InteractiveFly: GeneBrief

Transmembrane protein 63: Biological Overview | References

Gene name - Transmembrane protein 63

Synonyms -

Cytological map position - 44A4-44A4

Function - channel

Keywords - expressed in multidendritic neurons in the fly tongue (proboscis) - mechanically activated Ca2+ channel - confers the ability to discriminate particle sizes in food and uses this information to decide whether a food is appealing - required for humidity response in Drosophila olfactory sensory neurons

Symbol - Tmem63

FlyBase ID: FBgn0033259

Genetic map position - chr2R:8,098,684-8,102,306

NCBI classification - RSN1_7TM: Calcium-dependent channel, 7TM region, putative phosphate

Cellular location - surface transmembrane

NCBI links: EntrezGene, Nucleotide, Protein

GENE orthologs: Biolitmine

An animal's decision to accept or reject a prospective food is based only, in part, on its chemical composition. Palatability is also greatly influenced by textural features including smoothness versus grittiness, which is influenced by particle sizes. This study demonstrates that Drosophila melanogaster is endowed with the ability to discriminate particle sizes in food and uses this information to decide whether a food is appealing. The decision depends on a mechanically activated channel, OSCA/TMEM63, which is conserved from plants to humans. tmem63 is expressed in a multidendritic neuron (md-L) in the fly tongue (proboscis). Loss of tmem63 impairs the activation of md-L by mechanical stimuli and the ability to choose food based on particle size. These findings reveal the first role for this evolutionarily conserved, mechanically activated TMEM63 channel in an animal and provide an explanation of how flies can sense and behaviorally respond to the texture of food provided by particles (Li, 2021).

Food selection is among the most critical and ancient behaviors exhibited by animals. Multiple classes of chemosensory receptors have been defined in flies, mice, and other animals that contribute to the discrimination of nutritious from noxious foods. In humans and other mammals, the textural features of food also have a major impact on food appeal. The texture of food is influenced by its hardness and viscosity, as well as the size of food particles. These features comprise the mouthfeel of food and illustrate the importance of somatosensation for assessing palatability. Nevertheless, the receptors that detect food texture are unknown in mammals. Recently, several groups have begun to exploit the fruit fly, Drosophila melanogaster, to unravel the receptors and neurons that allow animals to evaluate palatability based on physical features of food including hardness viscosity and temperature (Li, 2021).

For humans, one of the key physical features of food that impacts food appeal is particle size. For example, ice crystals of 10-20 μm cause ice cream to be perceived as very smooth, whereas larger crystals of 50 μm confer a perception of grittiness Particle sizes in chocolate also influence the evaluation of desirability. In experiments with human volunteers, the addition of garnet, polyethylene, or mica particles of different sizes to flavored syrups impacts food appeal (Li, 2021).

The influence of particle size on food appeal might be conserved throughout the animal kingdom. Drosophila feed on many types of fruit and are especially attracted to ripened and decaying fruit. The size of starch granules in mango and kiwifruit range from ~10-15 μm. In addition, decaying fruit is laden with yeast, which is also a food source for flies. The size of budding yeast is ~8-10 μm. Thus, flies might prefer foods with particle sizes that are typically found in fruits and budding yeast. However, the underlying molecular and cellular basis through which the size of particles in food alters acceptance or rejection remains unexplored (Li, 2021).

This study established Drosophila as an animal model for revealing the mechanism through which particle size influences food attraction. Flies prefer sucrose-containing food with particles of a particular size. This ability to discriminate between foods based on particle size depends on TMEM63, a member of a recently discovered family of mechanically activated channels that are conserved from plants (OSCA) to animals including humans (TMEM63). OSCA/TMEM63 represents the only protein family shown to be mechanically gated ion channels in both plants and animals. However, their roles in animals are unknown. This study found that the role of TMEM63 in sensing particles in food is dependent on its expression in a pair of mechanically activated multidendritic neurons (md-L) in the labellum, which is the fly's major taste organ. Although TMEM63 is required for sensing small particles in food, which exert subtle mechanical forces, it is dispensable for detecting other textural features of food such as hardness and viscosity, which cause stronger mechanical interactions with the labellum. Another mechanosensitive channel, TMC, is also expressed in md-L neurons. However, in contrast to TMEM63, the TMC protein functions in detecting the hardness and viscosity of food (Zhang, 2016) but is dispensable for particle sensation. These results reveal the first molecular and cellular underpinning for sensing particle sizes in food and a requirement for a TMEM63 channel in an animal (Li, 2021).

