Calmodulin regulation of TMEM16A and 16B Ca2+-activated chloride channels

Ca²⁺-activated chloride channels encoded by TMEM16A and 16B are important for regulating epithelial mucus secretion, cardiac and neuronal excitability, smooth muscle contraction, olfactory transduction, and cell proliferation. Whether and how the ubiquitous Ca²⁺ sensor calmodulin (CaM) regulates the activity of TMEM16A and 16B channels has been controversial and the subject of an ongoing debate. Recently, using a bioengineering approach termed ChIMP (Channel Inactivation induced by Membrane-tethering of an associated Protein) we argued that Ca²⁺-free CaM (apoCaM) is pre-associated with functioning TMEM16A and 16B channel complexes in live cells. Further, the pre-associated apoCaM mediates Ca²⁺-dependent sensitization of activation (CDSA) and Ca²⁺-dependent inactivation (CDI) of some TMEM16A splice variants. In this review, we discuss these findings in the context of previous and recent results relating to Ca²⁺-dependent regulation of TMEM16A/16B channels and the putative role of CaM. We further discuss potential future directions for these nascent ideas on apoCaM regulation of TMEM16A/16B channels, noting that such future efforts will benefit greatly from the pioneering work of Dr. David T. Yue and colleagues on CaM regulation of voltage-dependent calcium channels.     

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.     

Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner

TMEM16A (Transmembrane protein 16A or Anoctamin1) is a calcium-activated chloride channel.(CaCC),that exerts critical roles in epithelial secretion. However, its localization, function, and regulation in intestinal chloride (Cl^?) secretion remain obscure. Here, we show that TMEM16A protein abundance correlates with Cl^? secretion in different regions of native intestine activated by the Ca²⁺-elevating muscarinic agonist carbachol (CCH). Basal, as well as both cAMP- and CCH-stimulated Isc, was largely reduced in Ano1 ± mouse intestine. We found CCH was not able to increase Isc in the presence of apical to serosal Cl^? gradient, strongly supporting TMEM16A as primarily a luminal Cl^? channel. Immunostaining demonstrated apical localization of TMEM16A where it colocalized with NHERF1 in mouse colonic tissue. Cellular depletion of NHERF1 in human colonic T84 cells caused a significant reduction of both cAMP- and CCH-stimulated Isc. Immunoprecipitation experiments revealed that NHERF1 forms a complex with TMEM16A through a PDZ-based interaction. We conclude that TMEM16A is a luminal Cl^? channel in the intestine that functionally interacts with CFTR via PDZ-based interaction of NHERF1 for efficient and specific cholinergic stimulation of intestinal Cl^? secretion.     

Anoctamin 1 in secretory epithelia

Fluid and electrolyte releasing from secretory epithelia are elaborately regulated by orchestrated activity of ion channels. The activity of chloride channel at the apical membrane decides on the direction and the rate of secretory fluid and electrolyte. Chloride-dependent secretion is conventionally associated with intracellular increases in two second messengers, cAMP and Ca²⁺, responding to luminal purinergic and basolateral adrenergic or cholinergic stimulation. While it is broadly regarded that cAMP-dependent Cl⁻ secretion is regulated by cystic fibrosis transmembrane conductance regulator (CFTR), Ca²⁺-activated Cl⁻ channel (CaCC) had been veiled for quite some time. Now, Anoctamin 1 (ANO1 or TMEM16A) confers Ca²⁺-activated Cl⁻ currents. Ano 1 and its paralogs have been actively investigated for multiple functions underlying Ca²⁺-activated Cl⁻ efflux and fluid secretion in a variety of secretory epithelial cells. In this review, we will discuss recent advances in the secretory function and signaling of ANO1 in the secretory epithelia, such as airways, intestines, and salivary glands.     

