Abstract
TMEM16A Ca2+-activated chloride channel (CaCC) plays an essential role in vascular homeostasis. In this study we investigated the molecular mechanisms underlying downregulation of TMEM16A CaCC activity during hypertension. In cultured basilar artery smooth muscle cells (BASMCs) isolated from 2k2c renohypertesive rats, treatment with angiotensin II (0.125−1 μM) dose-dependently increased endophilin A2 levels and decreased TMEM16A expression. Similar phenomenon was observed in basilar artery isolated from 2k2c rats. We then used whole-cell recording to examine whether endophilin A2 could regulate TMEM16A CaCC activity in BASMCs and found that knockdown of endophilin A2 significantly enhanced CaCC activity, whereas overexpression of endophilin A2 produced the opposite effect. Overexpression of endophilin A2 did not affect the TMEM16A mRNA level, but markedly decreased TMEM16A protein level in BASMCs by inducing ubiquitination and autophagy of TMEM16A. Ubiquitin-binding receptor p62 (SQSTM1) could bind to ubiquitinated TMEM16A and resulted in a process of TMEM16A proteolysis in autophagosome/lysosome. These data provide new insights into the regulation of TMEM16A CaCC activity by endophilin A2 in BASMCs, which partly explains the mechanism of angiotensin-II-induced TMEM16A inhibition during hypertension-induced vascular remodeling.
Similar content being viewed by others
Login or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, Sondo E, et al. TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science. 2008;322:590–4.
Schroeder BC, Cheng T, Jan YN, Jan LY. Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell. 2008;134:1019–29.
Yang YD, Cho H, Koo JY, Tak MH, Cho Y, Shim WS, et al. TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature. 2008;455:1210–5.
Pedemonte N, Galietta LJ. Structure and function of TMEM16 proteins (anoctamins). Physiol Rev. 2014;94:419–59.
Liu B, Linley JE, Du X, Zhang X, Ooi L, Zhang H, et al. The acute nociceptive signals induced by bradykinin in rat sensory neurons are mediated by inhibition of M-type K+ channels and activation of Ca2+-activated Cl− channels. J Clin Invest. 2010;120:1240–52.
Cho H, Yang YD, Lee J, Lee B, Kim T, Jang Y, et al. The calcium-activated chloride channel anoctamin 1 acts as a heat sensor in nociceptive neurons. Nat Neurosci. 2012;15:1015–21.
Hwang SJ, Blair PJ, Britton FC, O’Driscoll KE, Hennig G, Bayguinov YR, et al. Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles. J Physiol. 2009;587:4887–904.
Jin X, Shah S, Liu Y, Zhang H, Lees M, Fu Z, et al. Activation of the Cl− channel ANO1 by localized calcium signals in nociceptive sensory neurons requires coupling with the IP3 receptor. Sci Signal. 2013;6:ra73.
Huang F, Zhang H, Wu M, Yang H, Kudo M, Peters CJ, et al. Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction. Proc Natl Acad Sci USA. 2012;109:16354–9.
Wang M, Yang H, Zheng LY, Zhang Z, Tang YB, Wang GL, et al. Downregulation of TMEM16A calcium-activated chloride channel contributes to cerebrovascular remodeling during hypertension by promoting basilar smooth muscle cell proliferation. Circulation. 2012;125:697–707.
Tien J, Peters CJ, Wong XM, Cheng T, Jan YN, Jan LY, et al. A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity. Elife. 2014;3:e02772.
Yang T, Hendrickson WA, Colecraft HM. Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl- channels. Proc Natl Acad Sci USA. 2014;111:18213–8.
Jung J, Nam JH, Park HW, Oh U, Yoon JH, Lee MG. Dynamic modulation of ANO1/TMEM16A HCO3− permeability by Ca2+/calmodulin. Proc Natl Acad Sci USA. 2013;110:360–5.
Sala-Rabanal M, Yurtsever Z, Nichols CG, Brett TJ. Secreted CLCA1 modulates TMEM16A to activate Ca2+-dependent chloride currents in human cells. Elife. 2015;4:e05875.
Perez-Cornejo P, Gokhale A, Duran C, Cui Y, Xiao Q, Hartzell HC, et al. Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin-radixin-moesin network. Proc Natl Acad Sci USA. 2012;109:10376–81.
Loll PJ, Swain E, Chen Y, Turner BT, Zhang JF. Structure of the SH3 ___domain of rat endophilin A2. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008;64:243–6.
Gortat A, San-Roman MJ, Vannier C, Schmidt AA. Single point mutation in Bin/Amphiphysin/Rvs (BAR) sequence of endophilin impairs dimerization, membrane shaping, and Src homology 3 ___domain-mediated partnership. J Biol Chem. 2012;287:4232–47.
Renard HF, Simunovic M, Lemiere J, Boucrot E, Garcia-Castillo MD, Arumugam S, et al. Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis. Nature. 2015;517:493–6.
Bai J, Hu Z, Dittman JS, Pym EC, Kaplan JM. Endophilin functions as a membrane-bending molecule and is delivered to endocytic zones by exocytosis. Cell. 2010;143:430–41.
Milosevic I, Giovedi S, Lou X, Raimondi A, Collesi C, Shen H, et al. Recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission. Neuron. 2011;72:587–601.
Chen Y, Deng L, Maeno-Hikichi Y, Lai M, Chang S, Chen G, et al. Formation of an endophilin-Ca2+ channel complex is critical for clathrin-mediated synaptic vesicle endocytosis. Cell. 2003;115:37–48.
