Functional Divergence and Emerging Roles of the ANO–TMC–TMEM63 Channel Families in Olfaction and Gustation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript reviews the roles of the Anoctamin (ANO/TMEM16), Transmembrane Channel‑like (TMC), and TMEM63/OSCA channel families across chemosensory systems, with emphasis on mammalian olfaction and taste. It presents the diverse roles of ANOs in  the mammalian olfactory system (as well as the role of ANO1 in taste) and discusses the contribution of TMC4 to high‑salt taste via a putative chloride conductance and broader excitability modulation. Importantly it goes on to explore the olfactory/gustatory roles of this superfamily  across species showing deep evolutionary conservation from C. elegans and insects to basal chordates (Ciona) and to mammals. The manuscript further highlights some of the roles of TMCs and TMEM63 in mechanosensory and hygrosensory behaviors. The review also highlights potential scramblase‑linked epithelial pathology (e.g., ANO6 during SARS‑CoV‑2 infection).

Overall, the topic is timely, the review is rather comprehesive  in terms of covering the existing literate (obviously it is impossible to cover every relevant manuscript out there) and drawing evidence from many different experimental directions. I am particlarly happy to see that the author also leveraged a comparitive angle using multiple species to illustrate the adoption of TMC, Anoctamin and TMEM63 as a shared toolkit for sensory modulation across multiple modalities and species. I think this will make the review fairly citable and appealing for a broad(er) audience.

The review is very easy to read and the figures are very clear (but see below). I only have relatively minor issues that I would like to raise before this manuscript is suitable for publication.  

Minor points:

1. There are several incosistencies in the use of superscripts e.g. line 48: “resulting influx of Na+ and Ca2+ depolarizes…” vs line 49: “Ca²⁺-activated chloride currents (CaCCs)…” Please address all these minor incosistencies across the entire text.
2. Reference 89: ( Murata, M.; Saito, Y.; Kasahara, Y.; Fujii, W.; Misaka, T.; Asakura, T.; Narukawa, M. TMC4 localizes to multiple taste cell types in the mouse taste papillae. FEBS Open Bio 2025, in press.) is identified as in press however it is now published:

  (https://febs.onlinelibrary.wiley.com/doi/full/10.1002/2211-5463.70159)

3. In the Table 2 the species are itelisized but elsewhere they are not! Please italisize the different species in the main text.
4. For the figures I would suggest if this is ok with the editorial team to include panel letters (A, B, C), because the use of left panel, top right panel etc is a bit unusual and not so convenient. Also if you have made use of biorender please acknowledge that (I could be wrong).
5. I agree with the author view that TMC4 is very likely an important player in high salt sensing. However more work needs to be done to consolidate this so for example in line 304: “In this context, TMC4 has emerged as a key candidate involved in salt taste perception.” I would prefer if the author used “additional, new” or something to that effect.
6. Please format the tables in boxes so that they look a bit more appealing and organised.
7. I am not super convinced about the necessity of discussing the role of ANO6 in SARS-CoV-2 but if the author and editors consider this suitable for the review I am not opposing to it. It just feels a bit out of place.
8. Please standardize also gene/protein annotation: (e.g., human proteins ANO1/ANO2/ANO9 in caps; mouse genes Ano1/Ano2/Ano9 in italics).

Author Response

Comments 1: The manuscript reviews the roles of the Anoctamin (ANO/TMEM16), Transmembrane Channel‑like (TMC), and TMEM63/OSCA channel families across chemosensory systems, with emphasis on mammalian olfaction and taste. It presents the diverse roles of ANOs in  the mammalian olfactory system (as well as the role of ANO1 in taste) and discusses the contribution of TMC4 to high‑salt taste via a putative chloride conductance and broader excitability modulation. Importantly it goes on to explore the olfactory/gustatory roles of this superfamily  across species showing deep evolutionary conservation from C. elegans and insects to basal chordates (Ciona) and to mammals. The manuscript further highlights some of the roles of TMCs and TMEM63 in mechanosensory and hygrosensory behaviors. The review also highlights potential scramblase‑linked epithelial pathology (e.g., ANO6 during SARS‑CoV‑2 infection).