TMEM16A alternative splicing isoforms in Xenopus tropicalis: Distribution and functional properties

Oocytes of Xenopus tropicalis elicit a Ca²⁺-dependent outwardly rectifying, low-activating current (I_Cl,Ca) that is inhibited by Cl⁻ channel blockers. When inactivated, I_Cl,Ca shows an exponentially decaying tail current that is related to currents generated by TMEM16A ion channels. Accordingly, RT-PCR revealed the expression of five alternatively spliced isoforms of TMEM16A in oocytes, which, after expression in HEK-293 cells, gave rise to fully functional Cl⁻ channels. Upon hyperpolarization to −80 mV a transient current was observed only in isoforms that carry the exon 1d, coding for two potentially phosphorylatable Threonine residues. The identified isoforms are differentially expressed in several tissues of the frog. Thus, it appears that X. tropicalis oocytes express TMEM16A that gives rise to a Ca²⁺-dependent Cl⁻ current, which is different from the previously reported voltage-dependent outwardly rectifying Cl⁻ current.     

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.     

Regulation of Basolateral Cl− Channels in Airway Epithelial Cells: The Role of Nitric Oxide

The presence of basolateral Cl⁻ channels in airway epithelium has been reported in several studies, but little is known about their role in the regulation of anion secretion. The purpose of this study was to characterize regulation of these channels by nitric oxide (NO) in Calu-3 cells. Transepithelial measurements revealed that NO donors activated a basolateral Cl⁻ conductance sensitive to 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid. Apical membrane permeabilization studies confirmed the basolateral localization of NO-activated Cl⁻ channels. Experiments using 8-bromo cyclic guanosine monophosphate (8Br-cGMP) and selective inhibitors of soluble guanylyl cyclase and inducible NO synthase (1H-[1, 2, 4] oxadiazolol-[4, 3-a] quinoxalin-1-one [ODQ] and 1400W [N-(3-Aminomethyl)benzyl)acetamidine], respectively) demonstrated that NO activated Cl⁻ channels via a cGMP-dependent pathway. Anion replacement and ³⁶Cl⁻ flux studies showed that NO affected both Cl⁻ and HCO ₃ ⁻ secretion. Two different types of Cl⁻ channels are known to be present in the basolateral membrane of epithelial cells: Zn²⁺-sensitive ClC-2 and DIDS-sensitive bestrophin channels. S-Nitrosoglutathione (GSNO) activated Cl⁻ conductance in the presence of Zn²⁺ ions, indicating that ClC-2 channel function was not affected by GSNO. In contrast, DIDS completely inhibited GSNO-activated Cl⁻ conductance. Bestrophin immunoprecipitation studies showed that under control conditions bestrophin channels were not phosphorylated but became phosphorylated after GSNO treatment. The presence of bestrophin in airway epithelia was confirmed using immunohistochemistry. We conclude that basolateral Cl⁻ channels play a major role in the NO-dependent regulation of anion secretion in Calu-3 cells.     

Loss of TMEM16A Causes a Defect in Epithelial Ca2+-dependent Chloride Transport

Molecular identification of the Ca²⁺-dependent chloride channel TMEM16A (ANO1) provided a fundamental step in understanding Ca²⁺-dependent Cl⁻ secretion in epithelia. TMEM16A is an intrinsic constituent of Ca²⁺-dependent Cl⁻ channels in cultured epithelia and may control salivary output, but its physiological role in native epithelial tissues remains largely obscure. Here, we demonstrate that Cl⁻ secretion in native epithelia activated by Ca²⁺-dependent agonists is missing in mice lacking expression of TMEM16A. Ca²⁺-dependent Cl⁻ transport was missing or largely reduced in isolated tracheal and colonic epithelia, as well as hepatocytes and acinar cells from pancreatic and submandibular glands of TMEM16A^−/− animals. Measurement of particle transport on the surface of tracheas ex vivo indicated largely reduced mucociliary clearance in TMEM16A^−/− mice. These results clearly demonstrate the broad physiological role of TMEM16A^−/− for Ca²⁺-dependent Cl⁻ secretion and provide the basis for novel treatments in cystic fibrosis, infectious diarrhea, and Sjöegren syndrome.Electrolyte secretion in epithelial tissues is based on the major second messenger pathways cAMP and Ca²⁺, which activate the cystic fibrosis transmembrane conductance regulator (CFTR)² Cl⁻ channels and Ca²⁺-dependent Cl⁻ channels, respectively (1–3). CFTR conducts Cl⁻ in epithelial cells of airways, intestine, and the ducts of pancreas and sweat gland, while Ca²⁺-dependent Cl⁻ channels secrete Cl⁻ in pancreatic acini and salivary and sweat glands (4–6). Controversy exists as to the contribution of these channels to Cl⁻ secretion in submucosal glands of airways and the relevance for cystic fibrosis (7–9). While cAMP-dependent Cl⁻ secretion by CFTR is well examined, detailed analysis of epithelial Ca²⁺-dependent Cl⁻ secretion is hampered by the lack of a molecular counterpart. Although bestrophins may form Ca²⁺-dependent Cl⁻ channels and facilitate Ca²⁺-dependent Cl⁻ secretion in epithelial tissues (10, 11), they are unlikely to form secretory Cl⁻ channels in the apical cell membrane, because Ca²⁺-dependent Cl⁻ secretion is still present in epithelia of mice lacking expression of bestrophin (12). Bestrophins may rather have an intracellular function by facilitating receptor mediated Ca²⁺ signaling and activation of membrane localized channels (13). With the discovery that TMEM16A produces Ca²⁺-activated Cl⁻ currents with biophysical and pharmacological properties close to those in native epithelial tissues, these proteins are now very likely candidates for endogenous Ca²⁺-dependent Cl⁻ channels (14–17). In cultured airway epithelial cells, small interfering RNA knockdown of endogenous TMEM16A largely reduced calcium-dependent chloride secretion (16). However, apart from preliminary studies of airways and salivary glands, the physiological significance of TMEM16A in native epithelia, particularly in glands, is unclear (14, 17).     

