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  1. Evaluation Summary:

    This study investigates the role of acid sensing channels in pH homeostasis required for normal rhythmic muscle contractions in the defecation cycle of C .elegans. It is of importance to scientists with interest in ASIC channel function, pH homeostasis and the cellular mechanisms underlying generation of ultradian rhythms.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)”

  2. Reviewer #1 (Public Review):

    Kaulich and colleagues investigated the role DEG/ENaCs - ASICs channel family members in pH homeostasis underlying the rhythmic defecation cycle of C. elegans, using a combination of in vitro electrophysiology, in vivo imaging and behavioural genetics. They show evidence, from heterologous expression in oocytes, that ACD-5 is sensitive as a homomer to a specific range of pH, while FLR-1 requires co-expression of either ACD-3 or DEL-5 to be sensitive to pH, suggesting a heteromeric complex being inhibited by protons. acd-5 mutants have a strong effect on pH dynamics / proton concentration oscillations in the intestinal lumen, while showing rather subtle effects on rhythmic intestinal Ca++ oscillations and the rhythmic defecation pattern. flr-1 single mutants or RNAi and acd-3/del-5 double mutants show stronger effects on Ca++ oscillations and behaviour. Together with localization studies, the authors propose a model in which ACD-5 acts on the apical membrane facing the intestinal lumen controlling luminal pH, while acid sensitivity of a FLR-1/ACD-3/DEL-5 complex from the basolateral membrane controls intracellular Intestinal Ca++ oscillations. Finally, the overall effect of channel mutants on growth and fat-metabolisms is assessed documenting a functional implication of the degree by which the defecation cycle is impaired. Altogether, the work is interesting, and experiments are carefully performed and controlled. The model could be substantiated by tissue specific rescue experiments of flr-1, acd-3, del-5 mutants where possible.

  3. Reviewer #2 (Public Review):

    The authors show that ACD-5 channel is a homomeric proton-sensing channel by performing electrophysiology experiments in ACD-5 injected Xenopus oocytes. The results are solid and strongly support the idea that ACD-5 is an acid-sensing cation channel. They show ACD-5 localizes to the apical membrane of intestine by fluorescent imaging of translational reporters. Co-localization with different markers strongly supports that ACD-5 is concentrated on the apical membrane of the intestine. They further show that ACD-5 controls proton concentrations in the lumen through Maximum Anterior Transition (MAT) experiments. The disrupted intestinal lumen pH was rescued by expressing ACD-5 cDNA in the intestine, further supporting ACD-5's function as a pH regulator of the intestinal lumen. The authors show that ACD-5 may have a very minor role in in regulating intestinal calcium oscillations and defecation behavior, because null mutants were normal for cycle length and only exhibited small changes in calcium dynamics. On the other hand, larger (albeit subtle) differences in some parameters were seen in the dominant acd-5(ok2657) mutant, suggesting that this unusual allele is a dominant negative with some additional (neomorphic?) characteristics, raising questions about how useful this allele is in attributing functions to acd-5 .

    In the second part of paper, the authors show that FLR-1 forms a pH-sensitive channel with ACD-3 and/or DEL-5. They further show a strong link between FLR-1/ACD-3/DEL-5 channels with calcium oscillation in the intestine and defecation behavior. The loss of function phenotype of flr-1 has been described previously and this study extends that characterization by showing that flr-1 regulates intestinal calcium oscillations. However, the study does not establish a clear subcellular site-of-action for these channels, nor does it provide a mechanistic link between intestinal acid sensing and release of calcium from internal stores. This would require a more complete exploration of how the FLR-1/ACD-3/DEL-5 channels interact with the other players known to regulate calcium signaling in the intestine.

  4. Reviewer #3 (Public Review):

    The authors have identified two acid-sensing DEG/ENaC channels that act in C. elegans intestine to regulate the defecation motor program (DMP). The first channel ACD-5 is a homomeric channel that localizes to the apical membrane, is inhibited by high and low pH, and is important for maintaining the pH oscillations in the lumen but not for Ca2+ oscillations in the intestine. The other channel is a heteromeric channel formed by FLR-1 with ACD-3 and/or DEL-5. This channel localizes to the basolateral membrane, is inhibited by acidic pH, controls Ca2+ oscillations, and is important for worm development and lipid metabolism. The authors have proposed a model to explain the differential roles of the two ASIC channels in regulating DMP.

    Overall, the work is interesting, revealing two ASIC channels with distinct biophysical properties and physiological functions. The experiments were well designed and executed.