International Journal of Molecular Sciences

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6 pages, 2566 KiB

Open AccessEditorial

Ca²⁺-Activated Chloride Channels and Phospholipid Scramblases

by Simone Pifferi and Anna Boccaccio

Int. J. Mol. Sci. 2022, 23(4), 2158; https://doi.org/10.3390/ijms23042158 - 15 Feb 2022

Cited by 2 | Viewed by 1390

Abstract

The functional characterization of the TMEM16 protein family unexpectedly brought together two different research fields in membrane biology: anion channel and membrane lipid organization [...] Full article

(This article belongs to the Special Issue Ca²⁺-Activated Chloride Channels and Phospholipid Scramblases)

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20 pages, 7105 KiB

Open AccessArticle

Anion and Cation Permeability of the Mouse TMEM16F Calcium-Activated Channel

by Stefano Stabilini, Anna Menini and Simone Pifferi

Int. J. Mol. Sci. 2021, 22(16), 8578; https://doi.org/10.3390/ijms22168578 - 09 Aug 2021

Cited by 11 | Viewed by 2923

Abstract

TMEM16F is involved in several physiological processes, such as blood coagulation, bone development and virus infections. This protein acts both as a Ca²⁺-dependent phospholipid scramblase and a Ca²⁺-activated ion channel but several studies have reported conflicting results about the [...] Read more.

TMEM16F is involved in several physiological processes, such as blood coagulation, bone development and virus infections. This protein acts both as a Ca²⁺-dependent phospholipid scramblase and a Ca²⁺-activated ion channel but several studies have reported conflicting results about the ion selectivity of the TMEM16F-mediated current. Here, we have performed a detailed side-by-side comparison of the ion selectivity of TMEM16F using the whole-cell and inside-out excised patch configurations to directly compare the results. In inside-out configuration, Ca²⁺-dependent activation was fast and the TMEM16F-mediated current was activated in a few milliseconds, while in whole-cell recordings full activation required several minutes. We determined the relative permeability between Na⁺ and Cl¯ (P_Na/P_Cl) using the dilution method in both configurations. The TMEM16F-mediated current was highly nonselective, but there were differences depending on the configuration of the recordings. In whole-cell recordings, P_Na/P_Cl was approximately 0.5, indicating a slight preference for Cl¯ permeation. In contrast, in inside-out experiments the TMEM16F channel showed a higher permeability for Na⁺ with P_Na/P_Cl reaching 3.7. Our results demonstrate that the time dependence of Ca²⁺ activation and the ion selectivity of TMEM16F depend on the recording configuration. Full article

(This article belongs to the Special Issue Ca²⁺-Activated Chloride Channels and Phospholipid Scramblases)

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18 pages, 3423 KiB

Open AccessArticle

Anoctamin1 Induces Hyperproliferation of HaCaT Keratinocytes and Triggers Imiquimod-Induced Psoriasis-Like Skin Injury in Mice

by Mi Ran Choi, Hae Dong Kim, Sinyoung Cho, Seong Ho Jeon, Dong Hyun Kim, Jungwon Wee and Young Duk Yang

Int. J. Mol. Sci. 2021, 22(13), 7145; https://doi.org/10.3390/ijms22137145 - 01 Jul 2021

Cited by 10 | Viewed by 3554

Abstract

Psoriasis, a long-lasting and multifactorial skin disease, is related to comorbidities such as metabolic disease, depression, and psoriatic arthritis. Psoriasis occurs due to a variety of factors including keratinocyte hyperproliferation, inflammation, and abnormal differentiation. Proinflammatory cytokines upregulated by increased activation of keratinocytes and [...] Read more.

