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.

Many physiological processes occur with a predictable periodicity. The maintenance of such rhythms can rely on fluctuating gene expression, hormone concentrations, or homeostatic oscillations in signalling molecules within cellular compartments, depending on the timescale of the clock (Hastings et al., 2018; Rensing et al., 2001; Branicky and Hekimi, 2006; Thomas, 1990). Disruption of biological clocks can have devastating consequences on homeostasis or behaviour (Marcheva et al., 2013). Here, we examine the physiological role of acid-sensing ion channels (ASICs) in the defecation motor program (DMP) of the nematode Caenorhabditis elegans, an ultradian rhythm that occurs about every 50 s (Thomas, 1990; Branicky and Hekimi, 2006). We take advantage of this short-period cellular oscillator to relate ion channel physiology in vivo to organismal metabolism and behaviour.

ASICs, the acid-sensing members of the Degenerin/Epithelial Sodium Channel (DEG/ENaC) superfamily of cation channels, are thought to be the main proton receptors in vertebrates (Soto et al., 2018; Du et al., 2017). They are an important drug target because acidosis is a feature of painful inflammatory and ischemic conditions. ASICs are widely expressed in the central and peripheral nervous system, where their roles in inflammation, ischemia, pain perception, and learning are extensively studied (Waldmann et al., 1997; Wemmie et al., 2002; Wemmie et al., 2003; Voglis and Tavernarakis, 2008; Kreple et al., 2014; Du et al., 2014). However, less is known about their physiological role in non-neuronal tissue such as the intestinal epithelium, despite cross-phyla evidence for gastrointestinal expression and the role of acidity therein (Dong et al., 2011; Schaefer et al., 2000; Take-uchi et al., 1998; Levanti et al., 2011). The extreme acidity of the stomach requires an elaborate system of mucosal protective mechanisms, failure of which results in conditions such as gastritis, ulceration, and dyspepsia. Acidification of the intestinal tract is also a hallmark of conditions such as irritable bowel syndrome and cystic fibrosis, so understanding the role of acid sensors, of which the ASICs are prime candidates, is of wide-ranging therapeutic importance (Holzer, 2007).

The DMP depends on inositol 1,4,5-trisphosphate (IP₃)-dependent Ca²⁺ oscillations in the intestinal epithelial cells. IP₃ signalling loss-of-function mutations cause dramatic increases in both period length and variability, whereas overexpression of the IP₃ receptor, ITR-1, reduces period length (Dal Santo et al., 1999; Walker et al., 2002; Espelt et al., 2005). Increased cytosolic Ca²⁺ results in the rhythmic release of protons from the basolateral membrane, into the pseudocoelom. This triggers the posterior body wall muscles to contract (pBoc), via activation of a proton-gated cation channel PBO-5/PBO-6 (Beg et al., 2008; Pfeiffer et al., 2008). Propagation of the Ca²⁺ wave from the posterior to anterior then initiates contraction of the body wall muscle at the anterior of the intestine (aBoc) a few seconds later, via activation of the motor neuron AVL, and then finally, GABA release from both AVL and DVB triggers the enteric muscle contraction (EMC), expelling gut contents (Beg and Jorgensen, 2003; Mclntire et al., 1993). Premature termination of the Ca²⁺ wave or pharmacologically blocking the Ca²⁺ wave propagation before it can reach the anterior disrupts aBoc and EMC (Teramoto and Iwasaki, 2006), further supporting the idea that Ca²⁺ is the master regulator.

In addition to Ca²⁺ and pseudocoelomic pH, proton oscillations in the intestinal lumen are also integral to the DMP. The luminal pH fluctuates between ~4.4 and 6.5 during the cycle and the pH transitions in a wave that travels from posterior to anterior (Beg et al., 2008; Allman et al., 2009; Pfeiffer et al., 2008; Bender et al., 2013; Chauhan et al., 2013), dependent on proton uptake and release at the apical membrane. Proton gradients are fundamental to nutrient absorption, and some DMP-affecting mutations can result in aberrant fat metabolism and deficient nutrient uptake (Sheng et al., 2015; Allman et al., 2009). Loss of proton regulation can also disrupt immunity and pathogen resistance, as seen in mutants for C. elegans CHP1 homolog, PBO-1, in which the lumen is constantly acidic (Benomar et al., 2020). Thus, dysregulation of components of this ultradian clock can have far reaching organismal consequences.

