Yang Tingting, Hendrickson Wayne A, Colecraft Henry M
Departments of Biochemistry and Molecular Biophysics and
Departments of Biochemistry and Molecular Biophysics and Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columbia University, New York, NY 10032
Proc Natl Acad Sci U S A. 2014 Dec 23;111(51):18213-8. doi: 10.1073/pnas.1420984111. Epub 2014 Dec 8.
Ca(2+)-activated chloride currents carried via transmembrane proteins TMEM16A and TMEM16B regulate diverse processes including mucus secretion, neuronal excitability, smooth muscle contraction, olfactory signal transduction, and cell proliferation. Understanding how TMEM16A/16B are regulated by Ca(2+) is critical for defining their (patho)/physiological roles and for rationally targeting them therapeutically. Here, using a bioengineering approach--channel inactivation induced by membrane-tethering of an associated protein (ChIMP)--we discovered that Ca(2+)-free calmodulin (apoCaM) is preassociated with TMEM16A/16B channel complexes. The resident apoCaM mediates two distinct Ca(2+)-dependent effects on TMEM16A, as revealed by expression of dominant-negative CaM1234. These effects are Ca(2+)-dependent sensitization of activation (CDSA) and Ca(2+)-dependent inactivation (CDI). CDI and CDSA are independently mediated by the N and C lobes of CaM, respectively. TMEM16A alternative splicing provides a mechanism for tuning apoCaM effects. Channels lacking splice segment b selectively lost CDI, and segment a is necessary for apoCaM preassociation with TMEM16A. The results reveal multidimensional regulation of TMEM16A/16B by preassociated apoCaM and introduce ChIMP as a versatile tool to probe the macromolecular complex and function of Ca(2+)-activated chloride channels.
通过跨膜蛋白TMEM16A和TMEM16B携带的钙离子激活氯离子电流调节多种生理过程，包括黏液分泌、神经元兴奋性、平滑肌收缩、嗅觉信号转导和细胞增殖。了解TMEM16A/16B如何被钙离子调节对于明确它们的（病理）/生理作用以及合理地将它们作为治疗靶点至关重要。在这里，我们使用一种生物工程方法——通过相关蛋白的膜拴系诱导通道失活（ChIMP）——发现无钙钙调蛋白（脱钙钙调蛋白，apoCaM）与TMEM16A/16B通道复合物预先结合。常驻的apoCaM对TMEM16A介导两种不同的钙离子依赖性效应，如显性负性CaM1234的表达所揭示。这些效应是激活的钙离子依赖性致敏（CDSA）和钙离子依赖性失活（CDI）。CDI和CDSA分别由钙调蛋白的N叶和C叶独立介导。TMEM16A的可变剪接提供了一种调节apoCaM效应的机制。缺乏剪接片段b的通道选择性地丧失了CDI，并且片段a是apoCaM与TMEM16A预先结合所必需的。这些结果揭示了预先结合的apoCaM对TMEM16A/16B的多维调节，并引入ChIMP作为一种通用工具来探测钙离子激活氯离子通道的大分子复合物和功能。
