Engineering of channelrhodopsins with improved ion selectivity as next-generation optogenetic tools and their applications

2015 Fiscal Year Final Research Report

• Project/Area Number: 25290002
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Neurophysiology / General neuroscience
• Research Institution: Tohoku University
• Principal Investigator: ISHIZUKA Toru 東北大学, 生命科学研究科, 講師 (10344714)
• Co-Investigator(Renkei-kenkyūsha): YAWO Hiromu 東北大学, 大学院生命科学研究科, 教授 (00144353)
• Research Collaborator: HOSOSHIMA Shoko ; WATANABE Shota ; ZAMANI Alemeh ; HOQUE Mohammad Razuanul
• Project Period (FY): 2013-04-01 – 2016-03-31
• Keywords: オプトジェネティクス / チャネルロドプシン / 光遺伝学 / 微生物型ロドプシン / ナトリウムイオン

Outline of Final Research Achievements

The five glutamate residues in the second transmembrane helix of channelrhodopsin-2 (ChR2) are conserved among various ChRs. One of them, E97, interacts with cations to facilitate their permeation and is also a primary binding site for Gd(3+). However, the counterpart of MChR1 is alanine. We investigated the ion flux and the Gd(3+)-dependent channel block of MChR1. We found that the high-affinity binding site for Gd(3+) was absent, but was dependent on the negativity. The ion flux was markedly interfered with a negative charge at the position. Based on these findings, it is suggested that the channel pore of MChR1 is differently organized from that of ChR2.
Krokinobacter rhodopsin 2 (KR2) is a light-driven Na(+) pump. We introduced a codon-optimized KR2 fused with eYFP and membrane trafficking signals into cultured cortical neurons. The generation of action potential was completely blocked while KR2 activation with green light. KR2 would be useful as a next-generation optogenetic tool.

Free Research Field

分子・細胞神経生物学