The precise relationship between luminal proton oscillations, intestinal Ca²⁺ and the DMP is unclear; but the mutant phenotypes of some of the known transporters and exchangers that control lumen pH provide some clues. Knockdown of VHA-6, a subunit of the apical proton-transporting V-type ATPase, leads to more neutral lumen pH and an increase in cycle length (Allman et al., 2009). For mutations in the Na⁺/H⁺ exchanger PBO-4, the effect on lumen pH is controversial, since measurement using KR35 dye (Bender et al., 2013; Benomar et al., 2020) reveals a defect in anterior acidification, whereas measurement using Oregon Green 488 (Pfeiffer et al., 2008) reveals that global pH oscillations are normal; cycle length and Ca²⁺ oscillations are unaffected (Thomas, 1990; Pfeiffer et al., 2008; Benomar et al., 2020; Bender et al., 2013; Beg et al., 2008). Mutants of pbo-1, on the other hand, have a highly acidic lumen and no proton wave, but Ca²⁺ oscillations and cycle length are largely unaffected (Wagner et al., 2011; Thomas, 1990; Pfeiffer et al., 2008; Benomar et al., 2020). So, there is some evidence that the periodicity of the DMP can be independent of luminal pH, and thus that proton dynamics would be downstream of the Ca²⁺ oscillator.

We and others hypothesized that a proton wave transitioning from the posterior to the anterior of the lumen could lead to sequential activation of proton sensors in the apical membrane (Chauhan et al., 2013; Bender et al., 2013; Benomar et al., 2020), and acid-sensing DEG/ENaCs are good candidates. Compared to mammals, the C. elegans genome encodes a vastly expanded array of 30 DEG/ENaCs. One of these, FLR-1 (FLuoride-Resistant phenotype), functions in DMP rhythmicity and execution (Kwan et al., 2008; Take-uchi et al., 1998), although, the mechanistic basis of this role is unclear. With the exception of ACD-1, the first acid-inactivated DEG/ENaC to be identified (Wang et al., 2008), the proton sensing properties of the C. elegans DEG/ENaCs remained uncharacterised.

Here, we address the physiological role of C. elegans acid-sensing DEG/ENaCs in the intestine. We explore how Ca²⁺ and proton oscillations maintain periodicity and execution of the DMP, and find that disruption of Ca²⁺ and proton homeostasis are detrimental to metabolism and organismal health. We show that four intestinal DEG/ENaC subunits form two acid-inactivated channels with distinct properties. The first contains ACD-5 and localises to the luminal face of the intestinal cells and acts as a proton sensor, contributing to the establishment of low luminal pH and thus enabling cyclic pH oscillations. The second channel, containing FLR-1, ACD-3, and/or DEL-5, localises to the basolateral membrane, facing the pseudocoelom, and is essential for the Ca²⁺ oscillations that control the timing and execution of the DMP and thus is a key component of this ultradian clock. We thus provide direct evidence linking the channel properties of these proton sensors to their roles in pH regulation and Ca²⁺ signalling in vivo, which can provide insight into potential cellular roles for other acid-sensing members of this important channel family.

Using a combination of electrophysiology, behavioural analysis, genetic manipulations, and pH and Ca²⁺ indicator imaging, we have obtained evidence for two acid-sensitive DEG/ENaC channels with distinct functions (Figure 9). One includes ACD-5, which localises to the luminal membrane and is the main proton sensing subunit of the channel. This proton sensor is required for maintenance and/or establishment of acidic luminal pH and yet only subtly influences the DMP, and the Ca²⁺ oscillator. The second, composed of FLR-1, ACD-3, and/or DEL-5, localises to the basolateral membrane and is essential for timing and execution of DMP muscle contractions, with consequences for fat metabolism and growth.

Figure 9

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ACD-5 and FLR-1-containing channels exhibit very different pH dependence and kinetics; ACD-5 is fully open at pH 6 and closed at pH 4, reflecting the range of pH oscillations in the lumen (Allman et al., 2009), whereas FLR-1-containing heteromers form a proton-inhibited cation leak channel, similar to ACD-1, TadNaC6 and vertebrate ASIC5 (Wang et al., 2008; Elkhatib et al., 2019; Wiemuth and Gründer, 2010). This presumably reflects their different physiological contexts, just as the pH dependence of human ENaCs reflects the pH range in epithelia where they are expressed (Collier and Snyder, 2009). Neither ACD-5 nor FLR-1 currents desensitise; the channel remains open for the duration of the pH stimulus (although we see some evidence of partial desensitisation of FLR-1 at certain pHs). This contrasts with the fast desensitisation of mammalian ASICs and is more analogous to the slower kinetics of ENaCs. It coincides well with our evidence that ACD-5 is actually required for establishment or maintenance of acidic luminal pH; that is, its activation ensures a return to the condition (pH~4) at which it is closed. By contrast, the FLR-1/ACD-3/DEL-5 heteromeric channel on the basolateral membrane is predicted to be constitutively open as the extracellular resting pH is around 7.35 (Allman et al., 2009).