Mouthfeel results from the physical features of food including hardness, viscosity, and particle size. The human gustatory experience is influenced by particle size, because it impacts on whether a food is smooth or gritty. However, the mechanisms are unknown. This study established that flies are also able to discriminate between foods based on particle size. Their favorite size was 9.2 μm, which falls within the size range of the budding yeast in their diet and the starch granules in many of the fruit that they consume. The width of their pharynx is ~15 μm. Thus, there might be a selection for a preference for particles <15 μm. However, the width of the pharynx may not define the upper size limit of particles. Although flies do not chew food, they can process food extra-orally by expelling enzymes from the proboscis (Li, 2021).

The sensation of particle size in food is a type of somatosensation, which is proposed to depend on the detection of particles physically interacting with gustatory sensilla. Deflection of sensilla then activates a mechanically activated channel in md-L neurons that is critical for particle sensation. In support of this model, particles in food cause small deflections of gustatory sensilla. When the cuticle was depressed with a probe, thereby resulting in small angle deflections similar to those produced by particles, the md-L neurons displayed increased GCaMP6f responses and action potentials (Li, 2021).

The mechanically activated channel is TMEM63, because loss of this channel impairs food discrimination based on particle size. TMEM63 is expressed in md-L neurons, and mutation of tmem63 disrupts activation of md-L neurons by deflection of taste sensilla. Another mechanically activated channel, TMC, is also required in md-L neurons but for different aspects of texture sensation: hardness and viscosity. The overall structure and dimeric composition of OSCA/TMEM63 channels is similar to the structure and dimeric architecture of TMCs. Thus, this common structure may be well suited for sensing small physical differences in food texture (Li, 2021).

Despite the structural similarities and co-expression of TMEM63 and TMC in md-L neurons, it is suggested that it is unlikely that TMEM63 heterodimerizes with TMC. Expression of Drosophila TMEM63 in vitro is sufficient to produce a mechanically activated channel. Moreover, the phenotypes of tmem63 and tmc mutants are distinct. Although loss of tmem63 disrupts particle size sensation, the mutants can still discriminate between sugary foods with differences in hardness and viscosity. Conversely, mutation of tmc impairs the ability to detect the hardness and viscosity of sucrose-containing food but does not alter the behavioral preference for sucrose with particles (Li, 2021).

The observation that flies can discern different textural features of food, such as hardness from particle size, raises questions concerning the coding mechanism. This issue is particularly provocative given that the md-L neuron functions in the detection of these distinct textural features. It is suggested that one potential explanation is that md-L, as well as the MSNs that are associated with each taste hair, are all required for the detection of food hardness and viscosity, whereas only the md-L neuron functions in particle size discrimination. Indeed, hMSNs also contribute to food hardness detection (Li, 2021).

Alternatively, but not mutually exclusive, is that TMEM63 and TMC function in sensing different levels of deflection of gustatory hairs. The responses of md-L neurons to smaller deflections of a single sensillum are eliminated by the tmem63 mutation but not impacted by the tmc mutation. Conversely, the responses to larger deflections are impaired by the tmc mutation but not the tmem63 mutation. This suggests that TMC is required for larger mechanical stimulation caused by hard and viscous food, whereas TMEM63 is critical for more subtle mechanical stimulation that mimics the effects of particles in food. Indeed, TMEM63 could even provide md-L neurons with sensitivity to the smallest particles tested (1 μm), because 1 μm particles in food elicit small deflections of sensilla that are intermediate between those produced by the steps 1 and 2 cuticle depressions that induce action potentials in control, but not tmem63 mutants (Li, 2021).

