Статті в журналах: "TMEM16B"

1

Pedemonte, Nicoletta, and Luis J. V. Galietta. "Structure and Function of TMEM16 Proteins (Anoctamins)." Physiological Reviews 94, no. 2 (April 2014): 419–59. http://dx.doi.org/10.1152/physrev.00039.2011.

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TMEM16 proteins, also known as anoctamins, are involved in a variety of functions that include ion transport, phospholipid scrambling, and regulation of other membrane proteins. The first two members of the family, TMEM16A (anoctamin-1, ANO1) and TMEM16B (anoctamin-2, ANO2), function as Ca2+-activated Cl−channels (CaCCs), a type of ion channel that plays important functions such as transepithelial ion transport, smooth muscle contraction, olfaction, phototransduction, nociception, and control of neuronal excitability. Genetic ablation of TMEM16A in mice causes impairment of epithelial Cl−secretion, tracheal abnormalities, and block of gastrointestinal peristalsis. TMEM16A is directly regulated by cytosolic Ca2+as well as indirectly by its interaction with calmodulin. Other members of the anoctamin family, such as TMEM16C, TMEM16D, TMEM16F, TMEM16G, and TMEM16J, may work as phospholipid scramblases and/or ion channels. In particular, TMEM16F (ANO6) is a major contributor to the process of phosphatidylserine translocation from the inner to the outer leaflet of the plasma membrane. Intriguingly, TMEM16F is also associated with the appearance of anion/cation channels activated by very high Ca2+concentrations. Furthermore, a TMEM16 protein expressed in Aspergillus fumigatus displays both ion channel and lipid scramblase activity. This finding suggests that dual function is an ancestral characteristic of TMEM16 proteins and that some members, such as TMEM16A and TMEM16B, have evolved to a pure channel function. Mutations in anoctamin genes ( ANO3, ANO5, ANO6, and ANO10) cause various genetic diseases. These diseases suggest the involvement of anoctamins in a variety of cell functions whose link with ion transport and/or lipid scrambling needs to be clarified.

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2

Schreiber, Rainer, Jiraporn Ousingsawat, and Karl Kunzelmann. "Targeting of Intracellular TMEM16 Proteins to the Plasma Membrane and Activation by Purinergic Signaling." International Journal of Molecular Sciences 21, no. 11 (June 5, 2020): 4065. http://dx.doi.org/10.3390/ijms21114065.

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Anoctamins such as TMEM16A and TMEM16B are Ca2+-dependent Cl− channels activated through purinergic receptor signaling. TMEM16A (ANO1), TMEM16B (ANO2) and TMEM16F (ANO6) are predominantly expressed at the plasma membrane and are therefore well accessible for functional studies. While TMEM16A and TMEM16B form halide-selective ion channels, TMEM16F and probably TMEM16E operate as phospholipid scramblases and nonselective ion channels. Other TMEM16 paralogs are expressed mainly in intracellular compartments and are therefore difficult to study at the functional level. Here, we report that TMEM16E (ANO5), -H (ANO8), -J (ANO9) and K (ANO10) are targeted to the plasma membrane when fused to a C-terminal CAAX (cysteine, two aliphatic amino acids plus methionin, serine, alanin, cystein or glutamin) motif. These paralogs produce Ca2+-dependent ion channels. Surprisingly, expression of the TMEM16 paralogs in the plasma membrane did not produce additional scramblase activity. In contrast, endogenous scrambling induced by stimulation of purinergic P2X7 receptors was attenuated, in parallel with reduced plasma membrane blebbing. This could suggest that intracellular TMEM16 paralogs operate differently when compared to plasma membrane-localized TMEM16F, and may even stabilize intracellular membranes. Alternatively, CAAX tagging, which leads to expression in non-raft compartments of the plasma membrane, may antagonize phosphatidylserine exposure by endogenous raft-located TMEM16F. CAAX-containing constructs may be useful to further investigate the molecular properties of intracellular TMEM16 proteins.

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3

Thomas-Gatewood, Candice, Zachary P. Neeb, Simon Bulley, Adebowale Adebiyi, John P. Bannister, M. Dennis Leo, and Jonathan H. Jaggar. "TMEM16A channels generate Ca2+-activated Cl− currents in cerebral artery smooth muscle cells." American Journal of Physiology-Heart and Circulatory Physiology 301, no. 5 (November 2011): H1819—H1827. http://dx.doi.org/10.1152/ajpheart.00404.2011.

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Transmembrane protein (TMEM)16A channels are recently discovered membrane proteins that display electrophysiological properties similar to classic Ca2+-activated Cl− (ClCa) channels in native cells. The molecular identity of proteins that generate ClCa currents in smooth muscle cells (SMCs) of resistance-size arteries is unclear. Similarly, whether cerebral artery SMCs generate ClCa currents is controversial. Here, using molecular biology and patch-clamp electrophysiology, we examined TMEM16A channel expression and characterized Cl− currents in arterial SMCs of resistance-size rat cerebral arteries. RT-PCR amplified transcripts for TMEM16A but not TMEM16B–TMEM16H, TMEM16J, or TMEM16K family members in isolated pure cerebral artery SMCs. Western blot analysis using an antibody that recognized recombinant (r)TMEM16A channels detected TMEM16A protein in cerebral artery lysates. Arterial surface biotinylation and immunofluorescence indicated that TMEM16A channels are located primarily within the arterial SMC plasma membrane. Whole cell ClCa currents in arterial SMCs displayed properties similar to those generated by rTMEM16A channels, including Ca2+ dependence, current-voltage relationship linearization by an elevation in intracellular Ca2+ concentration, a Nerstian shift in reversal potential induced by reducing the extracellular Cl− concentration, and a negative reversal potential shift when substituting extracellular I− for Cl−. A pore-targeting TMEM16A antibody similarly inhibited both arterial SMC ClCa and rTMEM16A currents. TMEM16A knockdown using small interfering RNA also inhibited arterial SMC ClCa currents. In summary, these data indicate that TMEM16A channels are expressed, insert into the plasma membrane, and generate ClCa currents in cerebral artery SMCs.

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4

Gyobu, Sayuri, Haruhiko Miyata, Masahito Ikawa, Daiju Yamazaki, Hiroshi Takeshima, Jun Suzuki, and Shigekazu Nagata. "A Role of TMEM16E Carrying a Scrambling Domain in Sperm Motility." Molecular and Cellular Biology 36, no. 4 (December 14, 2015): 645–59. http://dx.doi.org/10.1128/mcb.00919-15.

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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 establishedTMEM16E-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 eggsin vitro. Our results suggest that TMEM16E may function as a phospholipid scramblase at inner membranes and that its defect affects sperm motility.

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5

Scudieri, Paolo, Elvira Sondo, Emanuela Caci, Roberto Ravazzolo, and Luis J. V. Galietta. "TMEM16A–TMEM16B chimaeras to investigate the structure–function relationship of calcium-activated chloride channels." Biochemical Journal 452, no. 3 (May 31, 2013): 443–55. http://dx.doi.org/10.1042/bj20130348.