Psoriasis, a long-lasting and multifactorial skin disease, is related to comorbidities such as metabolic disease, depression, and psoriatic arthritis. Psoriasis occurs due to a variety of factors including keratinocyte hyperproliferation, inflammation, and abnormal differentiation. Proinflammatory cytokines upregulated by increased activation of keratinocytes and immune cells in the skin trigger progression of psoriasis. This study aimed to investigate the effects of anoctamin1 (ANO1) on psoriasis development in vitro and in vivo. We analyzed the proliferation of HaCaT keratinocytes and ANO1-related ERK and AKT signaling pathways after ANO1 inhibitor (T16Ainh-A01 and Ani9) treatment and knock-down of ANO1. Furthermore, after applying imiquimod (IMQ) cream or coapplying IMQ cream and T16Ainh-A01 on mouse ears, we not only observed psoriatic symptoms, including ear thickening, but also quantified the effects of treatment on ERK and AKT signaling-involved proteins and proinflammatory cytokines. Inhibition of ANO1 attenuated the proliferation of HaCaT cells and induced reduction of pERK1/2. Coapplication of IMQ and T16Ainh-A01 on ears of mice reduced not only symptoms of IMQ-induced psoriasis such as thickening and erythema, but also expression of ANO1 and pERK1/2 compared to that of application of IMQ alone. In addition, the expression levels of IL-17A, IL-17F, IL-22, IL-23, IL-6, IL-1β, and TNF-α increased after applying IMQ and were significantly reduced by coapplying IMQ and T16Ainh-A01. These results aid in understanding the underlying mechanisms of ANO1 in epidermal layer keratinocyte hyperproliferation and suggest the potential of ANO1 as a target to treat psoriasis. Full article

(This article belongs to the Special Issue Ca²⁺-Activated Chloride Channels and Phospholipid Scramblases)

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13 pages, 2349 KiB

Open AccessArticle

Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1

by Yohan Seo, Sung Baek Jeong, Joo Han Woo, Oh-Bin Kwon, Sion Lee, Hye In Oh, Sungwoo Jo, Seon Ju Park, Wan Namkung, Uk Yeol Moon and Sungwoo Lee

Int. J. Mol. Sci. 2021, 22(13), 7100; https://doi.org/10.3390/ijms22137100 - 01 Jul 2021

Cited by 14 | Viewed by 3064

Abstract

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca²⁺-activated chloride channel (CaCC) overexpressed in [...] Read more.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca²⁺-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca²⁺ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent. Full article

(This article belongs to the Special Issue Ca²⁺-Activated Chloride Channels and Phospholipid Scramblases)

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17 pages, 5061 KiB

Open AccessArticle

CLCA1 Regulates Airway Mucus Production and Ion Secretion Through TMEM16A

by Raquel Centeio, Jiraporn Ousingsawat, Rainer Schreiber and Karl Kunzelmann

Int. J. Mol. Sci. 2021, 22(10), 5133; https://doi.org/10.3390/ijms22105133 - 12 May 2021

Cited by 18 | Viewed by 2804

Abstract

TMEM16A, a Ca²⁺-activated chloride channel (CaCC), and its regulator, CLCA1, are associated with inflammatory airway disease and goblet cell metaplasia. CLCA1 is a secreted protein with protease activity that was demonstrated to enhance membrane expression of TMEM16A. Expression of CLCA1 is [...] Read more.

TMEM16A, a Ca²⁺-activated chloride channel (CaCC), and its regulator, CLCA1, are associated with inflammatory airway disease and goblet cell metaplasia. CLCA1 is a secreted protein with protease activity that was demonstrated to enhance membrane expression of TMEM16A. Expression of CLCA1 is particularly enhanced in goblet cell metaplasia and is associated with various lung diseases. However, mice lacking expression of CLCA1 showed the same degree of mucous cell metaplasia and airway hyperreactivity as asthmatic wild-type mice. To gain more insight into the role of CLCA1, we applied secreted N-CLCA1, produced in vitro, to mice in vivo using intratracheal instillation. We observed no obvious upregulation of TMEM16A membrane expression by CLCA1 and no differences in ATP-induced short circuit currents (Iscs). However, intraluminal mucus accumulation was observed by treatment with N-CLCA1 that was not seen in control animals. The effects of N-CLCA1 were augmented in ovalbumin-sensitized mice. Mucus production induced by N-CLCA1 in polarized BCi-NS1 human airway epithelial cells was dependent on TMEM16A expression. IL-13 upregulated expression of CLCA1 and enhanced mucus production, however, without enhancing purinergic activation of Isc. In contrast to polarized airway epithelial cells and mouse airways, which express very low levels of TMEM16A, nonpolarized airway cells express large amounts of TMEM16A protein and show strong CaCC. The present data show an only limited contribution of TMEM16A to airway ion secretion but suggest a significant role of both CLCA1 and TMEM16A for airway mucus secretion. Full article

