Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools for manipulating neuronal activity. All currently known ChRs comprise a large cytoplasmic domain, whose function is elusive. Here, we report the cation channel properties of KnChR, one of the photoreceptors from a filamentous terrestrial alga Klebsormidium nitens, and demonstrate that the cytoplasmic domain of KnChR modulates the ion channel properties. KnChR is constituted of a 7-transmembrane domain forming a channel pore, followed by a C-terminus moiety encoding a peptidoglycan binding domain (FimV). Notably, the channel closure rate was affected by the C-terminus moiety. Truncation of the moiety to various lengths prolonged the channel open lifetime by more than 10-fold. Two Arginine residues (R287 and R291) are crucial for altering the photocurrent kinetics. We propose that electrostatic interaction between the rhodopsin domain and the C-terminus domain accelerates the channel kinetics. Additionally, maximal sensitivity was exhibited at 430 and 460 nm, the former making KnChR one of the most blue-shifted ChRs characterized thus far, serving as a novel prototype for studying the molecular mechanism of color tuning of the ChRs. Furthermore, KnChR would expand the optogenetics tool kit, especially for dual light applications when short-wavelength excitation is required.

视紫红质 (ChR) 是光门控离子通道，广泛用作操纵神经元活动的光遗传学工具。所有目前已知的 ChR 都包含一个大的细胞质结构域，其功能难以捉摸。在这里，我们报告了来自丝状陆生藻类Klebsormidium nitens的光感受器之一 KnChR 的阳离子通道特性，并证明 KnChR 的细胞质结构域调节离子通道特性。KnChR 由形成通道孔的 7 跨膜结构域和编码肽聚糖结合结构域 (FimV) 的 C 端部分组成。值得注意的是，通道关闭率受 C 端部分的影响。将该部分截断为不同长度可将通道开放寿命延长 10 倍以上。两个精氨酸残基（R287 和 R291）对于改变光电流动力学至关重要。我们建议视紫质域和 C 端域之间的静电相互作用加速了通道动力学。此外，在 430 和 460 nm 处表现出最大灵敏度，前者使 KnChR 成为迄今为止表征的最蓝移的 ChR 之一，作为研究 ChR 颜色调节分子机制的新原型。此外，KnChR 将扩展光遗传学工具包，特别是对于需要短波长激发的双光应用。