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TMEM16A and TMEM16B proteins are CaCCs (Ca2+-activated Cl− channels) with eight putative transmembrane segments. As shown previously, expression of TMEM16B generates CaCCs characterized by a 10-fold lower Ca2+ affinity and by faster activation and deactivation kinetics with respect to TMEM16A. To investigate the basis of the different properties, we generated chimaeric proteins in which different domains of the TMEM16A protein were replaced by the equivalent domains of TMEM16B. Replacement of the N-terminus, TMD (transmembrane domain) 1–2, the first intracellular loop and TMD3–4 did not change the channel's properties. Instead, replacement of intracellular loop 3 decreased the apparent Ca2+ affinity by nearly 8-fold with respect to wild-type TMEM16A. In contrast, the membrane currents derived from chimaeras containing TMD7–8 or the C-terminus of TMEM16B showed higher activation and deactivation rates without a change in Ca2+ sensitivity. Significantly accelerated kinetics were also found when the entire C-terminus of the TMEM16A protein (77 amino acid residues) was deleted. Our findings indicate that the third intracellular loop of TMEM16A and TMEM16B is the site involved in Ca2+-sensitivity, whereas the C-terminal part, including TMD7–8, affect the rate of transition between the open and the closed state.

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6

Agostinelli, Emilio, and Paolo Tammaro. "Polymodal Control of TMEM16x Channels and Scramblases." International Journal of Molecular Sciences 23, no. 3 (January 29, 2022): 1580. http://dx.doi.org/10.3390/ijms23031580.

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The TMEM16A/anoctamin-1 calcium-activated chloride channel (CaCC) contributes to a range of vital functions, such as the control of vascular tone and epithelial ion transport. The channel is a founding member of a family of 10 proteins (TMEM16x) with varied functions; some members (i.e., TMEM16A and TMEM16B) serve as CaCCs, while others are lipid scramblases, combine channel and scramblase function, or perform additional cellular roles. TMEM16x proteins are typically activated by agonist-induced Ca2+ release evoked by Gq-protein-coupled receptor (GqPCR) activation; thus, TMEM16x proteins link Ca2+-signalling with cell electrical activity and/or lipid transport. Recent studies demonstrate that a range of other cellular factors—including plasmalemmal lipids, pH, hypoxia, ATP and auxiliary proteins—also control the activity of the TMEM16A channel and its paralogues, suggesting that the TMEM16x proteins are effectively polymodal sensors of cellular homeostasis. Here, we review the molecular pathophysiology, structural biology, and mechanisms of regulation of TMEM16x proteins by multiple cellular factors.

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7

Betto, Giulia, O. Lijo Cherian, Simone Pifferi, Valentina Cenedese, Anna Boccaccio, and Anna Menini. "Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel." Journal of General Physiology 143, no. 6 (May 26, 2014): 703–18. http://dx.doi.org/10.1085/jgp.201411182.

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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 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.

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8

Shimizu, Takahiro, Takahiro Iehara, Kaori Sato, Takuto Fujii, Hideki Sakai, and Yasunobu Okada. "TMEM16F is a component of a Ca2+-activated Cl− channel but not a volume-sensitive outwardly rectifying Cl− channel." American Journal of Physiology-Cell Physiology 304, no. 8 (April 15, 2013): C748—C759. http://dx.doi.org/10.1152/ajpcell.00228.2012.

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TMEM16 (transmembrane protein 16) proteins, which possess eight putative transmembrane domains with intracellular NH2- and COOH-terminal tails, are thought to comprise a Cl− channel family. The function of TMEM16F, a member of the TMEM16 family, has been greatly controversial. In the present study, we performed whole cell patch-clamp recordings to investigate the function of human TMEM16F. In TMEM16F-transfected HEK293T cells but not TMEM16K- and mock-transfected cells, activation of membrane currents with strong outward rectification was found to be induced by application of a Ca2+ ionophore, ionomycin, or by an increase in the intracellular free Ca2+ concentration. The free Ca2+ concentration for half-maximal activation of TMEM16F currents was 9.6 μM, which is distinctly higher than that for TMEM16A/B currents. The outwardly rectifying current-voltage relationship for TMEM16F currents was not changed by an increase in the intracellular Ca2+ level, in contrast to TMEM16A/B currents. The Ca2+-activated TMEM16F currents were anion selective, because replacing Cl− with aspartate− in the bathing solution without changing cation concentrations caused a positive shift of the reversal potential. The anion selectivity sequence of the TMEM16F channel was I− > Br− > Cl− > F− > aspartate−. Niflumic acid, a Ca2+-activated Cl− channel blocker, inhibited the TMEM16F-dependent Cl− currents. Neither overexpression nor knockdown of TMEM16F affected volume-sensitive outwardly rectifying Cl− channel (VSOR) currents activated by osmotic swelling or apoptotic stimulation. These results demonstrate that human TMEM16F is an essential component of a Ca2+-activated Cl− channel with a Ca2+ sensitivity that is distinct from that of TMEM16A/B and that it is not related to VSOR activity.

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9

Cenedese, Valentina, Giulia Betto, Fulvio Celsi, O. Lijo Cherian, Simone Pifferi, and Anna Menini. "The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop." Journal of General Physiology 139, no. 4 (March 12, 2012): 285–94. http://dx.doi.org/10.1085/jgp.201110764.

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Ca2+-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 Ca2+ 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 Ca2+ concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates 386EEEEE390 and 399EYE401. The EYE deletion did not significantly modify the apparent Ca2+ dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca2+ affinity but modified the voltage dependence, shifting the conductance–voltage relations toward more positive voltages. These findings indicate that glutamates E367 and 386EEEEE390 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.

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10

Davis, Alison J., Abigail S. Forrest, Thomas A. Jepps, Maria L. Valencik, Michael Wiwchar, Cherie A. Singer, William R. Sones, Iain A. Greenwood, and Normand Leblanc. "Expression profile and protein translation of TMEM16A in murine smooth muscle." American Journal of Physiology-Cell Physiology 299, no. 5 (November 2010): C948—C959. http://dx.doi.org/10.1152/ajpcell.00018.2010.

