Biological clocks are fundamental to an organism's health, controlling periodicity of behaviour and metabolism. Here, we identify two acid-sensing ion channels, with very different proton sensing properties, and describe their role in an ultradian clock, the defecation motor program (DMP) of the nematode Caenorhabditis elegans. An ACD-5-containing channel, on the apical membrane of the intestinal epithelium, is essential for maintenance of luminal acidity, and thus the rhythmic oscillations in lumen pH. In contrast, the second channel, composed of FLR-1, ACD-3 and/or DEL-5, located on the basolateral membrane, controls the intracellular Ca2+ wave and forms a core component of the master oscillator that controls the timing and rhythmicity of the DMP. flr-1 and acd-3/del-5 mutants show severe developmental and metabolic defects. We thus directly link the proton-sensing properties of these channels to their physiological roles in pH regulation and Ca2+ signalling, the generation of an ultradian oscillator, and its metabolic consequences.
Keywords: C. elegans; acid sensing ion channel; calcium signalling; cell biology; defecation; intestine; proton signalling; ultradian rhythm.
Biological clocks regulate a myriad of processes that occur periodically, from sleeping and waking to how cells use nutrients and energy. One such clock is the one that controls intestinal movements and defecation in the nematode worm Caenorhabditis elegans, which consists of three muscle contractions occurring every 50 seconds. This rhythm is controlled by calcium and proton signalling in the cells of the intestine. The cells of the nematode intestine form a tube, through which gut contents pass. The inside of the tube is acidic, but acidity also plays a role on the outer face of the intestinal tube. In this area, nutrients are distributed and signals are conveyed to other tissues, such as muscles. In fact, acid – in the form of protons – secreted from the intestinal cells stimulates the muscles that contract in the biological clock that controls the worms’ defecation. However, it is poorly understood how the worms control the release of these protons. Kaulich et al. identified two ion channels on the membranes of intestinal cells that become inhibited when the levels of acid surrounding them are high. These channels play distinct roles in controlling the contractions that move the contents of the roundworms’ intestines along. The first channel contains a protein called ACD-5, and it is in the membrane of the intestinal cells that faces the inside of the intestinal tube. The second channel is formed by three proteins: FLR-1, ACD-3 and DEL-5. This channel is found on the other side of the intestinal cells, the region where nutrients are distributed and signals are conveyed to the rest of the body. To determine the role of each channel, Kaulich et al. genetically engineered the worms so they would not make the proteins that make up the channels, and imaged the live nematodes to see the effects of removing each channel. The inside of the intestines of worms lacking the ACD-5 containing channel was less acidic than that of normal worms, and the timing of the contractions that control defecation was also slightly altered. Removing the second channel (the one formed by three different proteins), however, had more dramatic effects: the worms were thin, developed more slowly, had less fat tissue and defecated very irregularly. Kaulich et al. imaged live worms to show that the second channel plays a major role in regulating oscillations in acidity both inside and outside cells, as well as controlling calcium levels. This demonstrates that this channel is responsible for the rhythmicity in the contractions that control defecation in the nematodes. Their findings provide important insights towards better understanding proton signalling and the role of acid-sensing ion channels in cellular contexts and biological clocks.
© 2022, Kaulich et al.