Erickson M G, Alseikhan B A, Peterson B Z, Yue D T
Department of Biomedical Engineering, Calcium Signals Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Neuron. 2001 Sep 27;31(6):973-85. doi: 10.1016/s0896-6273(01)00438-x.
Among the most intriguing forms of Ca(2+) channel modulation is the regulation of L-type and P/Q-type channels by intracellular Ca(2+), acting via unconventional channel-calmodulin (CaM) interactions. In particular, overexpressing Ca(2+)-insensitive mutant CaM abolishes Ca(2+)-dependent modulation, hinting that Ca(2+)-free CaM may "preassociate" with these channels to enhance detection of local Ca(2+). 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 Ca(2+)-independent associations between CaM and the pore-forming alpha(1) 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.
钙（Ca2+）通道调节最引人入胜的形式之一是细胞内Ca2+通过非常规的通道-钙调蛋白（CaM）相互作用对L型和P/Q型通道进行调节。特别是，过表达对Ca2+不敏感的突变型CaM会消除Ca2+依赖性调节，这表明无Ca2+的CaM可能与这些通道“预先结合”，以增强对局部Ca2+的检测。尽管这一观点具有深远影响，但测试预先结合的体外实验却给出了相互矛盾的结果。在此，我们开发了一种三滤光片盒式荧光共振能量转移方法（三盒式FRET），以直接探测单个活细胞中通道亚基与CaM之间的组成性结合。这种FRET分析检测到CaM与L型、P/Q型通道以及令人惊讶的R型通道的孔形成α1亚基之间存在不依赖Ca2+的结合。这些结果现在明确证明了静息细胞中通道与CaM的预先结合，并强调了三盒式FRET在探测蛋白质-蛋白质相互作用方面的潜力。