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Recently, overexpression of the genes TMEM16A and TMEM16B has been shown to produce currents qualitatively similar to native Ca2+-activated Cl− currents ( IClCa) in vascular smooth muscle. However, there is no information about this new gene family in vascular smooth muscle, where Cl− channels are a major depolarizing mechanism. Qualitatively similar Cl− currents were evoked by a pipette solution containing 500 nM Ca2+ in smooth muscle cells isolated from BALB/c mouse portal vein, thoracic aorta, and carotid artery. Quantitative PCR using SYBR Green chemistry and primers specific for transmembrane protein (TMEM) 16A or the closely related TMEM16B showed TMEM16A expression as follows: portal vein > thoracic aorta > carotid artery > brain. In addition, several alternatively spliced variant transcripts of TMEM16A were detected. In contrast, TMEM16B expression was very low in smooth muscle. Western blot analysis with different antibodies directed against TMEM16A revealed a number of products with a consistent band at ∼120 kDa, except portal vein, where an 80-kDa band predominated. TMEM16A protein was identified in the smooth muscle layers of 4-μm-thick slices of portal vein, thoracic aorta, and carotid artery. In isolated myocytes, fluorescence specific to a TMEM16A antibody was detected diffusely throughout the cytoplasm, as well as near the membrane. The same antibody used in Western blot analysis of lysates from vascular tissues also recognized an ∼147-kDa mouse TMEM16A-green fluorescent protein (GFP) fusion protein expressed in HEK 293 cells, which correlated to a similar band detected by a GFP antibody. Patch-clamp experiments revealed that IClCa generated by transfection of TMEM16A-GFP in HEK 293 cells displayed remarkable similarities to IClCa recorded in vascular myocytes, including slow kinetics, steep outward rectification, and a response similar to the pharmacological agent niflumic acid. This study shows that TMEM16A expression is robust in murine vascular smooth muscle cells, consolidating the view that this gene is a viable candidate for the native Ca2+-activated Cl− channel in this cell type.

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11

Nguyen, Dung M., Louisa S. Chen, Wei-Ping Yu, and Tsung-Yu Chen. "Comparison of ion transport determinants between a TMEM16 chloride channel and phospholipid scramblase." Journal of General Physiology 151, no. 4 (January 22, 2019): 518–31. http://dx.doi.org/10.1085/jgp.201812270.

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Two TMEM16 family members, TMEM16A and TMEM16F, have different ion transport properties. Upon activation by intracellular Ca2+, TMEM16A—a Ca2+-activated Cl− channel—is more selective for anions than cations, whereas TMEM16F—a phospholipid scramblase—appears to transport both cations and anions. Under saturating Ca2+ conditions, the current–voltage (I-V) relationships of these two proteins also differ; the I-V curve of TMEM16A is linear, while that of TMEM16F is outwardly rectifying. We previously found that mutating a positively charged lysine residue (K584) in the ion transport pathway to glutamine converted the linear I-V curve of TMEM16A to an outwardly rectifying curve. Interestingly, the corresponding residue in the outwardly rectifying TMEM16F is also a glutamine (Q559). Here, we examine the ion transport functions of TMEM16 molecules and compare the roles of K584 of TMEM16A and Q559 of TMEM16F in controlling the rectification of their respective I-V curves. We find that rectification of TMEM16A is regulated electrostatically by the side-chain charge on the residue at position 584, whereas the charge on residue 559 in TMEM16F has little effect. Unexpectedly, mutation of Q559 to aromatic amino acid residues significantly alters outward rectification in TMEM16F. These same mutants show reduced Ca2+-induced current rundown (or desensitization) compared with wild-type TMEM16F. A mutant that removes the rundown of TMEM16F could facilitate the study of ion transport mechanisms in this phospholipid scramblase in the same way that a CLC-0 mutant in which inactivation (or closure of the slow gate) is suppressed was used in our previous studies.

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12

Gyobu, Sayuri, Kenji Ishihara, Jun Suzuki, Katsumori Segawa, and Shigekazu Nagata. "Characterization of the scrambling domain of the TMEM16 family." Proceedings of the National Academy of Sciences 114, no. 24 (May 30, 2017): 6274–79. http://dx.doi.org/10.1073/pnas.1703391114.

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The TMEM16 protein family has 10 members, each of which carries 10 transmembrane segments. TMEM16A and 16B are Ca2+-activated Cl− channels. Several other members, including TMEM16F, promote phospholipid scrambling between the inner and outer leaflets of a cell membrane in response to intracellular Ca2+. However, the mechanism by which TMEM16 proteins translocate phospholipids in plasma membranes remains elusive. Here we show that Ca2+-activated, TMEM16F-supported phospholipid scrambling proceeds at 4 °C. Similar to TMEM16F and 16E, seven TMEM16 family members were found to carry a domain (SCRD; scrambling domain) spanning the fourth and fifth transmembrane segments that conferred scrambling ability to TMEM16A. By introducing point mutations into TMEM16F, we found that a lysine in the fourth transmembrane segment of the SCRD as well as an arginine in the third and a glutamic acid in the sixth transmembrane segment were important for exposing phosphatidylserine from the inner to the outer leaflet. However, their role in internalizing phospholipids was limited. Our results suggest that TMEM16 provides a cleft containing hydrophilic “stepping stones” for the outward translocation of phospholipids.

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13

Pietra, Gianluca, Michele Dibattista, Anna Menini, Johannes Reisert, and Anna Boccaccio. "The Ca2+-activated Cl− channel TMEM16B regulates action potential firing and axonal targeting in olfactory sensory neurons." Journal of General Physiology 148, no. 4 (September 12, 2016): 293–311. http://dx.doi.org/10.1085/jgp.201611622.

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The Ca2+-activated Cl− channel TMEM16B is highly expressed in the cilia of olfactory sensory neurons (OSNs). Although a large portion of the odor-evoked transduction current is carried by Ca2+-activated Cl− channels, their role in olfaction is still controversial. A previous report (Billig et al. 2011. Nat. Neurosci. http://dx.doi.org/10.1038/nn.2821) showed that disruption of the TMEM16b/Ano2 gene in mice abolished Ca2+-activated Cl− currents in OSNs but did not produce any major change in olfactory behavior. Here we readdress the role of TMEM16B in olfaction and show that TMEM16B knockout (KO) mice have behavioral deficits in odor-guided food-finding ability. Moreover, as the role of TMEM16B in action potential (AP) firing has not yet been studied, we use electrophysiological recording methods to measure the firing activity of OSNs. Suction electrode recordings from isolated olfactory neurons and on-cell loose-patch recordings from dendritic knobs of neurons in the olfactory epithelium show that randomly selected neurons from TMEM16B KO mice respond to stimulation with increased firing activity than those from wild-type (WT) mice. Because OSNs express different odorant receptors (ORs), we restrict variability by using a mouse line that expresses a GFP-tagged I7 OR, which is known to be activated by heptanal. In response to heptanal, we measure dramatic changes in the firing pattern of I7-expressing neurons from TMEM16B KO mice compared with WT: responses are prolonged and display a higher number of APs. Moreover, lack of TMEM16B causes a markedly reduced basal spiking activity in I7-expressing neurons, together with an alteration of axonal targeting to the olfactory bulb, leading to the appearance of supernumerary I7 glomeruli. Thus, TMEM16B controls AP firing and ensures correct glomerular targeting of OSNs expressing I7. Altogether, these results show that TMEM16B does have a relevant role in normal olfaction.

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14

Cruz-Rangel, Silvia, José J. De Jesús-Pérez, Juan A. Contreras-Vite, Patricia Pérez-Cornejo, H. Criss Hartzell, and Jorge Arreola. "Gating modes of calcium-activated chloride channels TMEM16A and TMEM16B." Journal of Physiology 593, no. 24 (December 7, 2015): 5283–98. http://dx.doi.org/10.1113/jp271256.