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 µM 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.     

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.     

Role of anoctamins in cancer and apoptosis

Anoctamin 1 (TMEM16A, Ano1) is a recently identified Ca²⁺-activated chloride channel and a member of a large protein family comprising 10 paralogues. Before Ano1 was identified as a chloride channel protein, it was known as the cancer marker DOG1. DOG1/Ano1 is expressed in gastrointestinal stromal tumours (GIST) and particularly in head and neck squamous cell carcinoma, at very high levels never detected in other tissues. It is now emerging that Ano1 is part of the 11q13 locus, amplified in several types of tumour, where it is thought to augment cell proliferation, cell migration and metastasis. Notably, Ano1 is upregulated through histone deacetylase (HDAC), corresponding to the known role of HDAC in HNSCC. As Ano1 does not enhance proliferation in every cell type, its function is perhaps modulated by cell-specific factors, or by the abundance of other anoctamins. Thus Ano6, by regulating Ca²⁺-induced membrane phospholipid scrambling and annexin V binding, supports cellular apoptosis rather than proliferation. Current findings implicate other cellular functions of anoctamins, apart from their role as Ca²⁺-activated Cl⁻ channels.     

Compartmentalized crosstalk of CFTR and TMEM16A (ANO1) through EPAC1 and ADCY1

Airway epithelial cells express both Ca²⁺ activated TMEM16A/ANO1 and cAMP activated CFTR anion channels. Previous work suggested a significant crosstalk of intracellular Ca²⁺ and cAMP signaling pathways, leading to activation of both chloride channels. We demonstrate that in airway epithelial cells, stimulation of purinergic or muscarinic G-protein coupled receptors (GPCRs) activates TMEM16A and CFTR. Additional expression of G_q/11 and phospholipase C coupled GPCRs strongly enhanced the crosstalk between Ca²⁺- and cAMP-dependent signaling. Knockdown of endogenous GRCRs attenuated crosstalk and functional coupling between TMEM16A and CFTR. The number of receptors did not affect expression or membrane localization of TMEM16A or CFTR, but controlled assembly of the local signalosome. GPCRs translocate Ca²⁺-sensitive adenylate cyclase type 1 (ADCY1) and exchange protein directly activated by cAMP (EPAC1) to particular plasma membrane domains containing GPCRs, CFTR and TMEM16A, thereby producing compartmentalized Ca²⁺ and cAMP signals and significant crosstalk. While biosynthesis and membrane trafficking of CFTR requires a functional Golgi apparatus, maturation and membrane trafficking of TMEM16A may occur independent of the Golgi. Because Ca²⁺ activated TMEM16A currents are only transient, continuous Cl⁻ secretion by airway epithelial cells requires CFTR. The present data also explain why receptor-dependent activation of TMEM16A is more efficient than direct stimulation by Ca²⁺.     

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.     

