Structural and Biophysical Analysis of the CLCA1 VWA Domain Suggests Mode of TMEM16A Engagement

Berry, Kayla N.; Brett, Tom J.

doi:10.1016/j.celrep.2019.12.059

Cited by 12 publications

(10 citation statements)

References 73 publications

(126 reference statements)

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“…CLCA1 is a secreted potentiator of the calcium-activated chloride channel. Its Willebrand factor type A domain elevates TMEM16A expression and activity within minutes, suggesting a dynamic association of TMEM16A with many partners that control its expression at various levels [36,37]. Thus, CLCA1 appears to work by an entirely different mechanism, namely by stabilizing expression of TMEM16A in the plasma membrane.…”

Section: Compoundmentioning

confidence: 99%

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

Centeio

Cabrita

Benedetto

et al. 2020

IJMS

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TMEM16A is a Ca 2+ 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 Ca 2+ increase by variable degrees, depending on the cell type. Niclosamide, while blocking Ca 2+ activated TMEM16A, also induced a subtle but significant Ca 2+ store release and inhibited store-operated Ca 2+ 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-PIP 2 are the reported activators of TMEM16A. However, the compounds were unable to activate endogenous TMEM16A in HT 29 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 Ca 2+ , which causes spontaneous activity even at basal intracellular Ca 2+ concentrations. Small molecules may therefore potentiate pre-stimulated TMEM16A currents, but may otherwise fail to activate silent endogenous TMEM16A.

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Section: Compoundmentioning

confidence: 99%

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

Centeio

Cabrita

Benedetto

et al. 2020

IJMS

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“…Von Willebrand factor also acts as a receptor for proteasome-mediated degradation for ubiquitin-like proteins. 26 - 28 Eukaryotic initiation factor 2B (EIF2B) is a GTP exchange factor necessary for protein synthesis. Stress responses lead to increase protein synthesis during which stress response proteins are produced, these proteins activate the kinases, which leads to phosphorylation of the eIF, and this reduces the activity of EIF2B.…”

Section: Discussionmentioning

confidence: 99%

Can Scoliotic Discs Be Controls for Molecular Studies in Intervertebral Disc Research? Insights From Proteomics

Rajasekaran

Tangavel

Anand

et al. 2020

Global Spine Journal

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Study Design: Proteomic analysis of human intervertebral discs. Objectives: To compare the characters of scoliotic discs and discs from magnetic resonance imaging (MRI)–normal voluntary organ donors controls used in disc research employing proteomics and establish “true controls” that can be utilized for future intervertebral disc (IVD) research. Methods: Eight MRI-normal discs from 8 brain-dead voluntary organ donors (ND) and 8 scoliotic discs (SD) from 3 patients who underwent anterior surgery for adolescent idiopathic scoliosis were subjected to tandem mass spectrometry, and further analysis was performed. Results: Mass spectrometry identified a total of 235 proteins in ND and 438 proteins in the SD group. Proteins involved in extracellular matrix integrity (Versican, keratins KRT6A, KRT14, KRT5, and KRT 13A1, A-kinase anchor protein 13, coagulation factor XIII A chain, proteoglycan 4) and proteins involved in transcription and DNA repair (Von Willebrand factor A domain-containing 3B, eukaryotic initiation factor 2B, histone H4, leukocyte cell–derived chemotaxin 2) were found to be downregulated in SD. Inflammatory proteins (C3, C1S), and oxidative stress response proteins (peroxiredoxin-2,6, catalase, myeloperoxidase, apolipoprotein E) were found to be upregulated in SD. These changes were reflected at the pathway level also. Conclusion: Findings of our study confirm that scoliotic discs have an abundance of inflammatory, oxidative stress response proteins, which are either absent or downregulated in the ND group indicating that scoliotic discs are not pathologically inert. Furthermore, this study has established MRI-normal discs from voluntary organ donors as the “true” control for molecular studies in IVD research.

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“…105 Expression of hCLCA1 in two mammalian cell lines led to stimulation of endogenous CaCCs. [198][199][200][201] first showed that this process involved the proteolytic self-cleavage of hCLCA1 due to the presence of a novel zincin metalloprotease domain at its N-terminal end. The stimulation of CaCCs was not linked to the proteolytic activity of hCLCA1 per se because exposure to an N-terminal mutant fragment lacking enzymatic activity still led to enhanced CaCC activity.…”

Section: Clca1 Andmentioning

confidence: 99%

“…Subsequent reports demonstrated that the von Willebrand factor type A (vWF) domain located in the N-terminus of hCLCA1 is responsible for the interaction with ANO1. 201,202 The engagement of ANO1 was speculated to involve Mg 2+ -dependent binding of the extracellular loop between TMD9 and 10 of ANO1 to a conserved metal ion-dependent adhesion site (MIDAS) motif on the vWF domain of hCLCA1. Finally, the effects described above are not unique to hCLCA1 as the expression of hCLCA2 in a HEK-293T cell line stably expressing hANO1 nearly doubled the magnitude of I Cl(Ca) evoked by the Ca 2+ ionophore ionomycin.…”

Section: Clca1 Andmentioning

confidence: 99%

“…This occurred by stabilization of ANO1 at the plasma membrane, most likely by decreasing internalization. Subsequent reports demonstrated that the von Willebrand factor type A (vWF) domain located in the N-terminus of hCLCA1 is responsible for the interaction with ANO1 [ 201 , 202 ]. The engagement of ANO1 was speculated to involve Mg 2+ -dependent binding of the extracellular loop between TMD9 and 10 of ANO1 to a conserved metal ion-dependent adhesion site (MIDAS) motif on the vWF domain of hCLCA1.…”

Section: Regulation Through Coupling Of Ano1 With Other Membrane Proteinsmentioning

confidence: 99%

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Molecular mechanisms of activation and regulation of ANO1-Encoded Ca²⁺-Activated Cl^- channels

et al. 2021

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Structural and Biophysical Analysis of the CLCA1 VWA Domain Suggests Mode of TMEM16A Engagement

Abstract: Highlights d A 2.00-Å crystal structure of the CLCA1 VWA domain that potentiates TMEM16A is reported d CLCA1 VWA MIDAS is in a high-affinity configuration with a disulfide bond nearby d Crystal lattice pseudoligand contacts mimic engagement with TMEM16A

Cited by 12 publications

References 73 publications

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

Can Scoliotic Discs Be Controls for Molecular Studies in Intervertebral Disc Research? Insights From Proteomics

Molecular mechanisms of activation and regulation of ANO1-Encoded Ca²⁺-Activated Cl^- channels

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Structural and Biophysical Analysis of the CLCA1 VWA Domain Suggests Mode of TMEM16A Engagement

Abstract: Highlights d A 2.00-Å crystal structure of the CLCA1 VWA domain that potentiates TMEM16A is reported d CLCA1 VWA MIDAS is in a high-affinity configuration with a disulfide bond nearby d Crystal lattice pseudoligand contacts mimic engagement with TMEM16A

Cited by 12 publications

References 73 publications

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

Can Scoliotic Discs Be Controls for Molecular Studies in Intervertebral Disc Research? Insights From Proteomics

Molecular mechanisms of activation and regulation of ANO1-Encoded Ca2+-Activated Cl- channels

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Molecular mechanisms of activation and regulation of ANO1-Encoded Ca²⁺-Activated Cl^- channels