Calcium Activated Chloride Channels In Olfactory Transduction

Ca2+-activated Cl \u304 channels are an important component of olfactory transduction. Odorant binding to odorant receptors in the cilia of olfactory sensory neurons (OSNs) leads to an increase of intraciliary Ca2+ concentration by Ca2+ entry through cyclic nucleotide-gated channels. Ca2+ activates a Cl \u304 channel that leads to an efflux of Cl \u304 from the cilia, contributing to the amplification of the OSN depolarization. The molecular identity of this Cl \u304 channel remains elusive. Recent evidences have indicated that bestrophins are able to form Ca2+-activated Cl \u304 channels channels in heterologous systems. Immunohistochemistry revealed that mBest2 was expressed on the cilia of OSNs, the site of olfactory transduction, and co-localized with the main subunit of cyclic nucleotide-gated channels, CNGA2. We performed a functional comparison of the properties of Ca2+-activated Cl \u304 channels from native channels expressed in dendritic knob/cilia of mouse OSNs with those induced by heterologous expression of mBest2 in HEK-293 cells. Even if the two channels did not display identical characteristics, they have many similar features such as the same anion permeability, the Ca2+ sensitivity in micromolar range and the same side-specific blockage of the two Cl \u304 channel blockers commonly used to inhibit the odorant-induced Ca2+-activated Cl \u304 channels in OSNs, niflumic acid and 4-acetamido-4\u2019-isothiocyanato-stilben-2,2\u2019-disulfonate (SITS). However electroolfactogram recording from mBest2 null mice showed a normal sensitivity to odorant stimulation. Therefore mBest2 is a good candidate for being a molecular component of the olfactory Ca2+-activated Cl \u304 channels but its precise role in olfactory transduction remains to be clarified

Ca2+-Activated Chloride Channels and Phospholipid Scramblases

none2no: The functional characterization of the TMEM16 protein family unexpectedly brought together two different research fields in membrane biology: anion channel and membrane lipid organization [...].openPifferi S.; Boccaccio A.Pifferi, S.; Boccaccio, A

The long tale of the calcium activated Cl(-) Channels in olfactory transduction

Ca(2+)-activated Cl(-) currents have been implicated in many cellular processes in different cells, but for many years, their molecular identity remained unknown. Particularly intriguing are Ca(2+)-activated Cl(-) currents in olfactory transduction, first described in the early 90s. Well characterized electrophysiologically, they carry most of the odorant-induced receptor current in the cilia of olfactory sensory neurons (OSNs). After many attempts to determine their molecular identity, TMEM16B was found to be abundantly expressed in the cilia of OSNs in 2009 and having biophysical properties like those of the native olfactory channel. A TMEM16B knock-out mouse confirmed that TMEM16B was indeed the olfactory Cl(-) channel but also suggested a limited role in olfactory physiology and behavior. The question then arises of what the precise role of TMEM16b in olfaction is. Here we review the long story of this channel and its possible roles. \ua9 2017 Taylor & Franci

The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop

Ca(2+)-activated Cl(-) channels (CaCCs) are involved in several physiological processes. Recently, TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs, but very little information is available on the structure-function relations of these channels. TMEM16B is expressed in the cilia of olfactory sensory neurons, in microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors. Here, we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage and Ca(2+) dependence. We have mutated amino acids in the first putative intracellular loop and measured the properties of the wild-type and mutant TMEM16B channels expressed in HEK 293T cells using the whole cell voltage-clamp technique in the presence of various intracellular Ca(2+) concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates (386)EEEEE(390) and (399)EYE(401). The EYE deletion did not significantly modify the apparent Ca(2+) dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca(2+) affinity but modified the voltage dependence, shifting the conductance-voltage relations toward more positive voltages. These findings indicate that glutamates E367 and (386)EEEEE(390) in the first intracellular putative loop play an important role in the voltage dependence of TMEM16B, thus providing an initial structure-function study for this channel. \ua9 2012 Cenedese et al

Sensory adaptation to chemical cues by vomeronasal sensory neurons

Sensory adaptation is a source of experience-dependent feedback that impacts responses to environmental cues. In the mammalian main olfactory system (MOS), adaptation influences sensory coding at its earliest processing stages. Sensory adaptation in the accessory olfactory system (AOS) remains incompletely explored, leaving many aspects of the phenomenon unclear. We investigated sensory adaptation in vomeronasal sensory neurons (VSNs) using a combination of in situ Ca2+ imaging and electrophysiology. Parallel studies revealed prominent short-term sensory adaptation in VSNs upon repeated stimulation with mouse urine and monomolecular bile acid ligands at interstimulus intervals (ISIs) less than 30 s. In such conditions, Ca2+ signals and spike rates were often reduced by more than 50%, leading to dramatically reduced chemosensory sensitivity. Short-term adaptation was reversible over the course of minutes. Population Ca2+ imaging experiments revealed the presence of a slower form of VSN adaptation that accumulated over dozens of stimulus presentations delivered over tens of minutes. Most VSNs showed strong adaptation, but in a substantial VSN subpopulation adaptation was diminished or absent. Investigation of same-and opposite-sex urine responses in male and female VSNs revealed that adaptation to same-sex cues occurred at ISIs up to 180 s, conditions that did not induce adaptation to opposite-sex cues. This result suggests that VSN sensory adaptation can be modulated by sensory experience. These studies comprehensively establish the presence of VSN sensory adaptation and provide a foundation for future inquiries into the molecular and cellular mechanisms of this phenomenon and its impact on mammalian behavior

