Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons

The rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of -261 pA was measured at -50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction.     

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 (P_X/P_Cl) was SCN⁻ > I⁻ > NO₃⁻ > Br⁻ > Cl⁻ > F⁻ > gluconate. When external Cl⁻ was substituted with other anions, TMEM16B activation and deactivation kinetics at 0.5 µM 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.     

Bestrophin and TMEM16—Ca activated Cl channels with different functions

In the past, a number of candidates have been proposed to form Ca²⁺ activated Cl⁻ currents, but it is only recently that two families of proteins, the bestrophins and the TMEM16-proteins, recapitulate reliably the properties of Ca²⁺ activated Cl⁻ currents. Bestrophin 1 is strongly expressed in the retinal pigment epithelium, but also at lower levels in other cell types. Bestrophin 1 may form Ca²⁺ activated chloride channels and, at the same time, affect intracellular Ca²⁺ signaling. In epithelial cells, bestrophin 1 probably controls receptor mediated Ca²⁺ signaling. It may do so by facilitating Ca²⁺ release from the endoplasmic reticulum, thereby indirectly activating membrane localized Ca²⁺-dependent Cl⁻ channels. In contrast to bestrophin 1, the Ca²⁺ activated Cl⁻ channel TMEM16A (anoctamin 1, ANO1) shows most of the biophysical and pharmacological properties that have been attributed to Ca²⁺-dependent Cl⁻ channels in various tissues. TMEM16A is broadly expressed in both mouse and human tissues and is of particular importance in epithelial cells. Thus exocrine gland secretion as well as electrolyte transport by both respiratory and intestinal epithelia requires TMEM16A. Because of its role for Ca²⁺-dependent Cl⁻ secretion in human airways, it is likely to become a prime target for the therapy of cystic fibrosis lung disease, caused by defective cAMP-dependent Cl⁻ secretion. It will be very exciting to learn, how TMEM16A and other TMEM16-proteins are activated upon increase in intracellular Ca²⁺, and whether the other nine members of the TMEM16 family also form Cl⁻ channels with properties similar to TMEM16A.     

Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

TransMEMbrane 16A (TMEM16A) is a Ca²⁺-activated Cl⁻ channel that plays critical roles in regulating diverse physiologic processes, including vascular tone, sensory signal transduction, and mucosal secretion. In addition to Ca²⁺, TMEM16A activation requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). However, the structural determinants mediating this interaction are not clear. Here, we interrogated the parts of the PI(4,5)P₂ head group that mediate its interaction with TMEM16A by using patch- and two-electrode voltage-clamp recordings on oocytes from the African clawed frog Xenopus laevis, which endogenously express TMEM16A channels. During continuous application of Ca²⁺ to excised inside–out patches, we found that TMEM16A-conducted currents decayed shortly after patch excision. Following this rundown, we show that the application of a synthetic PI(4,5)P₂ analog produced current recovery. Furthermore, inducible dephosphorylation of PI(4,5)P₂ reduces TMEM16A-conducted currents. Application of PIP₂ analogs with different phosphate orientations yielded distinct amounts of current recovery, and only lipids that include a phosphate at the 4′ position effectively recovered TMEM16A currents. Taken together, these findings improve our understanding of how PI(4,5)P₂ binds to and potentiates TMEM16A channels.     

Anoctamin-6 Controls Bone Mineralization by Activating the Calcium Transporter NCX1

Anoctamin-6 (Ano6, TMEM16F) belongs to a family of putative Ca²⁺-activated Cl⁻ channels and operates as membrane phospholipid scramblase. Deletion of Ano6 leads to reduced skeleton size, skeletal deformities, and mineralization defects in mice. However, it remains entirely unclear how a lack of Ano6 leads to a delay in bone mineralization by osteoblasts. The Na⁺/Ca²⁺ exchanger NCX1 was found to interact with Ano6 in a two-hybrid split-ubiquitin screen. Using human osteoblasts and osteoblasts from Ano6^−/− and WT mice, we demonstrate that NCX1 requires Ano6 to efficiently translocate Ca²⁺ out of osteoblasts into the calcifying bone matrix. Ca²⁺-activated anion currents are missing in primary osteoblasts isolated from Ano6 null mice. Our findings demonstrate the importance of NCX1 for bone mineralization and explain why deletion of an ion channel leads to the observed mineralization defect: Ano6 Cl⁻ currents are probably required to operate as a Cl⁻ bypass channel, thereby compensating net Na⁺ charge movement by NCX1.     