Liu CZ, Li XY, Du RH, Gao M, Ma MM, Li FY, et al. Endophilin A2 influences volume-regulated chloride current by mediating ClC-3 trafficking in vascular smooth muscle cells. Circ J. 2016;80:2397–406.
Liu YJ, Wang XG, Tang YB, Chen JH, Lv XF, Zhou JG, et al. Simvastatin ameliorates rat cerebrovascular remodeling during hypertension via inhibition of volume-regulated chloride channel. Hypertension. 2010;56:445–52.
Ma MM, Lin CX, Liu CZ, Gao M, Sun L, Tang YB, et al. Threonine 532 phosphorylation in ClC-3 is required for angiotensin II-induced Cl current and migration in cultured vascular smooth muscle cells. Br J Pharmacol. 2015;173:529–44.
Trempe JF, Chen CX, Grenier K, Camacho EM, Kozlov G, McPherson PS, et al. SH3 domains from a subset of BAR proteins define a Ubl-binding ___domain and implicate parkin in synaptic ubiquitination. Mol Cell. 2009;36:1034–47.
Stamenova SD, Dunn R, Adler AS, Hicke L. The Rsp5 ubiquitin ligase binds to and ubiquitinates members of the yeast CIN85-endophilin complex, Sla1-Rvs167. J Biol Chem. 2004;279:16017–25.
Khan MM, Strack S, Wild F, Hanashima A, Gasch A, Brohm K, et al. Role of autophagy, SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors. Autophagy. 2014;10:123–36.
Kirkin V, Lamark T, Sou YS, Bjorkoy G, Nunn JL, Bruun JA, et al. A role for NBR1 in autophagosomal degradation of ubiquitinated substrates. Mol Cell. 2009;33:505–16.
Kraft C, Peter M, Hofmann K. Selective autophagy: ubiquitin-mediated recognition and beyond. Nat Cell Biol. 2010;12:836–41.
Tien J, Lee HY, Minor DL Jr, Jan YN, Jan LY. Identification of a dimerization ___domain in the TMEM16A calcium-activated chloride channel (CaCC). Proc Natl Acad Sci USA. 2013;110:6352–7.
Sheridan JT, Worthington EN, Yu K, Gabriel SE, Hartzell HC, Tarran R. Characterization of the oligomeric structure of the Ca2+-activated Cl- channel Ano1/TMEM16A. J Biol Chem. 2011;286:1381–8.
Ohshiro J, Yamamura H, Saeki T, Suzuki Y, Imaizumi Y. The multiple expression of Ca2+-activated Cl- channels via homo- and hetero-dimer formation of TMEM16A splicing variants in murine portal vein. Biochem Biophys Res Commun. 2014;443:518–23.
Takahashi Y, Coppola D, Matsushita N, Cualing HD, Sun M, Sato Y, et al. Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis. Nat Cell Biol. 2007;9:1142–51.
Takahashi Y, Meyerkord CL, Wang HG. BARgaining membranes for autophagosome formation: regulation of autophagy and tumorigenesis by Bif-1/Endophilin B1. Autophagy. 2008;4:121–4.
Takahashi Y, Meyerkord CL, Wang HG. Bif-1/endophilin B1: a candidate for crescent driving force in autophagy. Cell Death Differ. 2009;16:947–55.
Wong AS, Lee RH, Cheung AY, Yeung PK, Chung SK, Cheung ZH, et al. Cdk5-mediated phosphorylation of endophilin B1 is required for induced autophagy in models of Parkinson’s disease. Nat Cell Biol. 2011;13:568–79.
Moscat J, Diaz-Meco MT, Wooten MW. Signal integration and diversification through the p62 scaffold protein. Trends Biochem Sci. 2007;32:95–100.
Komatsu M, Waguri S, Koike M, Sou YS, Ueno T, Hara T, et al. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell. 2007;131:1149–63.
Kirkin V, Dikic I. Role of ubiquitin- and Ubl-binding proteins in cell signaling. Curr Opin Cell Biol. 2007;19:199–205.
Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem. 2007;282:24131–45.
Acknowledgements
YYG was supported by the National Natural Science Foundation of China (Key grant numbers 81230082, 81302771, 81525025, 81473206, 81573422, 81773721, and 81500226). XFL is supported by the Natural Science Foundation of Guangdong Province (2018A030310233), The Fundamental Research Funds for the Central Universities (number 18zxxt74).
Author information
Authors and Affiliations
Contributions
CZL and FYL conceived and designed the experiments. CZL, FYL, XFL, MMM, XYL, and CXL performed the experiments. CZL and FYL analyzed the data. CZL, FYL, GLW, and YYG wrote the paper.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Supplementary information
Rights and permissions
About this article
Cite this article
Liu, Cz., Li, Fy., Lv, Xf. et al. Endophilin A2 regulates calcium-activated chloride channel activity via selective autophagy-mediated TMEM16A degradation. Acta Pharmacol Sin 41, 208–217 (2020). https://doi.org/10.1038/s41401-019-0298-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41401-019-0298-5
Keywords
This article is cited by
-
Endophilin A2 Deficiency Impairs Antibody Production in Humans
Journal of Clinical Immunology (2025)
-
Endophilin A2 controls touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking
Military Medical Research (2024)
-
Adipose-specific deletion of the cation channel TRPM7 inhibits TAK1 kinase-dependent inflammation and obesity in male mice
Nature Communications (2023)
-
Hepatocyte-specific TMEM16A deficiency alleviates hepatic ischemia/reperfusion injury via suppressing GPX4-mediated ferroptosis
Cell Death & Disease (2022)
-
Aggravated ulcerative colitis caused by intestinal Metrnl deficiency is associated with reduced autophagy in epithelial cells
Acta Pharmacologica Sinica (2020)
