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

Download asset Open asset

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.

See Appendix 1 for Key resources table.

All data generated or analysed during this study are included in the manuscript.

1. 1. Adams CM
   2. Snyder PM
   3. Welsh MJ

   (1999) Paradoxical stimulation of a DEG/ENaC channel by amiloride

   The Journal of Biological Chemistry 274:15500–15504.

   https://doi.org/10.1074/jbc.274.22.15500

   • PubMed
   • Google Scholar
2. 1. Allman E
   2. Johnson D
   3. Nehrke K

   (2009) Loss of the apical V-ATPase a-subunit VHA-6 prevents acidification of the intestinal lumen during a rhythmic behavior in C. elegans

   American Journal of Physiology-Cell Physiology 297:C1071–C1081.

   https://doi.org/10.1152/ajpcell.00284.2009

   • PubMed
   • Google Scholar
3. 1. Allman E
   2. Waters K
   3. Ackroyd S
   4. Nehrke K

   (2013) Analysis of Ca2+ signalling motifs that regulate proton signalling through the Na+/H+ exchanger NHX-7 during a rhythmic behavior in Caenorhabditis elegans*

   Journal of Biological Chemistry 288:5886–5895.

   https://doi.org/10.1074/jbc.M112.434852

   • PubMed
   • Google Scholar
4. 1. Allman E
   2. Wang Q
   3. Walker RL
   4. Austen M
   5. Peters MA
   6. Nehrke K

   (2016) Calcineurin homologous proteins regulate the membrane localization and activity of sodium/proton exchangers in C. elegans

   American Journal of Physiology-Cell Physiology 310:C233–C242.

   https://doi.org/10.1152/ajpcell.00291.2015

   • PubMed
   • Google Scholar
5. 1. Awayda MS
   2. Subramanyam M

   (1998) Regulation of the epithelial Na+ channel by membrane tension

   The Journal of General Physiology 112:97–111.

   https://doi.org/10.1085/jgp.112.2.97

   • PubMed
   • Google Scholar
6. 1. Beg AA
   2. Jorgensen EM

   (2003) EXP-1 is an excitatory GABA-gated cation channel

   Nature Neuroscience 6:1145–1152.

   https://doi.org/10.1038/nn1136

   • PubMed
   • Google Scholar
7. 1. Beg AA
   2. Ernstrom GG
   3. Nix P
   4. Davis MW
   5. Jorgensen EM

   (2008) Protons act as a transmitter for muscle contraction in C. elegans

   Cell 132:149–160.

   https://doi.org/10.1016/j.cell.2007.10.058

   • PubMed
   • Google Scholar
8. 1. Bender A
   2. Woydziak ZR
   3. Fu L
   4. Branden M
   5. Zhou Z
   6. Ackley BD
   7. Peterson BR

   (2013) Novel acid-activated fluorophores reveal a dynamic wave of protons in the intestine of Caenorhabditis elegans

   ACS Chemical Biology 8:636–642.

   https://doi.org/10.1021/cb300396j

   • PubMed
   • Google Scholar
9. 1. Benomar S
   2. Lansdon P
   3. Bender AM
   4. Peterson BR
   5. Chandler JR
   6. Ackley BD

   (2020) The C. elegans CHP1 homolog, pbo-1, functions in innate immunity by regulating the pH of the intestinal lumen

   PLOS Pathogens 16:e1008134.

   https://doi.org/10.1371/journal.ppat.1008134

   • PubMed
   • Google Scholar
10. 1. Besson T
    2. Lingueglia E
    3. Salinas M

    (2017) Pharmacological modulation of acid-sensing ion channels 1a and 3 by amiloride and 2-guanidine-4-methylquinazoline (GMQ

    Neuropharmacology 125:429–440.

    https://doi.org/10.1016/j.neuropharm.2017.08.004

    • PubMed
    • Google Scholar
11. 1. Bianchi L
    2. Gerstbrein B
    3. Frøkjaer-Jensen C
    4. Royal DC
    5. Mukherjee G
    6. Royal MA
    7. Xue J
    8. Schafer WR
    9. Driscoll M

    (2004) The neurotoxic MEC-4(d) DEG/ENaC sodium channel conducts calcium: implications for necrosis initiation