Presynaptic Localization and Possible Function of Calcium-Activated Chloride Channel Anoctamin 1 in the Mammalian Retina

Calcium (Ca²⁺)-activated chloride (Cl⁻) channels (CaCCs) play a role in the modulation of action potentials and synaptic responses in the somatodendritic regions of central neurons. In the vertebrate retina, large Ca²⁺-activated Cl⁻ currents (I_Cl(Ca)) regulate synaptic transmission at photoreceptor terminals; however, the molecular identity of CaCCs that mediate I_Cl(Ca) remains unclear. The transmembrane protein, TMEM16A, also called anoctamin 1 (ANO1), has been recently validated as a CaCC and is widely expressed in various secretory epithelia and nervous tissues. Despite the fact that tmem16a was first cloned in the retina, there is little information on its cellular localization and function in the mammalian retina. In this study, we found that ANO1 was abundantly expressed as puncta in 2 synaptic layers. More specifically, ANO1 immunoreactivity was observed in the presynaptic terminals of various retinal neurons, including photoreceptors. I_Cl(Ca) was first detected in dissociated rod bipolar cells expressing ANO1. I_Cl(Ca) was abolished by treatment with the Ca²⁺ channel blocker Co²⁺, the L-type Ca²⁺ channel blocker nifedipine, and the Cl⁻ channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and niflumic acid (NFA). More specifically, a recently discovered ANO1-selective inhibitor, T16A_inh-A01, and a neutralizing antibody against ANO1 inhibited I_Cl(Ca) in rod bipolar cells. Under a current-clamping mode, the suppression of I_Cl(Ca) by using NPPB and T16A_inh-A01 caused a prolonged Ca²⁺ spike-like depolarization evoked by current injection in dissociated rod bipolar cells. These results suggest that ANO1 confers I_Cl(Ca) in retinal neurons and acts as an intrinsic regulator of the presynaptic membrane potential during synaptic transmission.     

Calcium-calmodulin does not alter the anion permeability of the mouse TMEM16A calcium-activated chloride channel

The transmembrane protein TMEM16A forms a Ca²⁺-activated Cl⁻ channel that is permeable to many anions, including SCN⁻, I⁻, Br⁻, Cl⁻, and HCO₃⁻, and has been implicated in various physiological functions. Indeed, controlling anion permeation through the TMEM16A channel pore may be critical in regulating the pH of exocrine fluids such as the pancreatic juice. The anion permeability of the TMEM16A channel pore has recently been reported to be modulated by Ca²⁺-calmodulin (CaCaM), such that the pore of the CaCaM-bound channel shows a reduced ability to discriminate between anions as measured by a shift of the reversal potential under bi-ionic conditions. Here, using a mouse TMEM16A clone that contains the two previously identified putative CaM-binding motifs, we were unable to demonstrate such CaCaM-dependent changes in the bi-ionic potential. We confirmed the activity of CaCaM used in our study by showing CaCaM modulation of the olfactory cyclic nucleotide–gated channel. We suspect that the different bi-ionic potentials that were obtained previously from whole-cell recordings in low and high intracellular [Ca²⁺] may result from different degrees of bi-ionic potential shift secondary to a series resistance problem, an ion accumulation effect, or both.     

The long tale of the calcium activated Cl− channels in olfactory transduction

Ca²⁺-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 Ca²⁺-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 knockout 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.     

Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels

Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca²⁺/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca²⁺/calmodulin, one at submicromolar Ca²⁺ concentrations and one in the micromolar Ca²⁺ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca²⁺/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca²⁺ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca²⁺ regulation in anoctamin Cl⁻ channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.     

Trigeminal Ganglion Neurons of Mice Show Intracellular Chloride Accumulation and Chloride-Dependent Amplification of Capsaicin-Induced Responses

Intracellular Cl⁻ concentrations ([Cl⁻]_i) of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG) and olfactory sensory neurons (OSNs), Cl⁻ is accumulated by the Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1), resulting in a [Cl⁻]_i above electrochemical equilibrium and a depolarizing Cl⁻ efflux upon Cl⁻ channel opening. Here, we investigate the [Cl⁻]_i and function of Cl⁻ in primary sensory neurons of trigeminal ganglia (TG) of wild type (WT) and NKCC1^−/− mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The [Cl⁻]_i of WT TG neurons indicated active NKCC1-dependent Cl⁻ accumulation. Gamma-aminobutyric acid (GABA)_A receptor activation induced a reduction of [Cl⁻]_i as well as Ca²⁺ transients in a corresponding fraction of TG neurons. Ca²⁺ transients were sensitive to inhibition of NKCC1 and voltage-gated Ca²⁺ channels (VGCCs). Ca²⁺ responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1) were diminished in NKCC1^−/− TG neurons, but elevated under conditions of a lowered [Cl⁻]_o suggesting a Cl⁻-dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS), we found expression of different Ca²⁺-activated Cl⁻ channels (CaCCs) in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca²⁺ imaging and electrophysiological recordings. In a behavioral paradigm, NKCC1^−/− mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a Ca²⁺-activated Cl⁻-dependent signal amplification mechanism in TG neurons that requires intracellular Cl⁻ accumulation by NKCC1 and the activation of CaCCs.     

