Bitter and sweet receptors (T2Rs and T1Rs) are expressed in many extra-oral tissues including upper and lower airways. To investigate if bitter tastants and artificial sweeteners could activate physiological responses in tracheal epithelial cells we performed confocal Ca 2+ imaging recordings on acute tracheal slices. We stimulated the cells with denatonium benzoate, a T2R agonist, and with the artificial sweeteners sucralose, saccharin and acesulfame-K. To test cell viability we measured responses to ATP. We found that 39% of the epithelial cells responding to ATP also responded to bitter stimulation with denatonium benzoate. Moreover, artificial sweeteners activated different percentages of the cells, ranging from 5% for sucralose to 26% for saccharin, and 27% for acesulfame-K. By using carbenoxolone, a gap junction blocker, we excluded that responses were mainly mediated by Ca 2+ waves through cell-to-cell junctions. Pharmacological experiments showed that both denatonium and artificial sweeteners induced a PLC-mediated release of Ca 2+ from internal stores. In addition, bitter tastants and artificial sweeteners activated a partially overlapping subpopulation of tracheal epithelial cells. Our results provide new evidence that a subset of ATP-responsive tracheal epithelial cells from rat are activated by both bitter tastants and artificial sweeteners.

Stomatin-like protein-3 (STOML3) is an integral membrane protein expressed in the cilia of olfactory sensory neurons, but its functional role in this cell type has never been addressed. STOML3 is also expressed in dorsal root ganglia neurons, where it has been shown to be required for normal touch sensation. Here, we extended previous results indicating that STOML3 is mainly expressed in the knob and proximal cilia of olfactory sensory neurons. We additionally showed that mice lacking STOML3 have a morphologically normal olfactory epithelium. Due to its presence in the cilia, together with known olfactory transduction components, we hypothesized that STOML3 could be involved in modulating odorant responses in olfactory sensory neurons. To investigate the functional role of STOML3, we performed loose patch recordings from wild type and Stoml3 KO olfactory sensory neurons. We found that spontaneous mean firing activity was lower with additional shift in interspike intervals distributions in Stoml3 KOs compared to wild type neurons. Moreover, the firing activity in response to stimuli was reduced both in spike number and duration in neurons lacking STOML3 compared to wildtype neurons. Control experiments suggested that the primary deficit in neurons lacking STOML3 was at the level of transduction and not at the level of action potential generation. We conclude that STOML3 has a physiological role in olfaction, being required for normal sensory encoding by olfactory sensory neurons. Significance Statement Olfactory transduction comprises a series of well-characterized molecular steps that take place in the cilia of olfactory sensory neurons (OSNs) terminating in action potential firing. Here, we introduce a possible new player: stomatin-like protein 3 (STOML3). Indeed, STOML3 is localized in olfactory cilia, and we show that STOML3 plays a role in OSN physiology. First, it allows OSNs to broaden the possible frequency range of their spontaneous activity. Second, STOML3 modulates odorant-evoked action potential firing by regulating both the number of spikes and response duration. These new findings call for a reconsideration of the patterns of the peripheral coding of sensory stimuli. MATERIALS AND METHODS Animals Mice were handled in accordance with the Italian Guidelines for the Use of Laboratory Animals and the European Union guidelines on animal research according to a protocol approved by the ethics committee [Author Institute]. Experiments were performed on tissues from C57BL/6 WT and Stoml3 KO mice of either sex (Wetzel et al., 2007). Immunohistochemistry The head containing the nasal cavity was fixed in 4% paraformaldehyde in PBS at pH 7.4 for 4 hours at 4°C. After fixing, the heads of the mice were incubated in 0.5 M EDTA for 2 days. The tissues were cryoprotected by incubation in 30% sucrose in PBS at pH 7.4 overnight. The tissue was immersed in cryostat embedding medium (BioOptica) and immediately frozen at −80°C. Coronal sections (16-18 μm) were cut on a cryostat and mounted on Superfrost Plus Adhesion Mic...

Taste transduction occurs in taste buds in the tongue epithelium -The Ca 2+ -activated Clchannels TMEM16A and TMEM16B play relevant physiological roles in several sensory systems -Here, we report that TMEM16A, but not TMEM16B, is expressed in the apical part of taste buds -Large Ca 2+ -activated Cl − currents blocked by Ani-9, a selective inhibitor of TMEM16A, are measured in type I, but not in type II or III taste cells -ATP indirectly activates Ca 2+ -activated Clcurrents in type I cells through TMEM16A channels -These results indicate that TMEM16A is functional in type I taste cells and contribute to understanding the largely unknown physiological roles of these cells

The mouse vomeronasal system controls several social behaviors. Pheromones and other social cues are detected by sensory neurons in the vomeronasal organ. Stimuli activate a transduction cascade that leads to membrane potential depolarization, increase in cytosolic Ca 2+ level, and increased firing. The Ca 2+ -activated chloride channels TMEM16A and TMEM16B are co-expressed within microvilli of vomeronasal neurons, but their physiological role remains elusive. Here, we investigate the contribution of each of these channels to vomeronasal neuron firing activity by comparing wild-type and knockout mice. Performing loose-patch recordings from neurons in acute vomeronasal organ slices, we show that spontaneous activity is modified by Tmem16a knockout, indicating that TMEM16A, but not TMEM16B, is active under basal conditions. Upon exposure to diluted urine, a rich source of mouse pheromones, we observe significant changes in activity. Vomeronasal sensory neurons from Tmem16a cKO and Tmem16b KO mice show shorter interspike intervals compared to WT mice, indicating that both TMEM16A and TMEM16B modulate the firing pattern of pheromone-evoked activity in VSNs. Significance StatementVomeronasal sensory neurons express two Ca 2+ -activated chloride channels TMEM16A and TMEM16B, however their physiological role is still unclear. Using a loss of function approach, we found that TMEM16A modulates the pattern of VSN spontaneous spike activity, while TMEM16A and TMEM16B reduced the instant frequency of pheromone-evoked activity. These new findings call for a reconsideration of the patterns of the peripheral coding of sensory stimuli.

Recent data show that Stomatin-like protein 3 (STOML3), a member of the stomatin-domain family, is expressed in the olfactory sensory neurons (OSNs) where it modulates both spontaneous and evoked action potential firing. The protein family is constituted by other 4 members (besides STOML3): STOM, STOML1, STOML2 and podocin. Interestingly, STOML3 with STOM and STOML1 are expressed in other peripheral sensory neurons: dorsal root ganglia. In here, they functionally interact and modulate the activity of the mechanosensitive Piezo channels and members of the ASIC family. Therefore, we investigated whether STOM and STOML1 are expressed together with STOML3 in the OSNs and whether they could interact. We found that all three are indeed expressed in ONSs, although STOML1 at very low level. STOM and STOML3 share a similar expression pattern and STOML3 is necessary for STOM to properly localize to OSN cilia. In addition, we extended our investigation to podocin and STOML2, and while the former is not expressed in the olfactory system, the latter showed a peculiar expression pattern in multiple cell types. In summary, we provided a first complete description of stomatin-domain protein family in the olfactory system, highlighting the precise compartmentalization, possible interactions and, finally, their functional implications.