A Protein Interaction Network for the Large Conductance Ca2+-activated K+ Channel in the Mouse Cochlea
A Protein Interaction Network for the Large Conductance Ca2+-activated K+ Channel in the Mouse Cochlea
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DOI：
10.1074/mcp.m800495-mcp200
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发表时间：
2009-08-01
影响因子：
7
通讯作者：
Sokolowski, Bernd
中科院分区：
文献类型：
--
作者：
Kathiresan, Thandavarayan;Harvey, Margaret;Sokolowski, Bernd
大电导Ca 2+激活的K+或BK通道在感觉/神经元兴奋、细胞内信号传导和代谢中起作用。在非哺乳动物耳蜗中，BK在发育过程中的发作与非哺乳动物的听觉灵敏度增加相关，并且是频率调谐的基础，而其在哺乳动物听觉中的作用不太清楚。为了深入了解BK在哺乳动物中的功能，免疫共沉淀和二维PAGE，结合质谱，被用来揭示174个推定的BKAPs从小鼠耳蜗的细胞质和膜/细胞骨架组分。使用相互免疫共沉淀法验证了11种BKAP，包括膜联蛋白、载脂蛋白、钙调蛋白、钙调蛋白和髓鞘P0等。这些蛋白与BK在感觉细胞和神经细胞中免疫共定位。一个生物信息学的方法被用来挖掘数据库，以揭示二进制的合作伙伴和由此产生的蛋白质网络，以及确定以前的离子通道隶属关系，亚细胞定位，和细胞过程。使用IntAct分子相互作用数据库搜索二元合作伙伴产生了一个假定的全球网络，该网络由160个节点连接188个边，包含12个主要枢纽。对数据库的进一步挖掘显示，超过50%的主要BKAP先前与K+和Ca 2+通道有联系。虽然大多数BKAP存在于细胞质或细胞膜中，并参与主要涉及代谢（30.5%）和运输/支架（23.6%）的细胞过程，但至少20%与神经系统相关。在BKAP中有伴侣蛋白，如钙网蛋白、GRP 78和HSP 60，当用siRNA还原时，改变CHO细胞中的BK α表达。线粒体中BK α的研究揭示了感觉细胞的区室化，而从耳蜗克隆的BK-DEC剪接变体的异源表达揭示了BK线粒体候选者。本文所述的研究提供了对BK相关功能的深入了解，这些功能不仅包括细胞兴奋，还包括细胞信号传导和凋亡，并涉及与Ca 2+调节、结构和听力损失有关的蛋白质。分子与细胞蛋白质组学8：1972-1987，2009。
The large conductance Ca2+-activated K+ or BK channel has a role in sensory/neuronal excitation, intracellular signaling, and metabolism. In the non-mammalian cochlea, the onset of BK during development correlates with increased hearing sensitivity and underlies frequency tuning in non-mammals, whereas its role is less clear in mammalian hearing. To gain insights into BK function in mammals, coimmunoprecipitation and two-dimensional PAGE, combined with mass spectrometry, were used to reveal 174 putative BKAPs from cytoplasmic and membrane/cytoskeletal fractions of mouse cochlea. Eleven BKAPs were verified using reciprocal coimmunoprecipitation, including annexin, apolipoprotein, calmodulin, hippocalcin, and myelin P0, among others. These proteins were immunocolocalized with BK in sensory and neuronal cells. A bioinformatics approach was used to mine databases to reveal binary partners and the resultant protein network, as well as to determine previous ion channel affiliations, subcellular localization, and cellular processes. The search for binary partners using the IntAct molecular interaction database produced a putative global network of 160 nodes connected with 188 edges that contained 12 major hubs. Additional mining of databases revealed that more than 50% of primary BKAPs had prior affiliations with K+ and Ca2+ channels. Although a majority of BKAPs are found in either the cytoplasm or membrane and contribute to cellular processes that primarily involve metabolism (30.5%) and trafficking/scaffolding (23.6%), at least 20% are mitochondrial-related. Among the BKAPs are chaperonins such as calreticulin, GRP78, and HSP60 that, when reduced with siRNAs, alter BK alpha expression in CHO cells. Studies of BK alpha in mitochondria revealed compartmentalization in sensory cells, whereas heterologous expression of a BK-DEC splice variant cloned from cochlea revealed a BK mitochondrial candidate. The studies described herein provide insights into BK-related functions that include not only cell excitation, but also cell signaling and apoptosis, and involve proteins concerned with Ca2+ regulation, structure, and hearing loss. Molecular & Cellular Proteomics 8: 1972-1987, 2009.