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.     

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.     

Direct or Indirect Regulation of Calcium-Activated Chloride Channel by Calcium

Calcium-activated chloride channels (CaCCs) play fundamental roles in numerous physiological processes. Despite their physiological importance, the molecular identity of CaCCs has not been fully investigated until now. Recently, transmembrane 16A (TMEM16A) was demonstrated by three independent research groups to be a strong candidate for the CaCC molecular basis. To further investigate the electrophysiological characteristics, we constructed TMEM16A (abcd) stably transfected HEK293 cell lines and carried out whole-cell and excised inside–out patch-clamp experiments. The TMEM16A channel was Ca²⁺-dependent in both patch configurations. The TMEM16A current could be strongly inhibited by niflumic acid, and when Cl⁻ was substituted by gluconate ions, the current was reduced considerably. In inside–out configuration, TMEM16A channel was time-independent but voltage-dependent, in which the half-maximum activating free Ca²⁺ concentration was 63 nM at 80 mV. While in whole-cell configuration, the current was both time- and voltage-dependent. About the rectification feature, the TMEM16A current also showed distinct characteristics in the two patch configurations. In whole cells, the TMEM16A channel expressed outward rectification at low Ca²⁺ concentration but when the Ca²⁺ concentration was high it became linear. On the contrary, in inside–out configuration, it always expressed outward rectification. Comparing the different characteristics in the two configurations, some underlying mechanisms remain to be identified, which is discussed with respect to direct or indirect activation. There was irreversible rundown in this channel.     

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.     

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.     

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.     

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.     

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⁺.     

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.     

CFTR and TMEM16A are separate but functionally related Cl- channels

Previous reports point out to a functional relationship of the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca(2+) activated Cl(-) channels (CaCC). Recent findings showing that TMEM16A forms the essential part of CaCC, prompted us to examine whether CFTR controls TMEM16A. Inhibition of endogenous CaCC by activation of endogenous CFTR was found in 16HBE human airway epithelial cells, which also express TMEM16A. In contrast, CFBE airway epithelial cells lack of CFTR expression, but express TMEM16A along with other TMEM16-proteins. These cells produce CaCC that is inhibited by overexpression and activation of CFTR. In HEK293 cells coexpressing TMEM16A and CFTR, whole cell currents activated by IMBX and forskolin were significantly reduced when compared with cells expressing CFTR only, while the halide permeability sequence of CFTR was not changed. Expression of TMEM16A, but not of TMEM16F, H or J, produced robust CaCC, which that were inhibited by CaCCinh-A01 and niflumic acid, but not by CFTRinh-172. TMEM16A-currents were attenuated by additional expression of CFTR, and were completely abrogated when additionally expressed CFTR was activated by IBMX and forskolin. On the other hand, CFTR-currents were attenuated by additional expression of TMEM16A. CFTR and TMEM16A were both membrane localized and could be coimmunoprecipitated. Intracellular Ca(2+) signals elicited by receptor-stimulation was not changed during activation of CFTR, while ionophore-induced rise in [Ca(2+)](i) was attenuated after stimulation of CFTR. The data indicate that both CFTR and TMEM16 proteins are separate molecular entities that show functional and molecular interaction.     

TMEM16A inhibitors reveal TMEM16A as a minor component of calcium-activated chloride channel conductance in airway and intestinal epithelial cells

TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). We developed pharmacological tools to investigate the contribution of TMEM16A to CaCC conductance in human airway and intestinal epithelial cells. A screen of ～110,000 compounds revealed four novel chemical classes of small molecule TMEM16A inhibitors that fully blocked TMEM16A chloride current with an IC(50) < 10 μM, without interfering with calcium signaling. Following structure-activity analysis, the most potent inhibitor, an aminophenylthiazole (T16A(inh)-A01), had an IC(50) of ～1 μM. Two distinct types of inhibitors were identified. Some compounds, such as tannic acid and the arylaminothiophene CaCC(inh)-A01, fully inhibited CaCC current in human bronchial and intestinal cells. Other compounds, including T16A(inh)-A01 and digallic acid, inhibited total CaCC current in these cells poorly, but blocked mainly an initial, agonist-stimulated transient chloride current. TMEM16A RNAi knockdown also inhibited mainly the transient chloride current. In contrast to the airway and intestinal cells, all TMEM16A inhibitors fully blocked CaCC current in salivary gland cells. We conclude that TMEM16A carries nearly all CaCC current in salivary gland epithelium, but is a minor contributor to total CaCC current in airway and intestinal epithelia. The small molecule inhibitors identified here permit pharmacological dissection of TMEM16A/CaCC function and are potential development candidates for drug therapy of hypertension, pain, diarrhea, and excessive mucus production.     