    Nature Neuroscience 7:1337–1344.

    https://doi.org/10.1038/nn1347

    • PubMed
    • Google Scholar
12. 1. Branicky R
    2. Hekimi S

    (2006) What keeps C. elegans regular: the genetics of defecation

    Trends in Genetics 22:571–579.

    https://doi.org/10.1016/j.tig.2006.08.006

    • PubMed
    • Google Scholar
13. 1. Brenner S

    (1974) The genetics of Caenorhabditis elegans

    Genetics 77:71–94.

    https://doi.org/10.1093/genetics/77.1.71

    • PubMed
    • Google Scholar
14. 1. C. elegans Deletion Mutant Consortium

    (2012) large-scale screening for targeted knockouts in the Caenorhabditis elegans genome

    G3: Genes, Genomes, Genetics 2:1415–1425.

    https://doi.org/10.1534/g3.112.003830

    • PubMed
    • Google Scholar
15. 1. Canessa CM
    2. Schild L
    3. Buell G
    4. Thorens B
    5. Gautschi I
    6. Horisberger JD
    7. Rossier BC

    (1994) Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits

    Nature 367:463–467.

    https://doi.org/10.1038/367463a0

    • PubMed
    • Google Scholar
16. 1. Chauhan VM
    2. Orsi G
    3. Brown A
    4. Pritchard DI
    5. Aylott JW

    (2013) Mapping the pharyngeal and intestinal pH of Caenorhabditis elegans and real-time luminal pH oscillations using extended dynamic range pH-sensitive nanosensors

    ACS Nano 7:5577–5587.

    https://doi.org/10.1021/nn401856u

    • PubMed
    • Google Scholar
17. 1. Collier DM
    2. Snyder PM

    (2009) Extracellular protons regulate human ENaC by modulating Na+ self-inhibition

    The Journal of Biological Chemistry 284:792–798.

    https://doi.org/10.1074/jbc.M806954200

    • PubMed
    • Google Scholar
18. 1. Dal Santo P
    2. Logan MA
    3. Chisholm AD
    4. Jorgensen EM

    (1999) The inositol trisphosphate receptor regulates a 50-second behavioral rhythm in C. elegans

    Cell 98:757–767.

    https://doi.org/10.1016/s0092-8674(00)81510-x

    • PubMed
    • Google Scholar
19. 1. Dokshin GA
    2. Ghanta KS
    3. Piscopo KM
    4. Mello CC

    (2018) Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in Caenorhabditis elegans

    Genetics 210:781–787.

    https://doi.org/10.1534/genetics.118.301532

    • PubMed
    • Google Scholar
20. 1. Dong X
    2. Ko KH
    3. Chow J
    4. Tuo B
    5. Barrett KE
    6. Dong H

    (2011) Expression of acid-sensing ion channels in intestinal epithelial cells and their role in the regulation of duodenal mucosal bicarbonate secretion

    Acta Physiologica 201:97–107.

    https://doi.org/10.1111/j.1748-1716.2010.02207.x

    • PubMed
    • Google Scholar
21. 1. Du J
    2. Reznikov LR
    3. Price MP
    4. Zha X
    5. Lu Y
    6. Moninger TO
    7. Wemmie JA
    8. Welsh MJ

    (2014) Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala

    PNAS 111:8961–8966.

    https://doi.org/10.1073/pnas.1407018111

    • PubMed
    • Google Scholar
22. 1. Du J
    2. Hossain Z
    3. Mandal J

    (2017) Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdalai

    PNAS 3:1–3.

    https://doi.org/10.1073/pnas.1407018113

    • Google Scholar
23. 1. Elkhatib W
    2. Smith CL
    3. Senatore A

    (2019) A Na+ leak channel cloned from Trichoplax adhaerens extends extracellular pH and Ca2+ sensing for the DEG/ENaC family close to the base of Metazoa

    Journal of Biological Chemistry 294:16320–16336.

    https://doi.org/10.1074/jbc.RA119.010542

    • Google Scholar
24. 1. Escorcia W
    2. Ruter DL
    3. Nhan J
    4. Curran SP

    (2018) Quantification of lipid abundance and evaluation of lipid distribution in Caenorhabditis elegans by nile red and oil red o staining

    Journal of Visualized Experiments 5:57352.