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15

Scudieri, Paolo, Elvira Sondo, Loretta Ferrera, and Luis J. V. Galietta. "The anoctamin family: TMEM16A and TMEM16B as calcium-activated chloride channels." Experimental Physiology 97, no. 2 (November 11, 2011): 177–83. http://dx.doi.org/10.1113/expphysiol.2011.058198.

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16

Dibattista, Michele, Asma Amjad, Devendra Kumar Maurya, Claudia Sagheddu, Giorgia Montani, Roberto Tirindelli, and Anna Menini. "Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons." Journal of General Physiology 140, no. 1 (June 25, 2012): 3–15. http://dx.doi.org/10.1085/jgp.201210780.

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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.

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Kim, Andrew Y., Huanghe Yang, Tovo David, Jason Tien, Shaun R. Coughlin, Yuh Nung Jan, and Lily Jan. "TMEM16F Ion Channel Regulates Calcium-Dependent PS Exposure, Hemostasis, and Thrombosis." Blood 120, no. 21 (November 16, 2012): 1111. http://dx.doi.org/10.1182/blood.v120.21.1111.1111.

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Abstract Abstract 1111 Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca2+-activated Cl− channels (CaCCs) is linked to Scott Syndrome with deficient Ca2+-dependent lipid scrambling. We generated TMEM16F-knockout mice that exhibit bleeding defects associated with platelet deficiency in Ca2+-dependent phosphatidylserine exposure and procoagulant activity, and lack a Ca2+-activated cation current in the platelet precursor megakaryocytes. TMEM16F channels permeate both monovalent and divalent cations including Ca2+, and exhibit synergistic gating by Ca2+ and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a novel Ca2+-activated channel permeable to Ca2+ and critical for Ca2+-dependent scramblase activity. Disclosures: No relevant conflicts of interest to declare.

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18

Nguyen, Dung, Hwoi Kwon, and Tsung-Yu Chen. "Divalent Cation Modulation of Ion Permeation in TMEM16 Proteins." International Journal of Molecular Sciences 22, no. 4 (February 23, 2021): 2209. http://dx.doi.org/10.3390/ijms22042209.

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Intracellular divalent cations control the molecular function of transmembrane protein 16 (TMEM16) family members. Both anion channels (such as TMEM16A) and phospholipid scramblases (such as TMEM16F) in this family are activated by intracellular Ca2+ in the low µM range. In addition, intracellular Ca2+ or Co2+ at mM concentrations have been shown to further potentiate the saturated Ca2+-activated current of TMEM16A. In this study, we found that all alkaline earth divalent cations in mM concentrations can generate similar potentiation effects in TMEM16A when applied intracellularly, and that manipulations thought to deplete membrane phospholipids weaken the effect. In comparison, mM concentrations of divalent cations minimally potentiate the current of TMEM16F but significantly change its cation/anion selectivity. We suggest that divalent cations may increase local concentrations of permeant ions via a change in pore electrostatic potential, possibly acting through phospholipid head groups in or near the pore. Monovalent cations appear to exert a similar effect, although with a much lower affinity. Our findings resolve controversies regarding the ion selectivity of TMEM16 proteins. The physiological role of this mechanism, however, remains elusive because of the nearly constant high cation concentrations in cytosols.

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Ta, Chau M., Kathryn E. Acheson, Nils J. G. Rorsman, Remco C. Jongkind, and Paolo Tammaro. "Contrasting effects of phosphatidylinositol 4,5-bisphosphate on cloned TMEM16A and TMEM16B channels." British Journal of Pharmacology 174, no. 18 (August 10, 2017): 2984–99. http://dx.doi.org/10.1111/bph.13913.

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20

Grigoriev, V. V. "Calcium-activated chloride channels: structure, properties, role in physiological and pathological processes." Biomeditsinskaya Khimiya 67, no. 1 (January 2021): 17–33. http://dx.doi.org/10.18097/pbmc20216701017.

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Анотація:

Ca2+-activated chloride channels (CaCC) are a class of intracellular calcium activated chloride channels that mediate numerous physiological functions. In 2008, the molecular structure of CaCC was determined. CaCC are formed by the protein known as anoctamine 1 (ANO1 or TMEM16A). CaCC mediates the secretion of Cl– in secretory epithelia, such as the airways, salivary glands, intestines, renal tubules, and sweat glands. The presence of CaCC has also been recognized in the vascular muscles, smooth muscles of the respiratory tract, which control vascular tone and hypersensitivity of the respiratory tract. TMEM16A is activated in many cancers; it is believed that TMEM16A is involved in carcinogenesis. TMEM16A is also involved in cancer cells proliferation. The role of TMEM16A in the mechanisms of hypertension, asthma, cystic fibrosis, nociception, and dysfunction of the gastrointestinal tract has been determined. In addition to TMEM16A, its isoforms are involved in other physiological and pathophysiological processes. TMEM16B (or ANO2) is involved in the sense of smell, while ANO6 works like scramblase, and its mutation causes a rare bleeding disorder, known as Scott syndrome. ANO5 is associated with muscle and bone diseases. TMEM16A interacts with various cellular signaling pathways including: epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPK), calmodulin (CaM) kinases, transforming growth factor TGF-β. The review summarizes existing information on known natural and synthetic compounds that can block/modulate CaCC currents and their effect on some pathologies in which CaCC is involved.

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Hernandez-Clavijo, Andres, Nicole Sarno, Kevin Y. Gonzalez-Velandia, Rudolf Degen, David Fleck, Jason R. Rock, Marc Spehr, Anna Menini, and Simone Pifferi. "TMEM16A and TMEM16B Modulate Pheromone-Evoked Action Potential Firing in Mouse Vomeronasal Sensory Neurons." eneuro 8, no. 5 (August 25, 2021): ENEURO.0179–21.2021. http://dx.doi.org/10.1523/eneuro.0179-21.2021.

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22

Vocke, Kerstin, Kristin Dauner, Anne Hahn, Anne Ulbrich, Jana Broecker, Sandro Keller, Stephan Frings, and Frank Möhrlen. "Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels." Journal of General Physiology 142, no. 4 (September 30, 2013): 381–404. http://dx.doi.org/10.1085/jgp.201311015.

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Анотація:

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.

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Amjad, Asma, Andres Hernandez-Clavijo, Simone Pifferi, Devendra Kumar Maurya, Anna Boccaccio, Jessica Franzot, Jason Rock, and Anna Menini. "Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons." Journal of General Physiology 145, no. 4 (March 16, 2015): 285–301. http://dx.doi.org/10.1085/jgp.201411348.

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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.

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Maurya, Devendra Kumar, and Anna Menini. "Developmental expression of the calcium-activated chloride channels TMEM16A and TMEM16B in the mouse olfactory epithelium." Developmental Neurobiology 74, no. 7 (December 17, 2013): 657–75. http://dx.doi.org/10.1002/dneu.22159.

