles to modulate body curvature and arousal responses. In Chapter 4, TWK-23, a channel that is broadly expressed in the nervous system, was comprehensively characterized in terms of general locomotion on food. Using three different LOF alleles, I found that loss of TWK-23 led to altered morphology, loopy body curvature, and less pausing of motion. Although TWK-23 is expressed in multiple neuron classes, including polymodal sensory neurons OLQ, pharyngeal neurons, motor neurons and the proprioceptive neuron DVA, rescue experiments suggested that TWK-23 in DVA neuron is sufficient to regulate multiple features. However, the mechanisms by which TWK-23 governs these locomotory features have not been elucidated. In Chapter 5, I focused on the K2P channel TWK-18, which is known to be activated by high temperature, but whose thermoregulatory modulation has not been fully elucidated. To decipher its possible function, a CRISPR full-knockout mutant was generated. Likewise, this twk-18 null mutant did not exhibit any locomotory deficiency during basal locomotion on food. Nevertheless, we found that this LOF mutant showed altered body posture in a thermo-shift assay where worms were shifted from 23°C to 37°C. Furthermore, the altered body posture observed in the twk-18 LOF mutant in the thermo-shift assay was rescued by expressing twk-18 in body wall muscles, suggesting that TWK-18 in body wall muscles is sufficient to maintain the normal body posture of C. elegans when encountering noxious heat. Overall, a number K2P channels have been functionally characterized in various behavioral paradigms using LOF mutants. I was unable to identify roles for TWK-26 and TWK-46 in egg-laying behaviors; however, TWK-28 and TWK-18 were found to exert different roles in body wall muscles under different environmental contexts, while TWK-23 was demonstrated to regulate multiple locomotory features via the proprioceptive neuron DVA. Altogether, these results have expanded our current knowledge of this greatly diversified K2P channel family, particularly regarding their biological functions.