Cell swelling activates K+ and Cl− channels as well as nonselective,stretch-activated cation channels in ehrlich ascites tumor cells

Summary Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba²⁺ and hence presumably to Ca²⁺. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca²⁺ permeability, as part of the volume-activated signal system. In isolated insideout patches a Ca²⁺-dependent, inwardly rectifying K⁺ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mm K⁺ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K⁺ channel takes place after an increase in Ca²⁺ from 10^–7 to 10^–6 m which is in the physiological range. Patch-clamp studies in cellattached mode show K⁺ channels with spontaneous activity and with characteristics similar to those of the K⁺ channel seen in excised patches. The single-channel conductance for outward current at 5 mm external K⁺ is estimated at about 7 pS. A K⁺ channel with similar properties can be activated in the cellattached mode by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K⁺ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K⁺ channels following addition of Ca²⁺ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K⁺ channels in the presence of 3 mm Ca²⁺ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca²⁺ and could not be activated by membrane stretch (suction). In cell-attached mode singlechannel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by hypotonic exposure with a single-channel conductance at 7 pS (or less) and with a time delay at about 1 min. The number of open channels during RVD is estimated at 80 per cell. Two other types of Cl^– channels were regularly recorded in excised inside-out patches: a voltage-activated 400-pS channel and a 34-pS Cl^– channel which show properties similar to the Cl^– channel in the apical membrane in human airway epithelial cells. There is no evidence for a role in RVD for either of these two channels.     

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⁺.     

Activation of Nicotinic Acetylcholine Receptors Augments Calcium Channel-mediated Exocytosis in Rat Pheochromocytoma (PC12) Cells

The functional effect of activating Ca²⁺-permeable neuronal nicotinic acetylcholine receptors (nAChRs) on vesicle secretion was studied in PC12 cells. Single cells were patch-clamped in the whole-cell configuration and stimulated with either brief pulses of nicotine to activate the Ca²⁺-permeable nAChRs or with voltage steps to activate voltage-dependent Ca²⁺ channels. Membrane capacitance was used as a measure of vesicle secretion. Activation of nAChRs by nicotine application to cells voltage clamped at −80 mV evoked secretion. This secretion was completely abolished by nicotinic antagonists. When the cells were voltage clamped at +20 mV in the presence of Cd²⁺ to block voltage-activated Ca²⁺ channels, nicotine elicited a small amount of secretion. Most interestingly, when the nAChRs were activated coincidentally with voltage-dependent Ca²⁺ channels, secretion was augmented approximately twofold over the secretion elicited with voltage-dependent Ca²⁺ channels alone. Our data suggest that Ca²⁺ influx via nAChRs affects Ca²⁺-dependent cellular functions, including vesicle secretion. In addition to the secretion evoked by nAChR activation at hyperpolarized potentials, we demonstrate that even at depolarized potentials, nAChRs provide an important Ca²⁺ entry pathway underlying Ca²⁺-dependent cellular processes such as exocytosis.     

A Role of TMEM16E Carrying a Scrambling Domain in Sperm Motility

Transmembrane protein 16E (TMEM16E) belongs to the TMEM16 family of proteins that have 10 transmembrane regions and appears to localize intracellularly. Although TMEM16E mutations cause bone fragility and muscular dystrophy in humans, its biochemical function is unknown. In the TMEM16 family, TMEM16A and -16B serve as Ca²⁺-dependent Cl⁻ channels, while TMEM16C, -16D, -16F, -16G, and -16J support Ca²⁺-dependent phospholipid scrambling. Here, we show that TMEM16E carries a segment composed of 35 amino acids homologous to the scrambling domain in TMEM16F. When the corresponding segment of TMEM16A was replaced by this 35-amino-acid segment of TMEM16E, the chimeric molecule localized to the plasma membrane and supported Ca²⁺-dependent scrambling. We next established TMEM16E-deficient mice, which appeared to have normal skeletal muscle. However, fertility was decreased in the males. We found that TMEM16E was expressed in germ cells in early spermatogenesis and thereafter and localized to sperm tail. TMEM16E^−/− sperm showed no apparent defect in morphology, beating, mitochondrial function, capacitation, or binding to zona pellucida. However, they showed reduced motility and inefficient fertilization of cumulus-free but zona-intact eggs in vitro. Our results suggest that TMEM16E may function as a phospholipid scramblase at inner membranes and that its defect affects sperm motility.     

