Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
1] Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan [2] OptoBioTechnology Research Center, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan [3] PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
Nature. 2015 May 7;521(7550):48-53. doi: 10.1038/nature14322. Epub 2015 Apr 6.
Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.
嗜酸红假单胞菌视紫红质 2（KR2）是首个被发现的光驱动 Na+泵，被视为下一代光遗传学工具。由于位于所有光驱动离子泵离子传导途径内的带正电荷的席夫碱质子被认为会阻止非质子阳离子的运输，因此 KR2 的发现提出了一个问题，即它如何实现 Na+运输。在这里，我们呈现了中性和酸性条件下 KR2 的晶体结构，它们分别代表静止和 M 样中间状态。结构和光谱分析揭示了门控机制，其中 Asp116 的翻转将席夫碱质子从传导途径隔离出来，以促进 Na+运输。结合对首个光驱动 K+泵的基于结构的工程改造、哺乳动物神经元中的电生理测定和线虫中的行为测定，我们的研究揭示了光驱动非质子阳离子泵的分子基础，从而为下一代光遗传学的发展提供了一个框架。