Overall, the topic is timely, the review is rather comprehesive  in terms of covering the existing literate (obviously it is impossible to cover every relevant manuscript out there) and drawing evidence from many different experimental directions. I am particlarly happy to see that the author also leveraged a comparitive angle using multiple species to illustrate the adoption of TMC, Anoctamin and TMEM63 as a shared toolkit for sensory modulation across multiple modalities and species. I think this will make the review fairly citable and appealing for a broad(er) audience.

The review is very easy to read and the figures are very clear (but see below). I only have relatively minor issues that I would like to raise before this manuscript is suitable for publication.  

Minor points:

There are several incosistencies in the use of superscripts e.g. line 48: “resulting influx of Na+ and Ca2+ depolarizes…” vs line 49: “Ca²⁺-activated chloride currents (CaCCs)…” Please address all these minor incosistencies across the entire text.

Responses 1: I sincerely thank the reviewer for the positive and insightful comments, and for recognizing the scope and significance of our work. I have carefully reviewed the entire manuscript and corrected all inconsistencies in the use of superscripts, ensuring that the notation of ionic species (e.g., Ca²⁺, Na⁺, Cl⁻) is consistent throughout the text.

 

Comments 2: Reference 89: ( Murata, M.; Saito, Y.; Kasahara, Y.; Fujii, W.; Misaka, T.; Asakura, T.; Narukawa, M. TMC4 localizes to multiple taste cell types in the mouse taste papillae. FEBS Open Bio 2025, in press.) is identified as in press however it is now published:  (https://febs.onlinelibrary.wiley.com/doi/full/10.1002/2211-5463.70159)

Responses 2: Thank you for pointing this out. The reference has been updated to reflect its published version, and the citation has been revised accordingly.

 

Comments 3: In the Table 2 the species are itelisized but elsewhere they are not! Please italisize the different species in the main text.

Responses 3: Thank you for pointing this out. I have carefully revised the manuscript to ensure that all species names are consistently italicized throughout, including Caenorhabditis elegans, Drosophila melanogaster, Ciona intestinalis, Fundulus heteroclitus, Danio rerio, Mus musculus, and Gallus gallus.

Comments 4: For the figures I would suggest if this is ok with the editorial team to include panel letters (A, B, C), because the use of left panel, top right panel etc is a bit unusual and not so convenient. Also if you have made use of biorender please acknowledge that (I could be wrong).

Responses 4: Thank you for this helpful suggestion. I have revised all figures to include panel labels (A, B, C) instead of directional descriptions for improved clarity. In addition, I have included an acknowledgment indicating that the figures were created using BioRender, and this has also been specified in the figure legends.

(Lines 602-603) "Acknowledgments: The author would like to acknowledge BioRender.com for assistance in creating the figures used in this manuscript."

 

Comments 5: I agree with the author view that TMC4 is very likely an important player in high salt sensing. However more work needs to be done to consolidate this so for example in line 304: “In this context, TMC4 has emerged as a key candidate involved in salt taste perception.” I would prefer if the author used “additional, new” or something to that effect.

Responses 5: Thank you for this valuable suggestion. I agree with the reviewer and have revised the sentence accordingly. Specifically, the statement in line 327 has been modified to: (Lines 327-328) “In this context, TMC4 has recently emerged as additional, new candidate involved in salt taste perception.”

 

Comments 6: Please format the tables in boxes so that they look a bit more appealing and organised.

Responses 6: Thank you for this suggestion. I have revised Tables 1 and 2 by adding borders and applying background shading to the header rows to improve clarity and overall presentation. In addition, newly incorporated content from the revised manuscript has been included in the tables to improve completeness and clarity.

 

Comments 7: I am not super convinced about the necessity of discussing the role of ANO6 in SARS-CoV-2 but if the author and editors consider this suitable for the review I am not opposing to it. It just feels a bit out of place.