NMR-based molecular ruler for determining the depth of intercalants within the lipid bilayer Part I. Discovering the guidelines

The development of “molecular rulers” would allow one to quantitatively locate intercalants within the liposomal bilayer. To this end, we have attempted to correlate the ¹³C NMR chemical shift of a polarizable “reporter” carbon (e.g., carbonyl) of the intercalant—with the E_T(30) polarity it experiences, and with its Angstrom distance from the interface. This requires families of molecules with the same two “reporter carbons” separated by a fixed distance, residing at various depths/polarities within the bilayer. The families studied included 4,4-dialkylcyclohexa-2,5-dienones 1, benzenediacetic esters 15, benzenedipropionic esters 17, 4-alkoxybenzaldehydes 19 and methyl 4-alkoxybenzoates 22. These compounds possessed the following characteristics: (1) a planar backbone; (2) polar/hydrophilic “head” groups; (3) modular hydrophobic tails; (4) large changes in the ¹³C NMR chemical shift (Δδ) of the reporter atoms with solvent polarity. These studies revealed a fifth requirement, namely: (5) the reporter carbons must not be strongly conjugated, lest it reflect the charge build-up at another site within the conjugated system.     

TRPC1 regulates calcium‐activated chloride channels in salivary gland cells

Calcium‐activated chloride channel (CaCC) plays an important role in modulating epithelial secretion. It has been suggested that in salivary tissues, sustained fluid secretion is dependent on Ca²⁺ influx that activates ion channels such as CaCC to initiate Cl^? efflux. However direct evidence as well as the molecular identity of the Ca²⁺ channel responsible for activating CaCC in salivary tissues is not yet identified. Here we provide evidence that in human salivary cells, an outward rectifying Cl^? current was activated by increasing [Ca²⁺]_i, which was inhibited by the addition of pharmacological agents niflumic acid (NFA), an antagonist of CaCC, or T16Ainh‐A01, a specific TMEM16a inhibitor. Addition of thapsigargin (Tg), that induces store‐depletion and activates TRPC1‐mediated Ca²⁺ entry, potentiated the Cl^? current, which was inhibited by the addition of a non‐specific TRPC channel blocker SKF96365 or removal of external Ca²⁺. Stimulation with Tg also increased plasma membrane expression of TMEM16a protein, which was also dependent on Ca²⁺ entry. Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3, inhibited CaCC especially upon store depletion. Moreover, primary acinar cells isolated from submandibular gland also showed outward rectifying Cl^? currents upon increasing [Ca²⁺]_i. These Cl^? currents were again potentiated with the addition of Tg, but inhibited in the presence of T16Ainh‐A01. Finally, acinar cells isolated from the submandibular glands of TRPC1 knockout mice showed significant inhibition of the outward Cl^? currents without decreasing TMEM16a expression. Together the data suggests that Ca²⁺ entry via the TRPC1 channels is essential for the activation of CaCC. J. Cell. Physiol. 9999: 2848–2856, 2015. © 2015 Wiley Periodicals, Inc.

     

Effect of NaCl and CaCl2 on the lateral diffusion of zwitterionic and anionic lipids in bilayers

The influence of addition of NaCl or CaCl₂ (0.3 and 0.1 M, respectively) on the lateral diffusion coefficient (D_L) of dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylglycerol (DOPG) was measured by the pulsed field gradient NMR technique. D_L of DOPC was unaffected, whereas the DOPG diffusion decreased with salt concentration. ²³Na NMR quadrupole splittings of DOPG between 20 and 60 °C and added NaCl between 0 and 15 wt% decreased only slightly with salt content, but increased with increasing temperature. Similar results were obtained for palmitoyloleoylphosphatidylglycerol, in which the palmitoyl chain order parameter increased slightly with salt. A model with free and “bound” ions was used to interpret the splitting data.With increasing salt content a decrease in the water permeability for DOPG was observed, but not for DOPC, as measured by water diffusion perpendicular to the oriented lipid bilayers.It was concluded that calcium and sodium ions interacted with the DOPG head-groups resulting in a decrease in the “free area” per lipid molecule due to a screening of the charged lipid head-groups. Thus, there was a closer packing of DOPG, leading to a decrease in D_L and water permeability. DOPC did not show any changes in the bilayer properties upon the addition of ions.     

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.     

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.