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25

Zhang, Yang, Zhushan Zhang, Shaohua Xiao, Jason Tien, Son Le, Trieu Le, Lily Y. Jan, and Huanghe Yang. "Inferior Olivary TMEM16B Mediates Cerebellar Motor Learning." Neuron 95, no. 5 (August 2017): 1103–11. http://dx.doi.org/10.1016/j.neuron.2017.08.010.

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26

Zhang, Yang, Zhushan Zhang, Shaohua Xiao, Trieu Le, Son Le, Lily Jan, Jason Tien, and Huanghe Yang. "Inferior Olivary TMEM16B Mediates Cerebellar Motor Learning." Biophysical Journal 114, no. 3 (February 2018): 132a—133a. http://dx.doi.org/10.1016/j.bpj.2017.11.752.

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27

Grubb, Søren, Kristian A. Poulsen, Christian Ammitzbøll Juul, Tania Kyed, Thomas K. Klausen, Erik Hviid Larsen, and Else K. Hoffmann. "TMEM16F (Anoctamin 6), an anion channel of delayed Ca2+ activation." Journal of General Physiology 141, no. 5 (April 29, 2013): 585–600. http://dx.doi.org/10.1085/jgp.201210861.

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Анотація:

Members of the TMEM16 (Anoctamin) family of membrane proteins have been shown to be essential constituents of the Ca2+-activated Cl− channel (CaCC) in many cell types. In this study, we have investigated the electrophysiological properties of mouse TMEM16F. Heterologous expression of TMEM16F in HEK293 cells resulted in plasma membrane localization and an outwardly rectifying ICl,Ca that was activated with a delay of several minutes. Furthermore, a significant Na+ current was activated, and the two permeabilities were correlated according to PNa = 0.3 PCl. The current showed an EC50 of 100 µM intracellular free Ca2+ concentration and an Eisenman type 1 anion selectivity sequence of PSCN > PI > PBr > PCl > PAsp. The mTMEM16F-associated ICl,Ca was abolished in one mutant of the putative pore region (R592E) but retained in two other mutants (K616E and R636E). The mutant K616E had a lower relative permeability to iodide, and the mutant R636E had an altered anion selectivity sequence (PSCN = PI = PBr = PCl > PAsp). Our data provide evidence that TMEM16F constitutes a Ca2+-activated anion channel or a pore-forming subunit of an anion channel with properties distinct from TMEM16A.

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Yamamura, Hisao, Kaori Nishimura, Yumiko Hagihara, Yoshiaki Suzuki, and Yuji Imaizumi. "TMEM16A and TMEM16B channel proteins generate Ca2+-activated Cl−current and regulate melatonin secretion in rat pineal glands." Journal of Biological Chemistry 293, no. 3 (November 29, 2017): 995–1006. http://dx.doi.org/10.1074/jbc.ra117.000326.

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29

Pifferi, Simone. "Permeation Mechanisms in the TMEM16B Calcium-Activated Chloride Channels." PLOS ONE 12, no. 1 (January 3, 2017): e0169572. http://dx.doi.org/10.1371/journal.pone.0169572.

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30

Hernandez, Adan, Alfredo Alaniz-Palacios, Juan A. Contreras-Vite, and Ataúlfo Martínez-Torres. "Positive modulation of the TMEM16B mediated currents by TRPV4 antagonist." Biochemistry and Biophysics Reports 28 (December 2021): 101180. http://dx.doi.org/10.1016/j.bbrep.2021.101180.

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31

Pifferi, Simone, Michele Dibattista, and Anna Menini. "TMEM16B induces chloride currents activated by calcium in mammalian cells." Pflügers Archiv - European Journal of Physiology 458, no. 6 (May 28, 2009): 1023–38. http://dx.doi.org/10.1007/s00424-009-0684-9.

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32

Pifferi, Simone. "Molecular Mechanisms of Permeation in TMEM16b Ca2+-Activated Cl− Channel." Biophysical Journal 110, no. 3 (February 2016): 291a. http://dx.doi.org/10.1016/j.bpj.2015.11.1572.

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Le, Trieu, Son C. Le, Yang Zhang, Pengfei Liang, and Huanghe Yang. "Evidence that polyphenols do not inhibit the phospholipid scramblase TMEM16F." Journal of Biological Chemistry 295, no. 35 (July 24, 2020): 12537–44. http://dx.doi.org/10.1074/jbc.ac120.014872.

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TMEM16 Ca2+-activated phospholipid scramblases (CaPLSases) mediate rapid transmembrane phospholipid flip-flop and as such play essential roles in various physiological and pathological processes such as blood coagulation, skeletal development, viral infection, cell-cell fusion, and ataxia. Pharmacological tools specifically targeting TMEM16 CaPLSases are urgently needed to understand these novel membrane transporters and their contributions to health and disease. Tannic acid (TA) and epigallocatechin gallate (EGCG) were recently reported as promising TMEM16F CaPLSase inhibitors. However, our present study shows that TA and EGCG do not inhibit the phospholipid-scrambling or ion conduction activities of the dual-functional TMEM16F. Instead, we found that TA and EGCG mainly acted as fluorescence quenchers that rapidly suppress the fluorophores conjugated to annexin V, a phosphatidylserine-binding probe commonly used to report on TMEM16 CaPLSase activity. These data demonstrate the false positive effects of TA and EGCG on inhibiting TMEM16F phospholipid scrambling and discourage the use of these polyphenols as CaPLSase inhibitors. Appropriate controls as well as a combination of both fluorescence imaging and electrophysiological validation are necessary in future endeavors to develop TMEM16 CaPLSase inhibitors.

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Ousingsawat, Jiraporn, Rainer Schreiber, and Karl Kunzelmann. "TMEM16F/Anoctamin 6 in Ferroptotic Cell Death." Cancers 11, no. 5 (May 5, 2019): 625. http://dx.doi.org/10.3390/cancers11050625.

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Ca2+ activated Cl− channels (TMEM16A; ANO1) support cell proliferation and cancer growth. Expression of TMEM16A is strongly enhanced in different types of malignomas. In contrast, TMEM16F (ANO6) operates as a Ca2+ activated chloride/nonselective ion channel and scrambles membrane phospholipids to expose phosphatidylserine at the cell surface. Both phospholipid scrambling and cell swelling induced through activation of nonselective ion currents appear to destabilize the plasma membrane thereby causing cell death. There is growing evidence that activation of TMEM16F contributes to various forms of regulated cell death. In the present study, we demonstrate that ferroptotic cell death, occurring during peroxidation of plasma membrane phospholipids activates TMEM16F. Ferroptosis was induced by erastin, an inhibitor of the cystine-glutamate antiporter and RSL3, an inhibitor of glutathione peroxidase 4 (GPX4). Cell death was largely reduced in the intestinal epithelium, and in peritoneal macrophages isolated from mice with tissue-specific knockout of TMEM16F. We show that TMEM16F is activated during erastin and RSL3-induced ferroptosis. In contrast, inhibition of ferroptosis by ferrostatin-1 and by inhibitors of TMEM16F block TMEM16F currents and inhibit cell death. We conclude that activation of TMEM16F is a crucial component during ferroptotic cell death, a finding that may be useful to induce cell death in cancer cells.