All four intestinal DEG/ENaC subunits influence the DMP, but the degree of influence differs dramatically, providing us with mechanistic information about the relationships between extracellular and intracellular events. Research to date indicates that intestinal IP₃-dependent Ca²⁺ signalling act as the master oscillator and is responsible for cycle length and timing. Disrupting IP₃ signalling results in a dramatic lengthening and disruption of rhythmicity of the DMP, and a direct relationship is seen between the timing of Ca²⁺ fluctuations and the DMP (Kwan et al., 2008; Walker et al., 2002; Espelt et al., 2005; Teramoto and Iwasaki, 2006; Dal Santo et al., 1999). Our acd-5 mutant data supports this hypothesis; the underlying Ca²⁺ fluctuations, and DMP rhythmicity, were only subtly affected, despite dramatic disruption of luminal pH, underlining the dominance of the Ca²⁺ oscillator. Where detectable, the MAT interval length was also not affected, suggesting that ACD-5 is not responsible for the timing of the proton wave, but acts downstream. This is supported by the phenotype of animals deficient in the V-ATPase, VHA-6; lumen pH remained more neutral, but robust Ca²⁺ oscillations were maintained, with only a small increase in cycle length (similar to acd-5(ok2657)) (Allman et al., 2009). This suggests a functional link between the two; a pH~6-activated cation channel and a presumed H⁺ pump whose effect would be to acidify the lumen, although the vha-6 phenotype is more severe than acd-5, interfering with nutrient uptake and growth rate, indicating that there is not a complete overlap in their roles. Indeed, such a coupling has been proposed in Na⁺ uptake in the Zebrafish larva (Zimmer et al., 2018). We could speculate that the dominant mutation could result in interference with VHA-6 function, or alternatively it could be interfering with localisation or function of the other DEG/ENaC subunits (as we have shown that it can do for ACD-5 itself, when expressed in oocytes). Altering the subunit composition of the FLR-1-containing channels, rather than disrupting it completely, could result in such a subtle extension. Whatever the mechanism, it is clear that the Ca²⁺ oscillator is largely unperturbed. We do nevertheless see some influence; in particular, when the Ca²⁺ oscillator is disrupted, in itr-1 deficient animals, the very long cycles observed are dependent on acd-5, revealing an otherwise constraining role. The Ca²⁺ oscillator is thus upstream, and/or largely independent, of lumen pH, and is the dominant force driving DMP rhythmicity.

In stark contrast to acd-5 mutants, flr-1 deficient animals exhibit a dramatic dysregulation of the DMP, with a combination of very long and very short cycles, underlining their importance for rhythmicity (as previous research showed; Kobayashi et al., 2011; Kwan et al., 2008; Katsura et al., 1994; Take-uchi et al., 1998), which is phenocopied by acd-3/del-5 double mutants. The lack of a clear phenotype for acd-3 and del-5 single mutants suggests that they act redundantly, in a heteromer, along with FLR-1. This correlates well with our observation that FLR-1 pH-sensitive currents in Xenopus oocytes were dependent on the presence of one or other of these subunits, and that acd-3/del-5 double mutant phenocopy other aspects of flr-1 loss of function: EMC and pBoC defects, developmental timing, growth, and fat accumulation. The severity of disruption to the DMP is reminiscent of that seen when Ca²⁺ signalling is disrupted (Kwan et al., 2008; Walker et al., 2002; Espelt et al., 2005; Dal Santo et al., 1999). Indeed, we found that in flr-1(syb3521) animals, the rhythmicity and magnitude of intestinal Ca²⁺ transients, measured in the posterior cells, where the Ca²⁺ wave initiates, were completely disrupted. This indicates that the FLR-1/ACD-3/DEL-5 channel(s) is upstream of the Ca²⁺ oscillator, perhaps forming part of the ‘master oscillator’, linking extracellular proton concentration to the initiation of the intracellular Ca²⁺ wave that controls periodicity and execution of the DMP (see Figure 9).