Responses 7: Thank you for this thoughtful comment. I agree that the discussion of ANO6 in the context of SARS-CoV-2 may appear somewhat tangential. However, I have retained this section because scramblase-mediated lipid remodeling represents a biologically relevant mechanism that is closely linked to the maintenance of olfactory epithelium homeostasis, particularly in sustentacular cells.

To address the reviewer’s concern, I have revised the paragraph to improve its connection to the preceding discussion and to adopt a more balanced and cautious tone. Specifically, the section has been reframed to emphasize its relevance in the context of olfactory epithelial homeostasis and potential pathological implications, rather than overinterpreting the experimental findings.

(Lines 550- 566)

Beyond these roles, emerging evidence suggests that scramblase-mediated lipid remodeling may also be involved in broader pathological contexts. One clinically relevant example is virus-associated cellular pathology. SARS-CoV-2 preferentially infects sustentacular cells in the olfactory epithelium that coexpress ACE2 and TMPRSS2 [113]. These supporting cells express relatively high levels of ANO6, and viral spike protein engagement has been associated with increased ANO6 scramblase activity [114]. Activated ANO6 disrupts membrane lipid asymmetry and promotes phosphatidylserine (PS) externalization, which can facilitate membrane fusion between neighboring cells. This process contributes to the formation of multinucleated syncytia and disruption of epithelial architecture [114,115]. In addition, the ANO6 scramblase inhibitor niclosamide has been reported to suppress SARS-CoV-2 spike–induced cell fusion, further supporting the functional relevance of this pathway [116].

Collectively, these findings suggest that regulated scramblase activity and membrane lipid remodeling play important roles in maintaining sensory cell structure and microenvironment organization. While their roles in physiological contexts are well supported, dysregulation of these processes may also influence cellular vulnerability and tissue integrity under pathological conditions.

 

Comments 8: Please standardize also gene/protein annotation: (e.g., human proteins ANO1/ANO2/ANO9 in caps; mouse genes Ano1/Ano2/Ano9 in italics).

Responses 8: I thank the reviewer for this helpful suggestion. Gene and protein nomenclature has been carefully reviewed and standardized throughout the manuscript, including both the main text and tables. In particular, gene symbols are now consistently formatted in italics according to species-specific conventions.

To improve clarity, I have also explicitly indicated the use of mouse-derived data in relevant sections of the manuscript. Specifically, the revised text now clearly states that the reported expression patterns and functional observations are based on analyses of mouse tissues, and appropriate gene and protein annotations have been applied accordingly.

(Lines 131-133)

High-resolution X-ray crystallography and cryo–electron microscopy structures of fungal nhTMEM16 and mouse Ano1 define a conserved anoctamin fold that serves as the structural core of this family [34,35].

(Lines 352-354)

In addition to TMC channels, anoctamin family members have been reported in taste buds. Both Ano1 and Ano2 transcripts have been detected in mouse taste cells, although their precise cell-type distribution remains controversial.

(Lines 356-359)

However, subsequent immunohistochemical and functional studies failed to detect Ano2 protein in mouse taste buds and instead identified Ano1 as the predominant anoctamin channel, with expression largely restricted to the apical microvilli of Type I supporting cells.

Reviewer 2 Report

Comments and Suggestions for Authors

This is a fine review article authored by a researcher with extensive expertise in the field. The manuscript is written in a clear, elegant, and accessible style, which is particularly commendable given the conceptual density of the topic. The narrative is well structured, and the synthesis of information is both comprehensive and engaging. The illustrative schemes constitute a particularly valuable contribution, as they condense complex mechanisms into clear and visually intuitive representations. The tables are also useful; however, their organizational criteria appear somewhat subjective, and in some instances they function more as illustrative examples than as systematic references.

In my opinion, the manuscript is suitable for publication. Nevertheless, I would like to raise several points that, in my view, should be addressed to strengthen its scope and balance.