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35

Rasche, Sebastian, Bastian Toetter, Jenny Adler, Astrid Tschapek, Julia F. Doerner, Stefan Kurtenbach, Hanns Hatt, Helmut Meyer, Bettina Warscheid, and Eva M. Neuhaus. "Tmem16b is Specifically Expressed in the Cilia of Olfactory Sensory Neurons." Chemical Senses 35, no. 3 (January 25, 2010): 239–45. http://dx.doi.org/10.1093/chemse/bjq007.

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36

Pifferi, Simone, Valentina Cenedese, and Anna Menini. "Anoctamin 2/TMEM16B: a calcium-activated chloride channel in olfactory transduction." Experimental Physiology 97, no. 2 (October 13, 2011): 193–99. http://dx.doi.org/10.1113/expphysiol.2011.058230.

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37

Kim, Hanggu, Eunyoung Kim, and Byoung-Cheol Lee. "Investigation of Phosphatidylserine-Transporting Activity of Human TMEM16C Isoforms." Membranes 12, no. 10 (October 17, 2022): 1005. http://dx.doi.org/10.3390/membranes12101005.

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Lipid scrambling is a rapid process that dissipates the asymmetrical distribution of phospholipids in the plasma membrane. It is involved in various physiological functions such as blood coagulation and apoptosis. Many TMEM16 members are recognized as Ca2+-activated phospholipid scramblases, which transport phospholipids between the two leaflets of the plasma membrane nonspecifically and bidirectionally; among these, TMEM16C is abundant in the brain, especially in neuronal cells. We investigated the scrambling activity of three human TMEM16C isoforms with different N-terminus lengths. After optimizing conditions to minimize endogenous scrambling activity, an annexin V-based imaging assay was used to detect phosphatidylserine (PS) scrambling in 293T cells. Unlike previous results, our data showed that human TMEM16C isoform 1 and isoform 3 exposed PS to the cell surface. A surface biotinylation assay showed that the surface expression of isoform 2, which did not show scrambling activity, was ~5 times lower than the other isoforms. In contrast to other TMEM16 proteins, flux assays and electrophysiology recording showed TMEM16C does not possess ion-transporting activity. We conclude that the N-terminus of TMEM16C determines whether TMEM16C can translocate to the plasma membrane and facilitate scrambling activity; membrane-localized TMEM16C isoforms 1 and 3 transport PS to the outer leaflet.

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Falzone, Maria E., Mattia Malvezzi, Byoung-Cheol Lee, and Alessio Accardi. "Known structures and unknown mechanisms of TMEM16 scramblases and channels." Journal of General Physiology 150, no. 7 (June 18, 2018): 933–47. http://dx.doi.org/10.1085/jgp.201711957.

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Анотація:

The TMEM16 family of membrane proteins is composed of both Ca2+-gated Cl− channels and Ca2+-dependent phospholipid scramblases. The functional diversity of TMEM16s underlies their involvement in numerous signal transduction pathways that connect changes in cytosolic Ca2+ levels to cellular signaling networks. Indeed, defects in the function of several TMEM16s cause a variety of genetic disorders, highlighting their fundamental pathophysiological importance. Here, we review how our mechanistic understanding of TMEM16 function has been shaped by recent functional and structural work. Remarkably, the recent determination of near-atomic-resolution structures of TMEM16 proteins of both functional persuasions has revealed how relatively minimal rearrangements in the substrate translocation pathway are sufficient to precipitate the dramatic functional differences that characterize the family. These structures, when interpreted in the light of extensive functional analysis, point to an unusual mechanism for Ca2+-dependent activation of TMEM16 proteins in which substrate permeation is regulated by a combination of conformational rearrangements and electrostatics. These breakthroughs pave the way to elucidate the mechanistic bases of ion and lipid transport by the TMEM16 proteins and unravel the molecular links between these transport activities and their function in human pathophysiology.

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Ji, Wanying, Donghong Shi, Sai Shi, Xiao Yang, Yafei Chen, Hailong An, and Chunli Pang. "TMEM16A Protein: Calcium-Binding Site and its Activation Mechanism." Protein & Peptide Letters 28, no. 12 (December 2021): 1338–48. http://dx.doi.org/10.2174/0929866528666211105112131.

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Abstract: TMEM16A mediates the calcium-activated transmembrane flow of chloride ions and a variety of physiological functions. The binding of cytoplasmic calcium ions of TMEM16A and the consequent conformational changes of it are the key issues to explore the structure-function relationship. In recent years, researchers have explored this issue through electrophysiological experiments, structure resolving, molecular dynamic simulation, and other methods. The structures of TMEM16 family members determined by cryo-Electron microscopy (cryo-EM) and X-ray crystallization provide the primary basis for the investigation of the molecular mechanism of TMEM16A. However, the binding and activation mechanism of calcium ions in TMEM16A are still unclear and controversial. This review discusses four Ca2+ sensing sites of TMEM16A and analyzes activation properties of TMEM16A by them, which will help understand the structure-function relationship of TMEM16A and throw light on the molecular design targeting the TMEM16A channel.

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40

Yarotskyy, Viktor, Arianna R. S. Lark, Sara R. Nass, Yun K. Hahn, Michael G. Marone, A. Rory McQuiston, Pamela E. Knapp, and Kurt F. Hauser. "Chloride channels with ClC-1-like properties differentially regulate the excitability of dopamine receptor D1- and D2-expressing striatal medium spiny neurons." American Journal of Physiology-Cell Physiology 322, no. 3 (March 1, 2022): C395—C409. http://dx.doi.org/10.1152/ajpcell.00397.2021.

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Dynamic chloride (Cl−) regulation is critical for synaptic inhibition. In mature neurons, Cl− influx and extrusion are primarily controlled by ligand-gated anion channels (GABAA and glycine receptors) and the potassium chloride cotransporter K+-Cl− cotransporter 2 (KCC2), respectively. Here, we report for the first time, to our knowledge, a presence of a new source of Cl− influx in striatal neurons with properties similar to chloride voltage-gated channel 1 (ClC-1). Using whole cell patch-clamp recordings, we detected an outwardly rectifying voltage-dependent current that was impermeable to the large anion methanesulfonate (MsO−). The anionic current was sensitive to the ClC-1 inhibitor 9-anthracenecarboxylic acid (9-AC) and the nonspecific blocker phloretin. The mean fractions of anionic current inhibition by MsO−, 9-AC, and phloretin were not significantly different, indicating that anionic current was caused by active ClC-1-like channels. In addition, we found that Cl− current was not sensitive to the transmembrane protein 16A (TMEM16A; Ano1) inhibitor Ani9 and that the outward Cl− rectification was preserved even at a very high intracellular Ca2+ concentration (2 mM), indicating that TMEM16B ( Ano2) did not contribute to the total current. Western blotting and immunohistochemical analyses confirmed the presence of ClC-1 channels in the striatum mainly localized to the somata of striatal neurons. Finally, we found that 9-AC decreased action potential firing frequencies and increased excitability in medium spiny neurons (MSNs) expressing dopamine type 1 (D1) and type 2 (D2) receptors in the brain slices, respectively. We conclude that ClC-1-like channels are preferentially located at the somata of MSNs, are functional, and can modulate neuronal excitability.