    https://doi.org/10.3791/57352

    • PubMed
    • Google Scholar
25. 1. Espelt MV
    2. Estevez AY
    3. Yin X
    4. Strange K

    (2005) Oscillatory Ca2+ signaling in the isolated Caenorhabditis elegans intestine: role of the inositol-1,4,5-trisphosphate receptor and phospholipases C beta and gamma

    The Journal of General Physiology 126:379–392.

    https://doi.org/10.1085/jgp.200509355

    • PubMed
    • Google Scholar
26. 1. Fechner S
    2. D’Alessandro I
    3. Wang L
    4. Tower C
    5. Tao L
    6. Goodman MB

    (2021) DEG/ENaC/ASIC channels vary in their sensitivity to anti-hypertensive and non-steroidal anti-inflammatory drugs

    The Journal of General Physiology 153:e202012655.

    https://doi.org/10.1085/jgp.202012655

    • PubMed
    • Google Scholar
27. 1. Fei YJ
    2. Kanai Y
    3. Nussberger S
    4. Ganapathy V
    5. Leibach FH
    6. Romero MF
    7. Singh SK
    8. Boron WF
    9. Hediger MA

    (1994) Expression cloning of a mammalian proton-coupled oligopeptide transporter

    Nature 368:563–566.

    https://doi.org/10.1038/368563a0

    • PubMed
    • Google Scholar
28. 1. Hammarlund M
    2. Hobert O
    3. Miller DM
    4. Sestan N

    (2018) The CeNGEN project: the complete gene expression map of an entire nervous system

    Neuron 99:430–433.

    https://doi.org/10.1016/j.neuron.2018.07.042

    • PubMed
    • Google Scholar
29. 1. Hardege I
    2. Xu S
    3. Gordon RD
    4. Thompson AJ
    5. Figg N
    6. Stowasser M
    7. Murrell-Lagnado R
    8. O’Shaughnessy KM

    (2015) Novel insertion mutation in KCNJ5 channel produces constitutive aldosterone release from H295R cells

    Molecular Endocrinology 29:1522–1530.

    https://doi.org/10.1210/me.2015-1195

    • PubMed
    • Google Scholar
30. 1. Hastings MH
    2. Maywood ES
    3. Brancaccio M

    (2018) Generation of circadian rhythms in the suprachiasmatic nucleus

    Nature Reviews. Neuroscience 19:453–469.

    https://doi.org/10.1038/s41583-018-0026-z

    • PubMed
    • Google Scholar
31. 1. Hesselager M
    2. Timmermann DB
    3. Ahring PK

    (2004) pH Dependency and desensitization kinetics of heterologously expressed combinations of acid-sensing ion channel subunits

    The Journal of Biological Chemistry 279:11006–11015.

    https://doi.org/10.1074/jbc.M313507200

    • PubMed
    • Google Scholar
32. 1. Holzer P

    (2007) Taste receptors in the gastrointestinal tract. V. Acid sensing in the gastrointestinal tract

    American Journal of Physiology. Gastrointestinal and Liver Physiology 292:G699–G705.

    https://doi.org/10.1152/ajpgi.00517.2006

    • PubMed
    • Google Scholar
33. 1. Imanikia S
    2. Sheng M
    3. Castro C
    4. Griffin JL
    5. Taylor RC

    (2019a) XBP-1 remodels lipid metabolism to extend longevity

    Cell Reports 28:581–589.

    https://doi.org/10.1016/j.celrep.2019.06.057

    • PubMed
    • Google Scholar
34. 1. Imanikia S
    2. Özbey NP
    3. Krueger C
    4. Casanueva MO
    5. Taylor RC

    (2019b) Neuronal XBP-1 activates intestinal lysosomes to improve proteostasis in C. elegans

    Current Biology 29:2322–2338.

    https://doi.org/10.1016/j.cub.2019.06.031

    • PubMed
    • Google Scholar
35. 1. Kamath R S
    2. Martinez-Campos M
    3. Zipperlen P
    4. Fraser AG
    5. Ahringer J

    (2001) Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans

    Genome Biology 2:RESEARCH0002.

    https://doi.org/10.1186/gb-2000-2-1-research0002

    • PubMed
    • Google Scholar
36. 1. Kamath RS
    2. Ahringer J

    (2003) Genome-wide RNAi screening in Caenorhabditis elegans

    Methods 30:313–321.

