Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin-radixin-moesin network
Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin-radixin-moesin network
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DOI：
10.1073/pnas.1200174109
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发表时间：
2012-06-26
影响因子：
11.1
通讯作者：
Faundez, Victor
中科院分区：
文献类型：
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作者：
Perez-Cornejo, Patricia;Gokhale, Avanti;Faundez, Victor
新发现的 Ca2+ 激活的 Cl- 通道 (CaCC) Anoctamin 1（Ano1 或 TMEM16A）与重要的生理功能有关，包括上皮液分泌、肠道蠕动和平滑肌张力。 HEK 细胞或非洲爪蟾卵母细胞中 Ano1 的过度表达足以产生 Ca2+ 激活的 Cl- 电流，但通道组成的细节和控制通道生物学的调节因素尚不完全清楚。我们使用高度灵敏的定量 SILAC 蛋白质组学方法来深入了解与 Ano1 通道相关的化学计量蛋白质网络。这些研究提供了假定的 Ano1 调控网络的全面足迹。我们发现 Ano1 与信号/支架蛋白 ezrin、radixin、moesin 和 RhoA 相关，这些蛋白以非常高的化学计量将质膜与细胞骨架连接起来。 Ano1、ezrin 和 moesin/radixin 在唾液腺上皮细胞顶部共定位，HEK 细胞中 moesin 和 Ano1 的过度表达会改变这两种蛋白的亚细胞定位。此外，moesin 的干扰 RNA 会改变 Ano1 电流，而不影响其表面表达水平。与 Ano1 相关的另一个网络包括 SNARE 和 SM 蛋白 VAMP3、突触融合蛋白 2 和 -4，以及突触融合蛋白结合蛋白 munc18b 和 munc18c，它们是囊泡易位到质膜的组成部分。 Ano1 复合物中富含许多其他调节蛋白，包括 GTP 酶、Ca2+ 结合蛋白、激酶和脂质相互作用蛋白。这些数据提供了有关调节 Ano1 功能和运输到质膜极化域的机制的化学计量优先信息。
The newly discovered Ca2+-activated Cl- channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca2+-activated Cl- currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca2+-binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane.