Preassociation of calmodulin with voltage-gated Ca2+ channels revealed by FRET in single living cells
Preassociation of calmodulin with voltage-gated Ca2+ channels revealed by FRET in single living cells
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DOI：
10.1016/s0896-6273(01)00438-x
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发表时间：
2001-09-27
期刊：
影响因子：
16.2
通讯作者：
Yue, DT
中科院分区：
文献类型：
--
作者：
Erickson, MG;Alseikhan, BA;Yue, DT
其中最有趣的形式的Ca 2+通道的调节是L型和P/Q型通道的细胞内Ca 2+的调节，通过非常规的通道-钙调蛋白（CaM）的相互作用。特别是，过表达钙不敏感的突变体钙调素废除钙依赖的调制，提示钙游离钙调素可能与这些通道“preassociate”，以提高检测本地钙。尽管这一建议的深远影响，在体外实验测试预关联提供了相互矛盾的结果。在这里，我们开发了一个三过滤立方体荧光共振能量转移方法（三立方体FRET）直接探测通道亚基和钙调素在单个活细胞的组成协会。该FRET测定检测CaM与L型、P/Q型和令人惊讶的R型通道的孔形成α亚基之间的Ca 2+非依赖性关联。这些结果现在明确地证明了静息细胞中的通道-钙调素预关联，并强调了三立方体FRET用于探测蛋白质-蛋白质相互作用的潜力。
Among the most intriguing forms of Ca2+ channel modulation is the regulation of L-type and P/Q-type channels by intracellular Ca2+, acting via unconventional channel-calmodulin (CaM) interactions. In particular, overexpressing Ca2+-insensitive mutant CaM abolishes Ca2+-dependent modulation, hinting that Ca2+-free CaM may "preassociate" with these channels to enhance detection of local Ca2+. Despite the far-reaching consequences of this proposal, in vitro experiments testing for preassociation provide conflicting results. Here, we develop a three filter-cube fluorescence resonance energy transfer method (three-cube FRET) to directly probe for constitutive associations between channel subunits and CaM in single living cells. This FRET assay detects Ca2+-independent associations between CaM and the pore-forming a, subunit of L-type, P/Q-type, and, surprisingly, R-type channels. These results now definitively demonstrate channel-CaM preassociation in resting cells and underscore the potential of three-cube FRET for probing protein-protein interactions.