It is also plausible that there are differences in the spatial and temporal deflections due to hardness/viscosity versus particles. Particles would cause only some adjacent sensilla in any group to be transiently deflected at any given time as they are contacted by the particles. In contrast, hardness/viscosity would result in neighboring sensilla (e.g., L-type) to be simultaneously deflected. In addition, TMEM63 may be more rapidly inactivated than TMC, enabling TMEM63 to be better suited to sense transient deflections from particles. However, a direct comparison between TMEM63 and TMC is not possible because most TMCs, including fly TMC, have been refractory to biophysical analyses in vitro (Li, 2021).

The finding that tmem63 is expressed in many GRNs raises future questions as to the roles of TMEM63 in GRNs. This study found that the GRNs do not function in particle sensation, and mutation of tmem63 does not cause defects in sensation of sucrose, caffeine, a carboxylic acid, or NaCl. Finally, this and a previous study on TMC7 raise questions as to whether TMEM63 and TMC function in food texture sensation in mammals (Li, 2021).

Humidity response in Drosophila olfactory sensory neurons requires the mechanosensitive channel TMEM63

Birds, reptiles and insects have the ability to discriminate humidity levels that influence their survival and geographic distribution. Insects are particularly susceptible to humidity changes due to high surface area to volume ratios, but it remains unclear how humidity sensors transduce humidity signals. This study identified Or42b-expressing olfactory sensory neurons, which are required for moisture attraction in Drosophila. The sensilla housing Or42b neurons show cuticular deformations upon moist air stimuli, indicating a conversion of humidity into mechanical force. Accordingly, this study found Or42b neurons directly respond to humidity changes and rely on the mechanosensitive ion channel TMEM63 to mediate humidity sensing (hygrosensation). Expressing human TMEM63B in Tmem63 mutant flies rescued their defective phenotype in moisture attraction, demonstrating functional conservation. Thus, these results reveal a role of Tmem63 in hygrosensation and support the strategy to detect humidity by transforming it into a mechanical stimulus, which is unique in sensory transduction (Li, 2022).

In existing concepts explaining the mechanism of hygrosensory transduction, humidity receptors are either mechanosensitive or thermosensitive molecules. Previous studies have revealed the essential roles of IR40a, IR68a, IR93a and IR25a in hygrosensation, but due to the difficulty of function analysis of theses molecules in heterologous expression system, how they respond to humidity still need further investigation. By neuron ablation methods and mutant analysis, this study revealed a mechanosensory pathway that specifically contributes to 70% relative humidity (RH)-induced attraction. The data provide structural and molecular evidence supporting the concept that humidity changes can be transformed into mechanical cues which evoke hygrosensory inputs via mechanosensitive molecules. Moisture changes the cuticular curvature of ab1 sensilla, which may alter the membrane tension of associated sensory cilia expressing the mechanosensitive channel TMEM63. Previous in vitro studies have shown that TMEM63/OSCAs are sensitive to both osmotic stress and negative pressure (Zhang, 2018; Murthy, 2018), the present study shows that Drosophila TMEM63 confers stretch-activated currents when transfected into S2 cells. Thus DmTMEM63 might be a potential primary molecular receptor that can sense the membrane deformation resulting from humidity changes, although it could not be ruled out that TMEM63 could mediate humidity perception by sensing osmotic pressure changes. Intriguingly, this study reveals an evolutionary strategy to detect the obscure humidity or water vapor by converting it into a defined mechanical deformation, which is unique in the sensory system (Li, 2022).

Moreover, distinct Drosophila species show diverse humidity preferences and therefore have different geographical distributions, for example, experiments showed that rainforest flies prefer a high level of humidity while desert-dwelling flies prefer 20% RH. Apart from insects, other poikilotherms also display similar humidity-related geographical distributions. The current findings may provide a clue to study how distinct animal species evolve to select their native habitats (Li, 2022).

The data show that TMEM63 mediates hygrosensation in Or42b neurons, a neuron group for food odor detection in Drosophila, suggesting an early integration of hygrosensory and olfactory inputs. This is not surprising since insects rely on multisensory integration for locating vital resources. Disease-transmitting mosquitoes also exploit multimodal cues, such as CO2, odors, body heat and moisture, for finding and selecting potential hosts. Molecular receptors for chemicals and temperature have been unraveled in mosquitoes, but the receptors that incorporate humidity into multimodal sensory inputs have remained elusive. It will be interesting to test if Tmem63 functions as a humidity sensor in mosquitoes and guide host attraction by detecting the elevated humidity levels at the proximity of the host, which might provide insights into the molecular underpinnings of host approach behavior in disease vectors (Li, 2022).