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Keckeis, Susanne, Nadine Reichhart, Christophe Roubeix, and Olaf Strauß. "Anoctamin2 (TMEM16B) forms the Ca2+-activated Cl− channel in the retinal pigment epithelium." Experimental Eye Research 154 (January 2017): 139–50. http://dx.doi.org/10.1016/j.exer.2016.12.003.

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42

Betto, Giulia, O. Lijo Cherian, Simone Pifferi, Valentina Cenedese, Anna Boccaccio, and Anna Menini. "Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel." Journal of General Physiology 144, no. 1 (June 30, 2014): 125. http://dx.doi.org/10.1085/jgp.20141118206192014c.

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43

Gallos, George, Kenneth E. Remy, Jennifer Danielsson, Hiromi Funayama, Xiao Wen Fu, Herng-Yu Sucie Chang, Peter Yim, Dingbang Xu, and Charles W. Emala. "Functional expression of the TMEM16 family of calcium-activated chloride channels in airway smooth muscle." American Journal of Physiology-Lung Cellular and Molecular Physiology 305, no. 9 (November 1, 2013): L625—L634. http://dx.doi.org/10.1152/ajplung.00068.2013.

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Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of eight members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiological recordings, which were blocked by using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiological properties of CaCCs, and they modulate contractile tone in airway smooth muscle. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma.

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Jeng, Grace, Muskaan Aggarwal, Wei-Ping Yu, and Tsung-Yu Chen. "Independent activation of distinct pores in dimeric TMEM16A channels." Journal of General Physiology 148, no. 5 (October 17, 2016): 393–404. http://dx.doi.org/10.1085/jgp.201611651.

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Анотація:

The TMEM16 family encompasses Ca2+-activated Cl− channels (CaCCs) and lipid scramblases. These proteins are formed by two identical subunits, as confirmed by the recently solved crystal structure of a TMEM16 lipid scramblase. However, the high-resolution structure did not provide definitive information regarding the pore architecture of the TMEM16 channels. In this study, we express TMEM16A channels constituting two covalently linked subunits with different Ca2+ affinities. The dose–response curve of the heterodimer appears to be a weighted sum of two dose–response curves—one corresponding to the high-affinity subunit and the other to the low-affinity subunit. However, fluorescence resonance energy transfer experiments suggest that the covalently linked heterodimeric proteins fold and assemble as one molecule. Together these results suggest that activation of the two TMEM16A subunits likely activate independently of each other. The Ca2+ activation curve for the heterodimer at a low Ca2+ concentration range ([Ca2+] < 5 µM) is similar to that of the wild-type channel—the Hill coefficients in both cases are significantly greater than one. This suggests that Ca2+ binding to one subunit of TMEM16A is sufficient to activate the channel and that each subunit contains more than one Ca2+-binding site. We also take advantage of the I-V curve rectification that results from mutation of a pore residue to address the pore architecture of the channel. By introducing the pore mutation and the mutation that alters Ca2+ affinity in the same or different subunits, we demonstrate that activation of different subunits appears to be associated with the opening of different pores. These results suggest that the TMEM16A CaCC may also adopt a “double-barrel” pore architecture, similar to that found in CLC channels and transporters.

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Talbi, Khaoula, Jiraporn Ousingsawat, Raquel Centeio, Rainer Schreiber, and Karl Kunzelmann. "Calmodulin-Dependent Regulation of Overexpressed but Not Endogenous TMEM16A Expressed in Airway Epithelial Cells." Membranes 11, no. 9 (September 21, 2021): 723. http://dx.doi.org/10.3390/membranes11090723.

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Анотація:

Regulation of the Ca2+-activated Cl− channel TMEM16A by Ca2+/calmodulin (CAM) is discussed controversially. In the present study, we compared regulation of TMEM16A by Ca2+/calmodulin (holo-CAM), CAM-dependent kinase (CAMKII), and CAM-dependent phosphatase calcineurin in TMEM16A-overexpressing HEK293 cells and TMEM16A expressed endogenously in airway and colonic epithelial cells. The activator of the Ca2+/CAM-regulated K+ channel KCNN4, 1-EBIO, activated TMEM16A in overexpressing cells, but not in cells with endogenous expression of TMEM16A. Evidence is provided that CAM-interaction with TMEM16A modulates the Ca2+ sensitivity of the Cl− channel. Enhanced Ca2+ sensitivity of overexpressed TMEM16A explains its activity at basal (non-elevated) intracellular Ca2+ levels. The present results correspond well to a recent report that demonstrates a Ca2+-unbound form of CAM (apo-CAM) that is pre-associated with TMEM16A and mediates a Ca2+-dependent sensitization of activation (and inactivation). However, when using activators or inhibitors for holo-CAM, CAMKII, or calcineurin, we were unable to detect a significant impact of CAM, and limit evidence for regulation by CAM-dependent regulatory proteins on receptor-mediated activation of endogenous TMEM16A in airway or colonic epithelial cells. We propose that regulatory properties of TMEM16A and and other members of the TMEM16 family as detected in overexpression studies, should be validated for endogenous TMEM16A and physiological stimuli such as activation of phospholipase C (PLC)-coupled receptors.

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Kunzelmann, Karl, Jiraporn Ousingsawat, Roberta Benedetto, Ines Cabrita, and Rainer Schreiber. "Contribution of Anoctamins to Cell Survival and Cell Death." Cancers 11, no. 3 (March 19, 2019): 382. http://dx.doi.org/10.3390/cancers11030382.

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Before anoctamins (TMEM16 proteins) were identified as a family of Ca2+-activated chloride channels and phospholipid scramblases, the founding member anoctamin 1 (ANO1, TMEM16A) was known as DOG1, a marker protein for gastrointestinal stromal tumors (GIST). Meanwhile, ANO1 has been examined in more detail, and the role of ANO1 in cell proliferation and the development of different types of malignomas is now well established. While ANO5, ANO7, and ANO9 may also be relevant for growth of cancers, evidence has been provided for a role of ANO6 (TMEM16F) in regulated cell death. The cellular mechanisms by which anoctamins control cell proliferation and cell death, respectively, are just emerging; however, the pronounced effects of anoctamins on intracellular Ca2+ levels are likely to play a significant role. Recent results suggest that some anoctamins control membrane exocytosis by setting Ca2+i levels near the plasma membrane, and/or by controlling the intracellular Cl− concentration. Exocytosis and increased membrane trafficking induced by ANO1 and ANO6 may enhance membrane expression of other chloride channels, such as CFTR and volume activated chloride channels (VRAC). Notably, ANO6-induced phospholipid scrambling with exposure of phosphatidylserine is pivotal for the sheddase function of disintegrin and metalloproteinase (ADAM). This may support cell death and tumorigenic activity of IL-6 by inducing IL-6 trans-signaling. The reported anticancer effects of the anthelminthic drug niclosamide are probably related to the potent inhibitory effect on ANO1, apart from inducing cell cycle arrest through the Let-7d/CDC34 axis. On the contrary, pronounced activation of ANO6 due to a large increase in intracellular calcium, activation of phospholipase A2 or lipid peroxidation, can lead to ferroptotic death of cancer cells. It therefore appears reasonable to search for both inhibitors and potent activators of TMEM16 in order to interfere with cancer growth and metastasis.