    https://doi.org/10.1016/s1046-2023(03)00050-1

    • PubMed
    • Google Scholar
37. 1. Kamath RS
    2. Fraser AG
    3. Dong Y
    4. Poulin G
    5. Durbin R
    6. Gotta M
    7. Kanapin A
    8. Le Bot N
    9. Moreno S
    10. Sohrmann M
    11. Welchman DP
    12. Zipperlen P
    13. Ahringer J

    (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi

    Nature 421:231–237.

    https://doi.org/10.1038/nature01278

    • PubMed
    • Google Scholar
38. 1. Katsura I
    2. Kondo K
    3. Amano T
    4. Ishihara T
    5. Kawakami M

    (1994) Isolation, characterization and epistasis of fluoride-resistant mutants of Caenorhabditis elegans

    Genetics 136:145–154.

    https://doi.org/10.1093/genetics/136.1.145

    • PubMed
    • Google Scholar
39. Preprint

    1. Kaulich E
    2. McCubbin PTN
    3. Schafer WR
    4. Walker DS

    (2022) Physiological Insight into the conserved properties of Caenorhabditis elegans acid-sensing DEG/ENaCs

    bioRxiv.

    https://doi.org/10.1101/2022.04.12.488049

    • Google Scholar
40. 1. Kobayashi Y
    2. Kimura KD
    3. Katsura I

    (2011) Ultradian rhythm in the intestine of Caenorhabditis elegans is controlled by the C-terminal region of the FLR-1 ion channel and the hydrophobic domain of the FLR-4 protein kinase

    Genes to Cells 16:565–575.

    https://doi.org/10.1111/j.1365-2443.2011.01508.x

    • PubMed
    • Google Scholar
41. 1. Kreple CJ
    2. Lu Y
    3. Taugher RJ
    4. Schwager-Gutman AL
    5. Du J
    6. Stump M
    7. Wang Y
    8. Ghobbeh A
    9. Fan R
    10. Cosme CV
    11. Sowers LP
    12. Welsh MJ
    13. Radley JJ
    14. LaLumiere RT
    15. Wemmie JA

    (2014) Acid-sensing ion channels contribute to synaptic transmission and inhibit cocaine-evoked plasticity

    Nature Neuroscience 17:1083–1091.

    https://doi.org/10.1038/nn.3750

    • PubMed
    • Google Scholar
42. 1. Kwan CSM
    2. Vázquez-Manrique RP
    3. Ly S
    4. Goyal K
    5. Baylis HA

    (2008) TRPM channels are required for rhythmicity in the ultradian defecation rhythm of C. elegans

    BMC Physiology 8:11.

    https://doi.org/10.1186/1472-6793-8-11

    • PubMed
    • Google Scholar
43. 1. Levanti MB
    2. Guerrera MC
    3. Calavia MG
    4. Ciriaco E
    5. Montalbano G
    6. Cobo J
    7. Germanà A
    8. Vega JA

    (2011) Acid-sensing ion channel 2 (ASIC2) in the intestine of adult zebrafish

    Neuroscience Letters 494:24–28.

    https://doi.org/10.1016/j.neulet.2011.02.046

    • PubMed
    • Google Scholar
44. 1. Li W-G
    2. Yu Y
    3. Huang C
    4. Cao H
    5. Xu T-L

    (2011) Nonproton ligand sensing domain is required for paradoxical stimulation of acid-sensing ion channel 3 (ASIC3) channels by amiloride

    The Journal of Biological Chemistry 286:42635–42646.

    https://doi.org/10.1074/jbc.M111.289058

    • PubMed
    • Google Scholar
45. 1. Liang R
    2. Fei Y-J
    3. Prasad PD
    4. Ramamoorthy S
    5. Han H
    6. Yang-Feng TL
    7. Hediger MA
    8. Ganapathy V
    9. Leibach FH

    (1995) Human intestinal H+/Peptide cotransporter

    Journal of Biological Chemistry 270:6456–6463.

    https://doi.org/10.1074/jbc.270.12.6456

    • PubMed
    • Google Scholar
46. 1. Liu DW
    2. Thomas JH

    (1994)

    Regulation of a periodic motor program in C. elegans

    The Journal of Neuroscience 14:1953–1962.