First, the absence of any reference to the vomeronasal system is somewhat striking. Given its well-established role in mammalian chemosensation, and considering that anoctamins are involved in signal amplification and transduction processes, its omission represents a notable gap in an otherwise comprehensive review. Even a brief discussion would significantly improve the integrative perspective of the manuscript.

Second, although the evolutionary dimension is appropriately presented as a central theme (section 5), its development remains somewhat limited. The references on vertebrates are short and focused on fish, while other vertebrate groups -particularly amphibians, reptiles, and birds- are largely overlooked. Even in fishes, the cited literature is restricted to taste-related functions, overlooking the involvement of anoctamins in the olfactory system, which is a central and highly developed chemosensory modality in this group. This omission is particularly relevant and should be addressed to provide a more complete and accurate overview of anoctamin functio, especially considering that these groups may offer more direct comparative insight into mammalian systems.

Overall, this is a high-quality and timely review, and the points raised above are intended to reinforce its already strong scientific contribution.

Minor issues:

Lines 46-53: The description of the signaling cascade is clear and accurate. However, you might consider explicitly mentioning adenylyl cyclase III (ACIII), as it plays a central role in linking Golf activation to cAMP production in olfactory sensory neurons.

Line 112: It should be specified that the term “TMEM” refers to “transmembrane protein” and represents an initial gene annotation rather than a functional classification. This clarification would be useful for readers unfamiliar with the nomenclature.

Line 127: “These spores have been implicated in the regulation of phosphatidylserine”

There is a typo in this sentence (“spores”), which should likely read “pore”, “proteins” or “channels.”

Figure 1: In the left panel, you may consider indicating the position of sustentacular cell nuclei in the apical half of the olfactory epithelium to improve anatomical accuracy.

Line 213: Gallus gallus should be italicized.

Author Response

Comments 1: This is a fine review article authored by a researcher with extensive expertise in the field. The manuscript is written in a clear, elegant, and accessible style, which is particularly commendable given the conceptual density of the topic. The narrative is well structured, and the synthesis of information is both comprehensive and engaging. The illustrative schemes constitute a particularly valuable contribution, as they condense complex mechanisms into clear and visually intuitive representations. The tables are also useful; however, their organizational criteria appear somewhat subjective, and in some instances they function more as illustrative examples than as systematic references.

In my opinion, the manuscript is suitable for publication. Nevertheless, I would like to raise several points that, in my view, should be addressed to strengthen its scope and balance.

First, the absence of any reference to the vomeronasal system is somewhat striking. Given its well-established role in mammalian chemosensation, and considering that anoctamins are involved in signal amplification and transduction processes, its omission represents a notable gap in an otherwise comprehensive review. Even a brief discussion would significantly improve the integrative perspective of the manuscript.

Responses 1: I greatly appreciate the reviewer’s positive and encouraging evaluation of my manuscript, as well as the many insightful suggestions. I fully agree that the vomeronasal system represents an important component of chemosensation that should be considered in this review.
In response, I have incorporated a new paragraph addressing the VNO. Specifically, I note that several anoctamin family members, including ANO1, ANO2, and ANO9, are expressed in the VNO, and that functional evidence has been reported for their involvement in Ca²⁺-activated Cl⁻ currents, sensory signal modulation, and behavioral responses. Accordingly, relevant references have been added, and the following paragraph has been included in the revised manuscript (Section 3).

(Lines 241-263).

