Ernst Oliver P, Murcia Pedro A Sánchez, Daldrop Peter, Tsunoda Satoshi P, Kateriya Suneel, Hegemann Peter
Institut für medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
Institut für medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
J Biol Chem. 2008 Jan 18;283(3):1637-1643. doi: 10.1074/jbc.M708039200. Epub 2007 Nov 9.
Channelrhodopsins (ChRs) are light-gated ion channels that control photomovement of microalgae. In optogenetics, ChRs are widely applied for light-triggering action potentials in cells, tissues, and living animals, yet the spectral properties and photocycle of ChR remain obscure. In this study, we cloned a ChR from the colonial alga Volvox carteri, VChR. After electrophysiological characterization in Xenopus oocytes, VChR was expressed in COS-1 cells and purified. Time-resolved UV-visible spectroscopy revealed a pH-dependent equilibrium of two dark species, D(470)/D(480). Laser flashes converted both with tau approximately 200 mus into major photointermediates P(510)/P(530), which reverted back to the dark states with tau approximately 15-100 ms. Both intermediates were assigned to conducting states. Three early intermediates P(500)/P(515) and P(390) were detected on a ns to mus time scale. The spectroscopic and electrical data were unified in a photocycle model. The functional expression of VChR we report here paves the way toward a broader structure/function analysis of the recently identified class of light-gated ion channels.
视紫红质通道蛋白（ChRs）是控制微藻光运动的光门控离子通道。在光遗传学中，ChRs被广泛应用于触发细胞、组织和活体动物中的动作电位，然而ChR的光谱特性和光循环仍不清楚。在本研究中，我们从团藻Volvox carteri中克隆了一种ChR，即VChR。在非洲爪蟾卵母细胞中进行电生理特性分析后，VChR在COS-1细胞中表达并纯化。时间分辨紫外可见光谱揭示了两种暗态D(470)/D(480)的pH依赖性平衡。激光闪光将两者都在约200微秒的时间常数内转化为主要光中间体P(510)/P(530)，它们又在约15 - 100毫秒的时间常数内恢复到暗态。这两种中间体都被归为传导态。在纳秒到微秒的时间尺度上检测到了三种早期中间体P(500)/P(515)和P(390)。光谱和电学数据在一个光循环模型中得到统一。我们在此报告的VChR的功能表达为最近鉴定的光门控离子通道类别的更广泛结构/功能分析铺平了道路。