(This article belongs to the Special Issue Ca²⁺-Activated Chloride Channels and Phospholipid Scramblases)

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14 pages, 2782 KiB

Open AccessArticle

Differential Regulation of Ca²⁺-Activated Cl⁻ Channel TMEM16A Splice Variants by Membrane PI(4,5)P₂

by Woori Ko and Byung-Chang Suh

Int. J. Mol. Sci. 2021, 22(8), 4088; https://doi.org/10.3390/ijms22084088 - 15 Apr 2021

Cited by 7 | Viewed by 2206

Abstract

TMEM16A is a Ca²⁺-activated Cl⁻ channel that controls broad cellular processes ranging from mucus secretion to signal transduction and neuronal excitability. Recent studies have reported that membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is an important cofactor that allosterically regulates [...] Read more.

TMEM16A is a Ca²⁺-activated Cl⁻ channel that controls broad cellular processes ranging from mucus secretion to signal transduction and neuronal excitability. Recent studies have reported that membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is an important cofactor that allosterically regulates TMEM16A channel activity. However, the detailed regulatory actions of PIP₂ in splice variants of TMEM16A remain unclear. Here, we demonstrated that the attenuation of membrane phosphoinositide levels selectively inhibited the current amplitude of the TMEM16A(ac) isoform by decreasing the slow, but not instantaneous, Cl⁻ currents, which are independent of the membrane potential and specific to PI(4,5)P₂ depletion. The attenuation of endogenous PI(4,5)P₂ levels by the activation of Danio rerio voltage-sensitive phosphatase (Dr-VSP) decreased the Cl⁻ currents of TMEM16A(ac) but not the TMEM16A(a) isoform, which was abolished by the co-expression of PIP 5-kinase type-1γ (PIPKIγ). Using the rapamycin-inducible dimerization of exogenous phosphoinositide phosphatases, we further revealed that the stimulatory effects of phosphoinositide on TMEM16A(ac) channels were similar in various membrane potentials and specific to PI(4,5)P₂, not PI4P and PI(3,4,5)P₃. Finally, we also confirmed that PI(4,5)P₂ resynthesis is essential for TMEM16A(ac) recovery from Dr-VSP-induced current inhibition. Our data demonstrate that membrane PI(4,5)P₂ selectively modulates the gating of the TMEM16A(ac) channel in an agonistic manner, which leads to the upregulation of TMEM16A(ac) functions in physiological conditions. Full article

(This article belongs to the Special Issue Ca²⁺-Activated Chloride Channels and Phospholipid Scramblases)

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23 pages, 3736 KiB

Open AccessArticle

Divalent Cation Modulation of Ion Permeation in TMEM16 Proteins

by Dung M. Nguyen, Hwoi Chan Kwon and Tsung-Yu Chen

Int. J. Mol. Sci. 2021, 22(4), 2209; https://doi.org/10.3390/ijms22042209 - 23 Feb 2021

Cited by 6 | Viewed by 2544

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Ca²⁺-Activated Chloride Channels and Phospholipid Scramblases)

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Review

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30 pages, 2541 KiB

Open AccessReview

Polymodal Control of TMEM16x Channels and Scramblases

by Emilio Agostinelli and Paolo Tammaro

Int. J. Mol. Sci. 2022, 23(3), 1580; https://doi.org/10.3390/ijms23031580 - 29 Jan 2022

Cited by 8 | Viewed by 4037

Abstract

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., [...] Read more.

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 G_q-protein-coupled receptor (G_qPCR) 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. Full article

(This article belongs to the Special Issue Ca²⁺-Activated Chloride Channels and Phospholipid Scramblases)

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