    • PubMed
    • Google Scholar
47. 1. Mackenzie B
    2. Loo DD
    3. Fei Y
    4. Liu WJ
    5. Ganapathy V
    6. Leibach FH
    7. Wright EM

    (1996) Mechanisms of the human intestinal H+-coupled oligopeptide transporter hPEPT1

    The Journal of Biological Chemistry 271:5430–5437.

    https://doi.org/10.1074/jbc.271.10.5430

    • PubMed
    • Google Scholar
48. Book

    1. Marcheva B
    2. Ramsey K
    3. Peek C
    4. Affinati A
    5. Maury E
    6. Bass J

    (2013)

    Circadian clocks and metabolism

    In: Reinke H, editors. Handbook of Experimental Pharmacology. Springer. pp. 127–155.

    • Google Scholar
49. 1. Matthewman C
    2. Miller-Fleming TW
    3. Miller DM
    4. Bianchi L

    (2016) Ca2+ permeability and Na+ conductance in cellular toxicity caused by hyperactive DEG/ENaC channels

    American Journal of Physiology. Cell Physiology 311:C920–C930.

    https://doi.org/10.1152/ajpcell.00247.2016

    • PubMed
    • Google Scholar
50. 1. Mclntire SL
    2. Jorgensen E
    3. Kaplan J
    4. Horvitz HR

    (1993) The GABAergic nervous system of Caenorhabditis elegans

    Nature 364:337–341.

    https://doi.org/10.1038/364337a0

    • PubMed
    • Google Scholar
51. 1. Mello CC
    2. Kramer JM
    3. Stinchcomb D
    4. Ambros V

    (1991) Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences

    The EMBO Journal 10:3959–3970.

    https://doi.org/10.1002/j.1460-2075.1991.tb04966.x

    • PubMed
    • Google Scholar
52. 1. Miesenböck G
    2. De Angelis DA
    3. Rothman JE

    (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins

    Nature 394:192–195.

    https://doi.org/10.1038/28190

    • PubMed
    • Google Scholar
53. 1. Nehrke K

    (2003) A reduction in intestinal cell pH due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases life span

    Journal of Biological Chemistry 278:44657–44666.

    https://doi.org/10.1074/jbc.M307351200

    • PubMed
    • Google Scholar
54. 1. O’Rourke EJ
    2. Soukas AA
    3. Carr CE
    4. Ruvkun G

    (2009) C. elegans major fats are stored in vesicles distinct from lysosome-related organelles

    Cell Metabolism 10:430–435.

    https://doi.org/10.1016/j.cmet.2009.10.002

    • PubMed
    • Google Scholar
55. 1. Paukert M
    2. Babini E
    3. Pusch M
    4. Gründer S

    (2004) Identification of the Ca2+ blocking site of acid-sensing ion channel (ASIC) 1: implications for channel gating

    The Journal of General Physiology 124:383–394.

    https://doi.org/10.1085/jgp.200308973

    • PubMed
    • Google Scholar
56. 1. Pfeiffer J
    2. Johnson D
    3. Nehrke K

    (2008) Oscillatory transepithelial H(+) flux regulates a rhythmic behavior in C. elegans

    Current Biology 18:297–302.

    https://doi.org/10.1016/j.cub.2008.01.054

    • PubMed
    • Google Scholar
57. 1. Rabinowitch I
    2. Chatzigeorgiou M
    3. Schafer WR

    (2013) A gap junction circuit enhances processing of coincident mechanosensory inputs

    Current Biology 23:963–967.

    https://doi.org/10.1016/j.cub.2013.04.030

    • PubMed
    • Google Scholar
58. Software

    1. Rabinowitch I

    (2020) NeuronTracker

    GitHub.

    https://github.com/ithairab/NeuronTracker
59. 1. Rensing L
    2. Meyer-Grahle U
    3. Ruoff P

    (2001) Biological timing and the clock metaphor: oscillatory and hourglass mechanisms

    Chronobiology International 18:329–369.

    https://doi.org/10.1081/cbi-100103961

    • PubMed
    • Google Scholar
60. 1. Rollins JA
    2. Howard AC
    3. Dobbins SK
    4. Washburn EH
    5. Rogers AN