Bitter tastants and artificial sweeteners activate a subset of epithelial cells in acute tissue slices of the rat trachea

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 Ca2+ 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 Ca2+ waves through cell-to-cell junctions. Pharmacological experiments showed that both denatonium and artificial sweeteners induced a PLC-mediated release of Ca2+ 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

A Role for STOML3 in Olfactory Sensory Transduction

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

TMEM16A and TMEM16B modulate pheromone-evoked action potential firing in mouse vomeronasal sensory neurons

The mouse vomeronasal system controls several social behaviors. Pheromones and other social cues are detected by sensory neurons in the vomeronasal organ (VNO). Stimuli activate a transduction cascade that leads to membrane potential depolarization, increase in cytosolic Ca2+ level, and increased firing. The Ca2+-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 (WT) and knock-out (KO) mice. Performing loosepatch recordings from neurons in acute VNO slices, we show that spontaneous activity is modified by Tmem16a KO, 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 (VSNs) from Tmem16a cKO and Tmem16b KO mice show shorter interspike intervals (ISIs) compared with WT mice, indicating that both TMEM16A and TMEM16B modulate the firing pattern of pheromone-evoked activity in VSNs

Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel

At least two members of the TMEM16/anoctamin family, TMEM16A (also known as anoctamin1) and TMEM16B (also known as anoctamin2), encode Ca2+-activated Cl- channels (CaCCs), which are found in various cell types and mediate numerous physiological functions. Here, we used whole-cell and excised inside-out patch-clamp to investigate the relationship between anion permeation and gating, two processes typically viewed as independent, in TMEM16B expressed in HEK 293T cells. The permeability ratio sequence determined by substituting Cl- with other anions (PX/PCl) was SCN- > I- > NO3 - > Br- > Cl- > F- > gluconate. When external Cl- was substituted with other anions, TMEM16B activation and deactivation kinetics at 0.5 \u3bcM Ca2+ were modified according to the sequence of permeability ratios, with anions more permeant than Cl- slowing both activation and deactivation and anions less permeant than Cl- accelerating them. Moreover, replacement of external Cl- with gluconate, or sucrose, shifted the voltage dependence of steady-state activation (G-V relation) to more positive potentials, whereas substitution of extracellular or intracellular Cl- with SCN- shifted G-V to more negative potentials. Dose-response relationships for Ca2+ in the presence of different extracellular anions indicated that the apparent affinity for Ca2+ at +100 mV increased with increasing permeability ratio. The apparent affinity for Ca2+ in the presence of intracellular SCN- also increased compared with that in Cl-. Our results provide the first evidence that TMEM16B gating is modulated by permeant anions and provide the basis for future studies aimed at identifying the molecular determinants of TMEM16B ion selectivity and gating. \ua9 2014 Betto et al

Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons.

Pheromones are substances released from animals that, when detected by the vomeronasal organ of other individuals of the same species, affect their physiology and behavior. Pheromone binding to receptors on microvilli on the dendritic knobs of vomeronasal sensory neurons activates a second messenger cascade to produce an increase in intracellular Ca2+concentration. Here, we used whole-cell and inside-out patch-clamp analysis to provide a functional characterization of currents activated by Ca2+in isolated mouse vomeronasal sensory neurons in the absence of intracellular K+. In whole-cell recordings, the average current in 1.5 \u3bcM Ca2+and symmetrical Cl-was -382 pA at -100 mV. Ion substitution experiments and partial blockade by commonly used Cl-channel blockers indicated that Ca2+activates mainly anionic currents in these neurons. Recordings from inside-out patches from dendritic knobs of mouse vomeronasal sensory neurons confirmed the presence of Ca2+-activated Cl-channels in the knobs and/or microvilli. We compared the electrophysiological properties of the native currents with those mediated by heterologously expressed TMEM16A/anoctamin1 or TMEM16B/anoctamin2 Ca2+-activated Cl-channels, which are coexpressed in microvilli of mouse vomeronasal sensory neurons, and found a closer resemblance to those of TMEM16A. We used the Cre-loxP system to selectively knock out TMEM16A in cells expressing the olfactory marker protein, which is found in mature vomeronasal sensory neurons. Immunohistochemistry confirmed the specific ablation of TMEM16A in vomeronasal neurons. Ca2+-activated currents were abolished in vomeronasal sensory neurons of TMEM16A conditional knockout mice, demonstrating that TMEM16A is an essential component of Ca2+-activated Cl-currents in mouse vomeronasal sensory neurons