"In addition to the MOE, the mouse nose contains a more specialized chemosensory organ, the vomeronasal organ (VNO). Vomeronasal sensory neurons (VSNs) are morpho-logically distinct from OSNs in the MOE and utilize different receptor repertoires and signal transduction cascades to detect pheromonal cues [56]. Notably, members of the ANO family are also expressed in the VNO. Both Ano1 and Ano2, which encode Ca²⁺-activated Cl⁻ channels, are localized to the apical microvillar region of the mouse VNO, where they co-localize with Trpc2, the primary ion channel mediating pheromone-evoked Ca²⁺ influx in VSNs [56, 63, 71]. This pattern contrasts with the MOE, where Ano1 and Ano2 are not co-expressed within the same cellular compartment. Consistent with this localization, Ca²⁺-activated Cl⁻ currents have been electrophysiologically recorded in VSNs. Inside-out patch-clamp recordings from dendritic knobs and microvilli confirmed that these currents are activated by intracellular Ca²⁺ [72]. Furthermore, selective deletion of Ano1 abolished Ca²⁺-activated Cl⁻ currents, indicating that ANO1 represents a major component of this conductance in the VNO [72, 73]. In contrast, Ca²⁺-activated Cl⁻ currents remain detectable in Ano2-deficient mice, whereas loss of Ano1 markedly reduces these currents regardless of Ano2 expression, further supporting a dominant contribution of ANO1 to CaCC activity in VSNs [73]. Functionally, loss of Ano1 and Ano2 leads to reduced spontaneous and pheromone-evoked firing, as well as altered firing patterns in VSNs [73, 74]. However, the absence of significant changes in VNO-dependent aggressive behavior indicates that these channels are not strictly essential for pheromone-driven behaviors, but instead play modulatory roles in shaping sensory responses [73]. In addition, Ano9 expression has recently been reported in the VNO, although its specific functional contribution remains to be clarified [62]."

 

Comments 2: Second, although the evolutionary dimension is appropriately presented as a central theme (section 5), its development remains somewhat limited. The references on vertebrates are short and focused on fish, while other vertebrate groups -particularly amphibians, reptiles, and birds- are largely overlooked. Even in fishes, the cited literature is restricted to taste-related functions, overlooking the involvement of anoctamins in the olfactory system, which is a central and highly developed chemosensory modality in this group. This omission is particularly relevant and should be addressed to provide a more complete and accurate overview of anoctamin functio, especially considering that these groups may offer more direct comparative insight into mammalian systems.

Responses 2: I agree with the reviewer’s insightful comment. In response, I have expanded the previously discussion by incorporating additional references covering a broader range of vertebrate groups, including amphibians, reptiles, and birds, thereby strengthening the evolutionary perspective. Specifically, I have added descriptions of their respective olfactory systems, the involvement of anoctamin family members, and transcriptomic evidence where available, to avoid overlooking the role of anoctamins in olfaction. Although studies in some of these groups remain limited, I have included the available evidence to provide a more balanced and comprehensive overview.

In addition, while earlier sections of the manuscript primarily focus on the roles of ANO/TMC channels in mammalian olfaction and taste, I have clarified that this section (Section 5) emphasizes their broader polymodal sensory functions and evolutionary expansion. To avoid potential confusion, I have added a clarifying statement at the beginning of this section.

(Lines 451-457)

“In teleost fish, ciliated olfactory receptor neurons are present and utilize a cAMP-dependent signaling pathway in response to odorant stimulation [104, 105]. In addition, studies in rainbow trout have shown that amino acid–evoked inward currents are partly mediated by Ca²⁺-activated Cl⁻ conductance, suggesting that Cl⁻-dependent signal amplification mechanisms are functionally conserved in fish olfactory neurons [105]. Beyond sensory transduction, anoctamin channels have also been implicated in osmoregulatory processes in aquatic vertebrates.”

(Lines 466-482)

“In amphibians, Ca²⁺-activated Cl⁻ conductance has been directly demonstrated in the cilia of frog OSNs using patch-clamp recordings [6]. In contrast, electrophysiological and functional studies of olfactory transduction in reptiles remain limited; however, in species such as snakes, both the main olfactory system and the accessory olfactory system are well developed and are mediated by OR and V1R/V2R receptors, respectively. Transcriptomic analyses have revealed the expression of key GPCR–cAMP signaling components, including Adcy3, suggesting that the fundamental olfactory transduction cascade is likely conserved [109]. Nevertheless, the expression and functional roles of anoctamin family channels in reptiles remain largely unknown.