Functional Swapping between Transmembrane Proteins TMEM16A and TMEM16F

The transmembrane proteins TMEM16A and -16F each carry eight transmembrane regions with cytoplasmic N and C termini. TMEM16A carries out Ca²⁺-dependent Cl⁻ ion transport, and TMEM16F is responsible for Ca²⁺-dependent phospholipid scrambling. Here we established assay systems for the Ca²⁺-dependent Cl⁻ channel activity using 293T cells and for the phospholipid scramblase activity using TMEM16F^−/− immortalized fetal thymocytes. Chemical cross-linking analysis showed that TMEM16A and -16F form homodimers in both 293T cells and immortalized fetal thymocytes. Successive deletion from the N or C terminus of both proteins and the swapping of regions between TMEM16A and -16F indicated that their cytoplasmic N-terminal (147 amino acids for TMEM16A and 95 for 16F) and C-terminal (88 amino acids for TMEM16A and 68 for 16F) regions were essential for their localization at plasma membranes and protein stability, respectively, and could be exchanged. Analyses of TMEM16A and -16F mutants with point mutations in the pore region (located between the fifth and sixth transmembrane regions) indicated that the pore region is essential for both the Cl⁻ channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F. Some chemicals such as epigallocatechin-3-gallate and digallic acid inhibited the Cl⁻ channel activity of TMEM16A and the scramblase activity of TMEM16F with an opposite preference. These results indicate that TMEM16A and -16F use a similar mechanism for sorting to plasma membrane and protein stabilization, but their functional domains significantly differ.     

Ca2+-Activated Cl? Channels of the ClCa Family Express in the Cilia of a Subset of Rat Olfactory Sensory Neurons

The Ca²⁺-activated Cl⁻ channel is considered a key constituent of odor transduction. Odorant binding to a specific receptor in the cilia of olfactory sensory neurons (OSNs) triggers a cAMP cascade that mediates the opening of a cationic cyclic nucleotide-gated channel (CNG), allowing Ca²⁺ influx. Ca²⁺ ions activate Cl⁻ channels, generating a significant Cl⁻ efflux, with a large contribution to the receptor potential. The Anoctamin 2 channel (ANO2) is a major constituent of the Cl⁻ conductance, but its knock-out has no impairment of behavior and only slightly reduces field potential odorant responses of the olfactory epithelium. Likely, an additional Ca²⁺-activated Cl⁻ channel of unknown molecular identity is also involved. In addition to ANO2, we detected two members of the ClCa family of Ca²⁺-activated Cl⁻ channels in the rat olfactory epithelium, ClCa4l and ClCa2. These channels, also expressed in the central nervous system, may correspond to odorant transduction channels. Whole Sprague Dawley olfactory epithelium nested RT-PCR and single OSNs established that the mRNAs of both channels are expressed in OSNs. Real time RT-PCR and full length sequencing of amplified ClCa expressed in rat olfactory epithelium indicated that ClCa4l is the most abundant. Immunoblotting with an antibody recognizing both channels revealed immunoreactivity in the ciliary membrane. Immunochemistry of olfactory epithelium and OSNs confirmed their ciliary presence in a subset of olfactory sensory neurons. The evidence suggests that ClCa4l and ClCa2 might play a role in odorant transduction in rat olfactory cilia.     

The bestrophin- and TMEM16A-associated Ca2+-activated Cl– channels in vascular smooth muscles

The presence of Ca²⁺-activated Cl^– currents (I_Cl(Ca)) in vascular smooth muscle cells (VSMCs) is well established. I_Cl(Ca) are supposedly important for arterial contraction by linking changes in [Ca²⁺]_i and membrane depolarization. Bestrophins and some members of the TMEM16 protein family were recently associated with I_Cl(Ca). Two distinct I_Cl(Ca) are characterized in VSMCs; the cGMP-dependent I_Cl(Ca) dependent upon bestrophin expression and the ‘classical’ Ca²⁺-activated Cl^– current, which is bestrophin-independent. Interestingly, TMEM16A is essential for both the cGMP-dependent and the classical I_Cl(Ca). Furthermore, TMEM16A has a role in arterial contraction while bestrophins do not. TMEM16A’s role in the contractile response cannot be explained however only by a simple suppression of the depolarization by Cl^– channels. It is suggested that TMEM16A expression modulates voltage-gated Ca²⁺ influx in a voltage-independent manner and recent studies also demonstrate a complex role of TMEM16A in modulating other membrane proteins.     