The TMEM63 proteins have been found to be evolutionarily conserved from Drosophila to mammals (Murthy, 2018; Hou, 2014; Yuan, 2014), but research on their physiological function is still in its infancy. This study showed that human TMEM63B can substitute for Drosophila TMEM63 to mediate moisture attraction behavior, an evolutionary solution to satisfy the internal needs for water. Further studies are necessary to determine whether mammalian Tmem63 genes function in behaviors related to osmotic regulation or water homeostasis (Li, 2022).

Functions of Tmem63 orthologs in other species

Distinct functions of TMC channels: a comparative overview

In the past two decades, transmembrane channel-like (TMC) proteins have attracted a significant amount of research interest, because mutations of Tmc1 lead to hereditary deafness. As evolutionarily conserved membrane proteins, TMC proteins are widely involved in diverse sensorimotor functions of many species, such as hearing, chemosensation, egg laying, and food texture detection. Interestingly, recent structural and physiological studies suggest that TMC channels may share a similar membrane topology with the Ca(2+)-activated Cl(-) channel TMEM16 and the mechanically activated OSCA1.2/TMEM63 channel. Namely, these channels form dimers and each subunit consists of ten transmembrane segments. Despite this important structural insight, a key question remains: what is the gating mechanism of TMC channels? The major technical hurdle to answer this question is that the reconstitution of TMC proteins as functional ion channels has been challenging in mammalian heterologous systems. Since TMC channels are conserved across taxa, genetic studies of TMC channels in model organisms such as C. elegans, Drosophila, and zebrafish may provide critical information on the physiological function and regulation of TMCs. This study presents a comparative overview on the diverse functions of TMC channels in different species (Yue, 2019).

Overexpression of Osmosensitive Ca2+-Permeable Channel TMEM63B Promotes Migration in HEK293T Cells

The recent discovery of the osmosensitive calcium (Ca2+) channel OSCA has revealed the potential mechanism by which plant cells sense diverse stimuli. Osmosensory transporters and mechanosensitive channels can detect and respond to osmotic shifts that play an important role in active cell homeostasis. Members of the TMEM63 family of proteins are described as the closest homologues of OSCAs. This study characterized TMEM63B, a mammalian homologue of OSCAs, recently classified as mechanosensitive. In HEK293T cells, TMEM63B localizes to the plasma membrane and is associated with F-actin. This Ca2+-permeable channel specifically induces Ca2+ influx across the membrane in response to extracellular Ca2+ concentration and hyperosmolarity. In addition, overexpression of TMEM63B in HEK293T cells significantly enhanced cell migration and wound healing. The link between Ca2+ osmosensitivity and cell migration might help to establish TMEM63B’s pathogenesis, for example, in cancer in which it is frequently overexpressed (Marques, 2019).

Cryo-EM structure of the mechanically activated ion channel OSCA1.2

Mechanically activated ion channels underlie touch, hearing, shear-stress sensing, and response to turgor pressure. OSCA/TMEM63s are a newly-identified family of eukaryotic mechanically activated ion channels opened by membrane tension. The structural underpinnings of OSCA/TMEM63 function are not explored. This study elucidated high resolution cryo-electron microscopy structures of OSCA1.2, revealing a dimeric architecture containing eleven transmembrane helices per subunit and surprising topological similarities to TMEM16 proteins. The ion permeation pathway was located within each subunit by demonstrating that a conserved acidic residue is a determinant of channel conductance. Molecular dynamics simulations reveal membrane interactions, suggesting the role of lipids in OSCA1.2 gating. These results lay a foundation to decipher how the structural organization of OSCA/TMEM63 is suited for their roles as MA ion channels (Jojoa-Cruz, 2018).