Calcium-dependent chloride current activated by hyposmotic stress in rat lacrimal acinar cells

We have identified a whole-cell Cl^– current activated by hyposmotic stress in rat lacrimal acinar cells using the patch-clamp technique. Superfusion of isolated single cells with hyposmotic solution (80% of control osmolarity) caused a gradual increase of the current, which was reversed on return to the control solution. The current-voltage relationship showed outward rectification, and the current showed time and voltage dependence: slowly activated by depolarizing voltages and rapidly inactivated by hyperpolarizing voltages. The increase in current was not observed when intracellular Ca²⁺ was chelated with EGTA. It was also inhibited by the absence of extracellular Ca²⁺, or the presence of gadolinium ions (20 m Gd³⁺). We conclude that in rat lacrimal acinar cells hyposmotic stress activates Ca²⁺-dependent Cl^– channels as a result of Ca²⁺ influx through a Gd³⁺-sensitive pathway. The Cl^– channels involved appear to be indistinguishable from those activated by muscarinic stimulation. The inhibitory effect of Gd³⁺ suggests that stretch-activated nonselective cation channels may be responsible for the Ca²⁺ influx.The authors are grateful to Prof. R.M. Case, Dr. A.C. Elliott and Dr. K.R. Lau for helpful discussion. This work was supported by the US Cystic Fibrosis Foundation, Wellcome Trust and Medical Research Council.     

Sinupret Activates CFTR and TMEM16A-Dependent Transepithelial Chloride Transport and Improves Indicators of Mucociliary Clearance

Introduction

We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl⁻) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid.

Methods

Primary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR^−/−], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl⁻ transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca²⁺]_i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis.

Results

Sinupret-mediated Cl⁻ secretion [ΔI_SC(µA/cm²)] was pronounced in WT MNSE (20.7+/−0.9 vs. 5.6+/−0.9(control), p<0.05), CFTR^−/− MNSE (10.1+/−1.0 vs. 0.9+/−0.3(control), p<0.05) and HSNE (20.7+/−0.3 vs. 6.4+/−0.9(control), p<0.05). The formulation activated Ca²⁺ signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl⁻ secretion. Sinupret also enhanced CBF and ASL depth.

Conclusion

Sinupret stimulates CBF, promotes transepithelial Cl⁻ secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca²⁺-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl⁻ secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.     

Role of voltage-dependent ionic currents in coupling glucose stimulation to insulin secretion in canine pancreatic islet B-Cells

Summary Glucose-induced electrical activity in canine pancreatic islet B cells is distinct from that in rodent islets, though both display Ca²⁺-dependent insulin secretion. Rodent islet B cells undergo regular bursts of Ca²⁺-dependent action potentials, while canine islet B cells generate isolated Na⁺-dependent action potentials which often give way to a plateau depolarization. Here we present evidence to reconcile the species difference in electrical activity with the similarity of Ca²⁺ dependence of secretion. (i) In canine B cells increasing glucose concentrations produce membrane depolarization and increasing frequency of Na_o-dependent action potentials until a background membrane potential (-40mV) is reached where Na⁺ currents are inactivated. (ii) Voltage-dependent Ca²⁺ currents are present which are activated over the voltage excursion of the action potential (–50 to +20 mV) and inactivate slowly, (over seconds) in the range of the plateau depolarization (–40 to –25 mV). Hence, they are available to contribute to both phases of depolarization. (iii) Tetrodotoxin (TTX) reduces by half an early transient phase of glucosestimulated insulin secretion but not a subsequent prolonged plateau phase. The transient phase of secretion often corresponds well in time to the period of initial high frequency action potential activity. These latter results suggest that in canine B cells voltagedependent Na⁺ and Ca²⁺ currents mediate biphasic glucose-induced insulin secretion. The early train of Na⁺-dependent action potentials, by transiently activating Ca²⁺ channels and allowing pulsatile Ca²⁺ entry, may promote an early transient phase of insulin secretion. The subsequent sustained plateau depolarization, by allowing sustained Ca²⁺ entry, may permit steady insulin release.