Expression and Function of Epithelial Anoctamins

The calcium-activated chloride channel anoctamin1 (ANO1; TMEM16A) is fundamental for the function of epithelial organs. Mice lacking ANO1 expression exhibit transport defects and a pathology similar to cystic fibrosis. They also show a general defect of epithelial electrolyte transport. Here we analyzed expression of all ten members (ANO1–ANO10) in a broad range of murine tissues and detected predominant expression of ANO1, 6, 7, 8, 9, 10 in epithelial tissues, while ANO2, 3, 4, 5 are common in neuronal and muscle tissues. When expressed in Fisher Rat Thyroid (FTR) cells, all ANO proteins localized to the plasma membrane but only ANO1, 2, 6, and 7 produced Ca²⁺-activated Cl⁻ conductance, as analyzed by ATP-induced iodide quenching of YFP fluorescence. In contrast ANO9 and ANO10 suppressed baseline Cl⁻ conductance and coexpression of ANO9 with ANO1 inhibited ANO1 activity. Patch clamping of ANO-expressing FRT cells indicated that apart from ANO1 also ANO6 and 10 produced chloride currents, albeit with very different Ca²⁺ sensitivity and activation time. We conclude that each tissue expresses a set of anoctamins that form cell- and tissue-specific Ca²⁺-dependent Cl⁻ channels.     

Multiple effects of anthracene-9-carboxylic acid on the TMEM16B/anoctamin2 calcium-activated chloride channel

Ca^2 +-activated Cl⁻ currents (CaCCs) play important roles in many physiological processes. Recent studies have shown that TMEM16A/anoctamin1 and TMEM16B/anoctamin2 constitute CaCCs in several cell types. Here we have investigated for the first time the extracellular effects of the Cl⁻ channel blocker anthracene-9-carboxylic acid (A9C) and of its non-charged analogue anthracene-9-methanol (A9M) on TMEM16B expressed in HEK 293T cells, using the whole-cell patch-clamp technique. A9C caused a voltage-dependent block of outward currents and inhibited a larger fraction of the current as depolarization increased, whereas the non-charged A9M produced a small, not voltage dependent block of outward currents. A similar voltage-dependent block by A9C was measured both when TMEM16B was activated by 1.5 and 13 μM Ca^2 +. However, in the presence of 1.5 μM Ca^2 + (but not in 13 μM Ca^2 +), A9C also induced a strong potentiation of tail currents measured at − 100 mV after depolarizing voltages, as well as a prolongation of the deactivation kinetics. On the contrary, A9M did not produce potentiation of tail currents, showing that the negative charge is required for potentiation. Our results provide the first evidence that A9C has multiple effects on TMEM16B and that the negative charge of A9C is necessary both for voltage-dependent block and for potentiation. Future studies are required to identify the molecular mechanisms underlying these complex effects of A9C on TMEM16B. Understanding these mechanisms will contribute to the elucidation of the structure and functional properties of TMEM16B channels.     

Divalent Cations Modulate TMEM16A Calcium-Activated Chloride Channels by a Common Mechanism

The gating of Ca²⁺-activated Cl^? channels is controlled by a complex interplay among [Ca²⁺]_i, membrane potential and permeant anions. Besides Ca²⁺, Ba²⁺ also can activate both TMEM16A and TMEM16B. This study reports the effects of several divalent cations as regulators of TMEM16A channels stably expressed in HEK293T cells. Among the divalent cations that activate TMEM16A, Ca²⁺ is most effective, followed by Sr²⁺ and Ni²⁺, which have similar affinity, while Mg²⁺ is ineffective. Zn²⁺ does not activate TMEM16A but inhibits the Ca²⁺-activated chloride currents. Maximally effective concentrations of Sr²⁺ and Ni²⁺ occluded activation of the TMEM16A current by Ca²⁺, which suggests that Ca²⁺, Sr²⁺ and Ni²⁺ all regulate the channel by the same mechanism.     

The TMEM16 Protein Family: A New Class of Chloride Channels?

Cl⁻ channels play important roles in many physiological processes, including transepithelial ion absorption and secretion, smooth and skeletal muscle contraction, neuronal excitability, sensory perception, and cell volume regulation. The molecular identity of many types of Cl⁻ channels is still unknown. Recently, three research groups have arrived independently at the identification of TMEM16A (also known as anoctamin-1) as a membrane protein strongly related to the activity of Ca²⁺-activated Cl⁻ channels (CaCCs). Site-specific mutagenesis of TMEM16A alters the properties of the channels, thus suggesting that TMEM16A forms, at least in part, the CaCC. TMEM16A is a member of a family that includes nine other membrane proteins. All TMEM16 proteins have a similar structure, with eight putative transmembrane domains and cytosolic amino- and carboxy-termini. TMEM16B expression also evokes the appearance of CaCCs, but with biophysical characteristics (voltage dependence, unitary conductance) different from those associated to TMEM16A. The roles of the other TMEM16 proteins are still unknown. The study of TMEM16 proteins may lead to identification of novel molecular mechanisms underlying ion transport and channel gating by voltage and Ca²⁺.