    (2017) Assessing health span in Caenorhabditis elegans: lessons from short-lived mutants

    The Journals of Gerontology 72:473–480.

    https://doi.org/10.1093/gerona/glw248

    • PubMed
    • Google Scholar
61. 1. Schaefer L
    2. Sakai H
    3. Mattei M-G
    4. Lazdunski M
    5. Lingueglia E

    (2000) Molecular cloning, functional expression and chromosomal localization of an amiloride-sensitive Na ⁺ channel from human small intestine

    FEBS Letters 471:205–210.

    https://doi.org/10.1016/S0014-5793(00)01403-4

    • PubMed
    • Google Scholar
62. 1. Schneider CA
    2. Rasband WS
    3. Eliceiri KW

    (2012) NIH Image to ImageJ: 25 years of image analysis

    Nature Methods 9:671–675.

    https://doi.org/10.1038/nmeth.2089

    • PubMed
    • Google Scholar
63. 1. Sheng M
    2. Hosseinzadeh A
    3. Muralidharan SV
    4. Gaur R
    5. Selstam E
    6. Tuck S
    7. Hart AC

    (2015) Aberrant fat metabolism in Caenorhabditis elegans mutants with defects in the defecation motor program

    PLOS ONE 10:e0124515.

    https://doi.org/10.1371/journal.pone.0124515

    • PubMed
    • Google Scholar
64. 1. Soto E
    2. Ortega-Ramírez A
    3. Vega R

    (2018) Protons as messengers of intercellular communication in the nervous system

    Frontiers in Cellular Neuroscience 12:342.

    https://doi.org/10.3389/fncel.2018.00342

    • PubMed
    • Google Scholar
65. 1. Suzuki H
    2. Kerr R
    3. Bianchi L
    4. Frøkjær-Jensen C
    5. Slone D
    6. Xue J
    7. Gerstbrein B
    8. Driscoll M
    9. Schafer WR

    (2003) In vivo imaging of C. elegans mechanosensory neurons demonstrates a specific role for the MEC-4 channel in the process of gentle touch sensation

    Neuron 39:1005–1017.

    https://doi.org/10.1016/j.neuron.2003.08.015

    • PubMed
    • Google Scholar
66. 1. Take-uchi M
    2. Kawakami M
    3. Ishihara T
    4. Amano T
    5. Kondo K
    6. Katsura I

    (1998) An ion channel of the degenerin/epithelial sodium channel superfamily controls the defecation rhythm in Caenorhabditis elegans

    PNAS 95:11775–11780.

    https://doi.org/10.1073/pnas.95.20.11775

    • PubMed
    • Google Scholar
67. 1. Taylor SR
    2. Santpere G
    3. Weinreb A
    4. Barrett A
    5. Reilly MB
    6. Xu C
    7. Varol E
    8. Oikonomou P
    9. Glenwinkel L
    10. McWhirter R
    11. Poff A
    12. Basavaraju M
    13. Rafi I
    14. Yemini E
    15. Cook SJ
    16. Abrams A
    17. Vidal B
    18. Cros C
    19. Tavazoie S
    20. Sestan N
    21. Hammarlund M
    22. Hobert O
    23. Miller DM III

    (2021) Molecular topography of an entire nervous system

    Cell 184:4329–4347.

    https://doi.org/10.1016/j.cell.2021.06.023

    • PubMed
    • Google Scholar
68. 1. Teramoto T
    2. Iwasaki K

    (2006) Intestinal calcium waves coordinate a behavioral motor program in C. elegans

    Cell Calcium 40:319–327.

    https://doi.org/10.1016/j.ceca.2006.04.009

    • PubMed
    • Google Scholar
69. 1. Thomas JH

    (1990) Genetic analysis of defecation in Caenorhabditis elegans

    Genetics 124:855–872.

    https://doi.org/10.1093/genetics/124.4.855

    • PubMed
    • Google Scholar
70. 1. Urra J
    2. Sandoval M
    3. Cornejo I
    4. Barros LF
    5. Sepúlveda FV
    6. Cid LP

    (2008) A genetically encoded ratiometric sensor to measure extracellular pH in microdomains bounded by basolateral membranes of epithelial cells