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47

Yan, Huifang, Shuyan Yang, Yiming Hou, Saima Ali, Adrian Escobar, Kai Gao, Ruoyu Duan, et al. "Functional Study of TMEM163 Gene Variants Associated with Hypomyelination Leukodystrophy." Cells 11, no. 8 (April 9, 2022): 1285. http://dx.doi.org/10.3390/cells11081285.

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Hypomyelinating leukodystrophies (HLDs) are a rare group of heterogeneously genetic disorders characterized by persistent deficit of myelin observed on magnetic resonance imaging (MRI). To identify a new disease-associated gene of HLD, trio-based whole exome sequencing was performed for unexplained patients with HLD. Functional studies were performed to confirm the phenotypic effect of candidate protein variants. Two de novo heterozygous variants, c.227T>G p.(L76R) or c.227T>C p.(L76P) in TMEM163 were identified in two unrelated HLD patients. TMEM163 protein is a zinc efflux transporter localized within the plasma membrane, lysosomes, early endosomes, and other vesicular compartments. It has not been associated with hypomyelination. Functional zinc flux assays in HeLa cells stably-expressing TMEM163 protein variants, L76R and L76P, revealed distinct attenuation or enhancement of zinc efflux, respectively. Experiments using a zebrafish model with knockdown of tmem163a and tmem163b (morphants) showed that loss of tmem163 causes dysplasia of the larvae, locomotor disability and myelin deficit. Expression of human wild type TMEM163 mRNAs in morphants rescues the phenotype, while the TMEM163 L76P and L76R mutants aggravated the condition. Moreover, poor proliferation, elevated apoptosis of oligodendrocytes, and reduced oligodendrocytes and neurons were also observed in zebrafish morphants. Our findings suggest an unappreciated role for TMEM163 protein in myelin development and add TMEM163 to a growing list of genes associated with hypomyelination leukodystrophy.

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Centeio, Raquel, Inês Cabrita, Roberta Benedetto, Khaoula Talbi, Jiraporn Ousingsawat, Rainer Schreiber, John K. Sullivan, and Karl Kunzelmann. "Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A." International Journal of Molecular Sciences 21, no. 7 (April 7, 2020): 2557. http://dx.doi.org/10.3390/ijms21072557.

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TMEM16A is a Ca2+ activated Cl− channel with important functions in airways, intestine, and other epithelial organs. Activation of TMEM16A is proposed as a therapy in cystic fibrosis (CF) to reinstall airway Cl− secretion and to enhance airway surface liquid (ASL). This CFTR-agnostic approach is thought to improve mucociliary clearance and lung function in CF. This could indeed improve ASL, however, mucus release and airway contraction may also be induced by activators of TMEM16A, particularly in inflamed airways of patients with asthma, COPD, or CF. Currently, both activators and inhibitors of TMEM16A are developed and examined in different types of tissues. Here we compare activation and inhibition of endogenous and overexpressed TMEM16A and analyze potential off-target effects. The three well-known blockers benzbromarone, niclosamide, and Ani9 inhibited both TMEM16A and ATP-induced Ca2+ increase by variable degrees, depending on the cell type. Niclosamide, while blocking Ca2+ activated TMEM16A, also induced a subtle but significant Ca2+ store release and inhibited store-operated Ca2+ influx. Niclosamide, benzbromarone and Ani9 also affected TMEM16F whole cell currents, indicating limited specificity for these inhibitors. The compounds Eact, cinnamaldehyde, and melittin, as well as the phosphatidylinositol diC8-PIP2 are the reported activators of TMEM16A. However, the compounds were unable to activate endogenous TMEM16A in HT29 colonic epithelial cells. In contrast, TMEM16A overexpressed in HEK293 cells was potently stimulated by these activators. We speculate that overexpressed TMEM16A might have a better accessibility to intracellular Ca2+, which causes spontaneous activity even at basal intracellular Ca2+ concentrations. Small molecules may therefore potentiate pre-stimulated TMEM16A currents, but may otherwise fail to activate silent endogenous TMEM16A.

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Zeng, Mengying, Ziyan Xie, Jiahao Zhang, Shicheng Li, Yanxiang Wu, and Xiaowei Yan. "Arctigenin Attenuates Vascular Inflammation Induced by High Salt through TMEM16A/ESM1/VCAM-1 Pathway." Biomedicines 10, no. 11 (October 31, 2022): 2760. http://dx.doi.org/10.3390/biomedicines10112760.

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Salt-sensitive hypertension is closely related to inflammation, but the mechanism is barely known. Transmembrane member 16A (TMEM16A) is the Ca2+-activated chloride channel in epithelial cells, smooth muscle cells, and sensory neurons. It can promote inflammatory responses by increasing proinflammatory cytokine release. Here, we identified a positive role of TMEM16A in vascular inflammation. The expression of TMEM16A was increased in high-salt-stimulated vascular smooth muscle cells (VSMCs), whereas inhibiting TMEM16A or silencing TMEM16A with small interfering RNA (siRNA) can abolish this effect in vitro or in vivo. Transcriptome analysis of VSMCs revealed some differential downstream genes of TMEM16A related to inflammation, such as endothelial cell-specific molecule 1 (ESM1) and CXC chemokine ligand 16 (CXCL16). Overexpression of TMEM16A in VSMCs was accompanied by high levels of ESM1, CXCL16, intercellular adhesion molecule-1 (ICAM-1), and vascular adhesion molecule-1 (VCAM-1). We treated VSMCs cultured with high salt and arctigenin (ARC), T16Ainh-A01 (T16), and TMEM16A siRNA (siTMEM16A), leading to greatly decreased ESM1, CXCL16, VCAM-1, and ICAM-1. Beyond that, silencing ESM1, the expression of VCAM-1 and ICAM-1, and CXCL16 was attenuated. In conclusion, our results outlined a signaling scheme that increased TMEM16 protein upregulated ESM1, which possibly activated the CXCL16 pathway and increased VCAM-1 and ICAM-1 expression, which drives VSMC inflammation. Beyond that, arctigenin, as a natural inhibitor of TMEM16A, can reduce the systolic blood pressure (SBP) of salt-sensitive hypertension mice and alleviate vascular inflammation.

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50

Cherian, O. Lijo, Anna Menini, and Anna Boccaccio. "Multiple effects of anthracene-9-carboxylic acid on the TMEM16B/anoctamin2 calcium-activated chloride channel." Biochimica et Biophysica Acta (BBA) - Biomembranes 1848, no. 4 (April 2015): 1005–13. http://dx.doi.org/10.1016/j.bbamem.2015.01.009.

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