Specific residues in the cytoplasmic domain modulate photocurrent kinetics of channelrhodopsin from Klebsormidium nitens

Rintaro Tashiro,Shoko Hososhima,Sunita Sharma,Hideki Kandori,Kumari Sushmita,Satoshi P Tsunoda,Suneel Kateriya

doi:10.1038/s42003-021-01755-5

Rintaro Tashiro, Shoko Hososhima + Show 5 more

Open Access

https://doi.org/10.1038/s42003-021-01755-5

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Abstract

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.

Highlights

Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools for manipulating neuronal activity
In this study, we report on the electrophysiological characterization of a newly identified multi-domain cation channelrhodopsin from Klebsormidium nitens (KnChR)
KnChR possesses a large cytoplasmic domain made of about 540 amino acids which might involve a peptidoglycan binding moiety (FimV)

Summary

Introduction

Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools for manipulating neuronal activity. Channelrhodopsin-1 and -2 from Chlamydomonas reinhardtii (CrChR1 and CrChR2) were the first ChRs to be discovered, characterized, and the latter was extensively utilized for optogenetic applications These proteins conduct cations such as H+, Na+, K+, and Ca2+. The initial reports of CrChR1 and CrChR2 described that truncation of the cytoplasmic C-terminal domain did not alter the ion channel function[2,3]. Researchers investigated their molecular properties by using truncated proteins carrying only the 7-TM domain. We performed extensive electrophysiological experiments using KnChR to reveal its ion channel function Its photocurrent properties such as channel kinetics, absorption maxima, light sensitivity, and ion selectivity were compared to those of well-characterized CrChR2. Functional characterization of ChR from an evolutionary terrestrial alga indicates the existence of ChR-mediated physiological responses beyond aquatic habitats (marine and freshwater systems)

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