Structure of the mechanosensitive OSCA channels

Mechanosensitive ion channels convert mechanical stimuli into a flow of ions. These channels are widely distributed from bacteria to higher plants and humans, and are involved in many crucial physiological processes. This study shows that two members of the OSCA protein family in Arabidopsis thaliana, namely AtOSCA1.1 and AtOSCA3.1, belong to a new class of mechanosensitive ion channels. The structure of the AtOSCA1.1 channel was solved at 3.5-Å resolution and AtOSCA3.1 at 4.8-Å resolution by cryo-electron microscopy. OSCA channels are symmetric dimers that are mediated by cytosolic inter-subunit interactions. Strikingly, they have structural similarity to the mammalian TMEM16 family proteins. This structural analysis accompanied with electrophysiological studies identifies the ion permeation pathway within each subunit and suggests a conformational change model for activation (Zhang, 2018).

OSCA/TMEM63 are an evolutionarily conserved family of mechanically activated ion channels

Mechanically activated (MA) ion channels convert physical forces into electrical signals, and are essential for eukaryotic physiology. Despite their importance, few bona-fide MA channels have been described in plants and animals. This study shows that various members of the OSCA and TMEM63 family of proteins from plants, flies, and mammals confer mechanosensitivity to naive cells. This study conclusively demonstrates that OSCA1.2, one of the Arabidopsis thaliana OSCA proteins, is an inherently mechanosensitive, pore-forming ion channel. The results suggest that OSCA/TMEM63 proteins are the largest family of MA ion channels identified, and are conserved across eukaryotes. These findings will enable studies to gain deep insight into molecular mechanisms of MA channel gating, and will facilitate a better understanding of mechanosensory processes in vivo across plants and animals (Murthy, 2018).

Co-expression of mouse TMEM63A, TMEM63B and TMEM63C confers hyperosmolarity activated ion currents in HEK293 cells

Osmoreception is essential for systemic osmoregulation, a process to stabilize the tonicity and volume of the extracellular fluid through regulating the ingestive behaviour, sympathetic outflow and renal function. The sensation of osmotic changes by osmoreceptor neurons is mediated by ion channels that detect the change of osmolarity in extracellular fluid. However, the molecular identity of these channels remains mysterious. AtCSC1and OSCA1, two closely related paralogues from Arabidopsis, have been demonstrated to form hyperosmolarity activated ion channels, which makes their mammalian orthologues-the members of TMEM63 proteins, possible candidates for osmoreceptor transduction channel. To test this possibility, in this study the cDNAs of all the three members of the mouse TMEM63 family, TMEM63A, TMEM63B and TMEM63C were cloned from the mRNA from mouse brain. When all of the three subtypes of TMEM63 proteins were co-expressed in HEK293 cells, membrane currents evoked by hypertonic stimulation in these cells were recorded. However, the cells expressing the combinations of any two subtypes of TMEM63 proteins could not exhibit any hyperosmolarity evoked currents. Thus, all the three members of TMEM63 proteins are required to constitute a hyperosmolarity activated ion channel. It is proposed that the TMEM63 proteins may serve as an osmolarity sensitive ion channel for the osmoreception (Zhao, 2016).

OSCA1 mediates osmotic-stress-evoked Ca2+ increases vital for osmosensing in Arabidopsis

Water is crucial to plant growth and development. Environmental water deficiency triggers an osmotic stress signalling cascade, which induces short-term cellular responses to reduce water loss and long-term responses to remodel the transcriptional network and physiological and developmental processes. Several signalling components that have been identified by extensive genetic screens for altered sensitivities to osmotic stress seem to function downstream of the perception of osmotic stress. It is known that hyperosmolality and various other stimuli trigger increases in cytosolic free calcium concentration ([Ca2+]i). Considering that in bacteria and animals osmosensing Ca2+ channels serve as osmosensors hyperosmolality-induced [Ca2+]i increases have been widely speculated to be involved in osmosensing in plants However, the molecular nature of corresponding Ca2+ channels remain unclear. This study describes a hyperosmolality-gated calcium-permeable channel and its function in osmosensing in plants. Using calcium-imaging-based unbiased forward genetic screens, Arabidopsis mutants were isolated that exhibit low hyperosmolality-induced [Ca2+]i increases. These mutants were rescreened for their cellular, physiological and developmental responses to osmotic stress, and those with clear combined phenotypes were selected for further physical mapping. One of the mutants, reduced hyperosmolality-induced [Ca2+]i increase 1 (osca1), displays impaired osmotic Ca2+ signalling in guard cells and root cells, and attenuated water transpiration regulation and root growth in response to osmotic stress. OSCA1 is identified as a previously unknown plasma membrane protein and forms hyperosmolality-gated calcium-permeable channels, revealing that OSCA1 may be an osmosensor. OSCA1 represents a channel responsible for [Ca2+]i increases induced by a stimulus in plants, opening up new avenues for studying Ca2+ machineries for other stimuli and providing potential molecular genetic targets for engineering drought-resistant crops (Yuan, 2014).