    Pflugers Archiv 457:233–242.

    https://doi.org/10.1007/s00424-008-0497-2

    • PubMed
    • Google Scholar
71. 1. Voglis G
    2. Tavernarakis N

    (2008) A synaptic DEG/ENaC ion channel mediates learning in C. elegans by facilitating dopamine signalling

    The EMBO Journal 27:3288–3299.

    https://doi.org/10.1038/emboj.2008.252

    • PubMed
    • Google Scholar
72. 1. Wagner J
    2. Allman E
    3. Taylor A
    4. Ulmschneider K
    5. Kovanda T
    6. Ulmschneider B
    7. Nehrke K
    8. Peters MA

    (2011) A calcineurin homologous protein is required for sodium-proton exchange events in the C. elegans intestine

    American Journal of Physiology. Cell Physiology 301:e00139.

    https://doi.org/10.1152/ajpcell.00139.2011

    • PubMed
    • Google Scholar
73. 1. Waldmann R
    2. Champigny G
    3. Bassilana F
    4. Heurteaux C
    5. Lazdunski M

    (1997) A proton-gated cation channel involved in acid-sensing

    Nature 386:173–177.

    https://doi.org/10.1038/386173a0

    • PubMed
    • Google Scholar
74. 1. Walker DS
    2. Gower NJD
    3. Ly S
    4. Bradley GL
    5. Baylis HA

    (2002) Regulated disruption of inositol 1,4,5-trisphosphate signaling in Caenorhabditis elegans reveals new functions in feeding and embryogenesis

    Molecular Biology of the Cell 13:1329–1337.

    https://doi.org/10.1091/mbc.01-08-0422

    • PubMed
    • Google Scholar
75. 1. Wang Y
    2. Apicella A Jr
    3. Lee S-K
    4. Ezcurra M
    5. Slone RD
    6. Goldmit M
    7. Schafer WR
    8. Shaham S
    9. Driscoll M
    10. Bianchi L

    (2008) A glial DEG/ENaC channel functions with neuronal channel DEG-1 to mediate specific sensory functions in C. elegans

    The EMBO Journal 27:2388–2399.

    https://doi.org/10.1038/emboj.2008.161

    • PubMed
    • Google Scholar
76. 1. Wemmie JA
    2. Chen J
    3. Askwith CC
    4. Hruska-Hageman AM
    5. Price MP
    6. Nolan BC
    7. Yoder PG
    8. Lamani E
    9. Hoshi T
    10. Freeman JH Jr
    11. Welsh MJ

    (2002) The acid-activated ion channel ASIC contributes to synaptic plasticity, learning, and memory

    Neuron 34:463–477.

    https://doi.org/10.1016/s0896-6273(02)00661-x

    • PubMed
    • Google Scholar
77. 1. Wemmie JA
    2. Askwith CC
    3. Lamani E
    4. Cassell MD
    5. Freeman JH Jr
    6. Welsh MJ

    (2003)

    Acid-sensing ion channel 1 is localized in brain regions with high synaptic density and contributes to fear conditioning

    The Journal of Neuroscience 23:5496–5502.

    • PubMed
    • Google Scholar
78. 1. Wiemuth D
    2. Gründer S

    (2010) A single amino acid tunes Ca2+ inhibition of brain liver intestine Na+ channel (BLINaC

    The Journal of Biological Chemistry 285:30404–30410.

    https://doi.org/10.1074/jbc.M110.153064

    • PubMed
    • Google Scholar
79. 1. Zhang P
    2. Canessa CM

    (2002) Single channel properties of rat acid-sensitive ion channel-1alpha, -2a, and -3 expressed in Xenopus oocytes

    The Journal of General Physiology 120:553–566.

    https://doi.org/10.1085/jgp.20028574

    • PubMed
    • Google Scholar
80. 1. Zimmer AM
    2. Dymowska AK
    3. Kumai Y
    4. Goss GG
    5. Perry SF
    6. Kwong RWM

    (2018) Assessing the role of the acid-sensing ion channel ASIC4b in sodium uptake by larval zebrafish

    Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology 226:1–10.

    https://doi.org/10.1016/j.cbpa.2018.06.012

    • PubMed
    • Google Scholar

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

• 1,281

  views

• 353

  downloads

• 8

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.