Interestingly, in contrast to itr-1, the flr-1 and acd-3/del-5 deficient animals also exhibit very short cycles, and ‘echos’ of the DMP as well as missed or weak EMC or pBoc muscle contractions which has previously been described for the unc-43 CaMK II mutant (Teramoto and Iwasaki, 2006) and cmd-1 (calmodulin) RNAi (Allman et al., 2013), which again links the phenotypes of these mutants to the disturbance in intestinal Ca²⁺ signalling. These shorter cycles, for which we also saw evidence in both intracellular pH and Ca²⁺ oscillations, indicate a role for calmodulin, CaMK II and the FLR-1 channel in retarding a shorter timescale oscillator. Cycle variability, the other effect of flr-1 or acd-3/del-5 mutations, presumably represents a loss of synchronicity between semi-independent oscillators, each of which has an innate homeostatic rhythm—but is checked by a delay in reaching permissive conditions provided by another. A channel that is open for most of the cycle is consistent with a role in maintaining cellular excitability, and thereby underpinning intracellular Ca²⁺ signalling; closing briefly following proton release into the pseudocoelom would serve to terminate the signal. This model fits well with the relatively low currents observed for the FLR-1 channel (e.g., compared with ACD-5) and the temporal relationship observed (Allman et al., 2009), where Ca²⁺ concentration precisely follows the decline in pseudocoelom pH.

Notwithstanding the challenges associated with imaging freely moving animals, two important barriers to dissecting the relationships between events in the intestinal cells and their extracellular environments are the dependent crosstalk between signalling events and the fact that some components are expressed on both the apical and basolateral membranes. PBO-1, for example, localises to both membranes, where it has distinct roles (Allman et al., 2016). Our data for ACD-3 and DEL-5 localisation to the basolateral membrane supports previous evidence for FLR-1 (Kobayashi et al., 2011). We did not detect apical localisation for ACD-3 or DEL-5 subunits. However, we did observe widespread, diffuse localisation within many cells, which might make any potential apical localisation difficult to distinguish, and Take-uchi et al., 1998 reported apical localisation (Take-uchi et al., 1998), so we cannot exclude the possibility of a dual role at the two membranes. Basolateral localisation fits well with the pH dependence of the channel. However, an important remaining question is how exactly the FLR-1 channel influences Ca²⁺ and calmodulin signalling, and the broad permeability of the channel does not help to shed light on this problem. Of key importance, and explaining why ACD-5 and FLR-1 have such distinct effects, may be the local nature of signalling events, with their activity thus specifically influencing components within their immediate vicinity.

We have shown that two acid-sensing DEG/ENaC channels with distinct properties play very different roles in the interplay between extracellular pH and intracellular Ca²⁺. ACD-5 is required to maintain acidic lumen pH and has a subtle impact on the Ca²⁺ oscillations that control DMP timing. In contrast, FLR-1, ACD-3, and/or DEL-5-containing channel is the essential link between pseudocoelomic pH and Ca²⁺, and thus timing and execution of the DMP. DEG/ENaCs are conserved across the animal kingdom, and the ASICs and ENaC members are relevant to a broad range of medical conditions, and thus of significant therapeutic importance. Our data provide an insight into the relationship between the acid-sensing properties of these channels and their physiological role in epithelia. However, the family contains a diversity of members with distinct properties, and vastly expanded numbers in some species. Better understanding the relevance of channel properties to function will help us to better understand their role, and the role of acid-sensing, in other cellular contexts.

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Author details

1. Eva Kaulich

   Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Performed all experiments except calcium and pH imaging, Resources, Visualization, Writing - original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0868-3702

2. Trae Carroll

   Department of Pathology and Lab Medicine, University of Rochester Medical Center, Rochester, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Performed and analysed intracellular and pseudocoelomic pH experiments, Writing – review and editing

   Competing interests

   No competing interests declared

3. Brian D Ackley

   Department of Molecular Biosciences, University of Kansas, Lawrence, United States

   Contribution

   Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Performed luminal pH experiments, Resources, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1257-2407

4. Yi-Quan Tang

   1. Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
   2. State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai, China

   Contribution

   Methodology, Supervision, Writing – review and editing, Provided significant technical guidance for electrophysiology

   Competing interests

   No competing interests declared

5. Iris Hardege

   Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Methodology, Supervision, Writing – review and editing, Provided significant technical guidance for electrophysiology

   Competing interests

   No competing interests declared

6. Keith Nehrke

   Department of Medicine, Nephrology Division, University of Rochester Medical Center, Rochester, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Performed and analysed intracellular and luminal pH measurements, Resources, Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

7. William R Schafer

   1. Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
   2. Department of Biology, KU Leuven, Leuven, Belgium

   Contribution

   Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Writing – review and editing

   For correspondence

   wschafer@mrc-lmb.cam.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6676-8034

8. Denise S Walker

   Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Performed calcium imaging experiments, Supervision, Writing - original draft, Writing – review and editing

   For correspondence

   dwalker@mrc-lmb.cam.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1534-1679

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