In contrast, although birds are generally considered to lack a functional VNO [110], recent studies have demonstrated that Or/Taar, Gnal, Adcy3, and Cng channel subunits, along with Ano2 mRNA, are expressed in the olfactory epithelium of Gallus gallus at the qPCR level. These findings suggest that the canonical olfactory transduction cascade is conserved in birds [111]. Interestingly, ANO9 cloned from the Gallus gallus was reported to lack functional activity in electrophysiological assays, implying potential functional divergence of anoctamin family members or alternative signaling mechanisms in the avian olfactory system [62].”

(Lines 399-407)

“In mammals, the roles of ANO/TMC superfamily members in olfactory and gustatory systems have been relatively well characterized, whereas accumulating evidence suggests that these channels function as polymodal sensory effectors across diverse biological contexts. Members of the ANO/TMC superfamily are conserved across a wide phylogenetic range, from protists and invertebrates to mammals, and are thought to represent ancient molecular solutions for converting environmental stimuli into electrical signals (Figure 3, Table 2). Recent work on genetically tractable model organisms, including C. elegans and Drosophila melanogaster, has suggested that these channels operate across multiple sensory modalities.”

 

Comments 3: Overall, this is a high-quality and timely review, and the points raised above are intended to reinforce its already strong scientific contribution.

Minor issues:

Lines 46-53: The description of the signaling cascade is clear and accurate. However, you might consider explicitly mentioning adenylyl cyclase III (ACIII), as it plays a central role in linking Golf activation to cAMP production in olfactory sensory neurons.

Responses 3: I thank the reviewer for this helpful suggestion. I have revised the manuscript accordingly to explicitly include adenylyl cyclase III (ACIII) in the signaling cascade. The following sentence has been added (Lines 46–53):

(Lines 46-49)

“Odorant binding to ORs activates the olfactory-specific G protein (Golf), which in turn stimulates adenylyl cyclase III (ACIII). This enzymatic activation leads to elevated intracellular cAMP levels and triggers the opening of cyclic nucleotide–gated (CNG) cation channels.”

Comments 4: Line 112: It should be specified that the term “TMEM” refers to “transmembrane protein” and represents an initial gene annotation rather than a functional classification. This clarification would be useful for readers unfamiliar with the nomenclature.

Responses 4:

I thank the reviewer for this helpful comment. To clarify the nomenclature, I have revised the beginning of Section 2 to explicitly state the origin of the term “TMEM.” The following sentence has been added:

(Lines 113-115)

“The anoctamin (ANO) family, also known as the TMEM16 (transmembrane protein 16) family—a nomenclature originally based on its predicted protein structure rather than function—was established following the identification of ANO1 (TMEM16A) as a Ca²⁺--activated Cl^- channel in 2008”

 

Comments 5: Line 127: “These spores have been implicated in the regulation of phosphatidylserine”

There is a typo in this sentence (“spores”), which should likely read “pore”, “proteins” or “channels.”

Responses 5: I appreciate the reviewer for pointing out this error. I have corrected “spores” to “proteins” in the revised manuscript.

(Lines 157-160)

“In addition to forming the ion-conducting pore, these proteins have been implicated in the regulation of phosphatidylserine (PS) at stereociliary membranes, suggesting a dual role in ion conduction and membrane lipid homeostasis [37].”

 

Comments 6: Figure 1: In the left panel, you may consider indicating the position of sustentacular cell nuclei in the apical half of the olfactory epithelium to improve anatomical accuracy.

Responses 6: I thank the reviewer for this helpful suggestion. I have revised Figure 1 accordingly by positioning the nuclei of sustentacular cells in the apical half of the olfactory epithelium to improve anatomical accuracy.

 

Comments 7: Line 213: Gallus gallus should be italicized.

Responses 7: I appreciate the reviewer’s comment. I have revised the manuscript to ensure that Gallus gallus, as well as other species names, are properly italicized throughout.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The author has addressed all comments in a thorough and systematic manner. In my opinion, what was already an excellent manuscript has improved considerably.

Author Response

Comment: The author has addressed all comments in a thorough and systematic manner. In my opinion, what was already an excellent manuscript has improved considerably.

Response: I thank the reviewer for the positive and encouraging feedback.