Search PubMed for articles about Drosophila Tmem63

Hou, C., Tian, W., Kleist, T., He, K., Garcia, V., Bai, F., Hao, Y., Luan, S. and Li, L. (2014). DUF221 proteins are a family of osmosensitive calcium-permeable cation channels conserved across eukaryotes. Cell Res 24(5): 632-635. PubMed ID: 24503647

Jojoa-Cruz, S., Saotome, K., Murthy, S. E., Tsui, C. C. A., Sansom, M. S., Patapoutian, A. and Ward, A. B. (2018). Cryo-EM structure of the mechanically activated ion channel OSCA1.2. Elife 7. PubMed ID: 30382939

Li, Q. and Montell, C. (2021). Mechanism for food texture preference based on grittiness. Curr Biol. PubMed ID: 33657409

Li, S., Li, B., Gao, L., Wang, J. and Yan, Z. (2022). Humidity response in Drosophila olfactory sensory neurons requires the mechanosensitive channel TMEM63. Nat Commun 13(1): 3814. PubMed ID: 35780140

Marques, M. C., Albuquerque, I. S., Vaz, S. H. and Bernardes, G. J. L. (2019). Overexpression of Osmosensitive Ca(2+)-Permeable Channel TMEM63B Promotes Migration in HEK293T Cells. Biochemistry 58(26): 2861-2866. PubMed ID: 31243992

Murthy, S. E., Dubin, A. E., Whitwam, T., Jojoa-Cruz, S., Cahalan, S. M., Mousavi, S. A. R., Ward, A. B. and Patapoutian, A. (2018). OSCA/TMEM63 are an Evolutionarily Conserved Family of Mechanically Activated Ion Channels. Elife 7. PubMed ID: 30382938

Yuan, F., Yang, H., Xue, Y., Kong, D., Ye, R., Li, C., Zhang, J., Theprungsirikul, L., Shrift, T., Krichilsky, B., Johnson, D. M., Swift, G. B., He, Y., Siedow, J. N. and Pei, Z. M. (2014). OSCA1 mediates osmotic-stress-evoked Ca2+ increases vital for osmosensing in Arabidopsis. Nature 514(7522): 367-371. PubMed ID: 25162526

Yue, X., Sheng, Y., Kang, L. and Xiao, R. (2019). Distinct functions of TMC channels: a comparative overview. Cell Mol Life Sci 76(21): 4221-4232. PubMed ID: 31584127

Zhang, Y. V., Aikin, T. J., Li, Z. and Montell, C. (2016). The basis of food texture sensation in Drosophila. Neuron 91(4): 863-877. PubMed ID: 27478019

Zhang, M., Wang, D., Kang, Y., Wu, J. X., Yao, F., Pan, C., Yan, Z., Song, C. and Chen, L. (2018). Structure of the mechanosensitive OSCA channels. Nat Struct Mol Biol 25(9): 850-858. PubMed ID: 30190597

Zhao, X., Yan, X., Liu, Y., Zhang, P. and Ni, X. (2016). Co-expression of mouse TMEM63A, TMEM63B and TMEM63C confers hyperosmolarity activated ion currents in HEK293 cells. Cell Biochem Funct 34(4): 238-241. PubMed ID: 27045885

Biological Overview

date revised: 26 July 2022

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