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

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.

Optogenetic manipulation of second messengers in neurons and cardiomyocytes with microbial rhodopsins and adenylyl cyclase

“…In addition to Gt CCR4, we also analyzed in vivo optogenetic activity of another cation channel Kn ChR, whose biophysical properties were previously analyzed using ND7/23 cells ( Tashiro et al, 2021 ). We expressed Gt CCR4 and Kn ChR in the hindbrain reticulospinal V2a neurons in zebrafish, which were reported to drive locomotion ( Kimura et al, 2013 ), by using a Gal4-UAS system.…”

“…We expressed Gt CCR4 and Kn ChR in the hindbrain reticulospinal V2a neurons in zebrafish, which were reported to drive locomotion ( Kimura et al, 2013 ), by using a Gal4-UAS system. As carboxy terminal truncations of Kn ChR were shown to prolong the channel open lifetime and give a stronger optogenetic activity ( Tashiro et al, 2021 ), a truncated Kn ChR containing 272 amino acids from the N-terminus was expressed as a fusion protein containing the membrane-trafficking signal, the ER-export signal, and EYFP ( Kn ChR-3.0-EYFP). We compared the activities of these channelrhodopsins with that of Cr ChR2[T159C]-mCherry.…”

“…We found that Kn ChR was the most potent optogenetic tool in zebrafish reticulospinal V2a neurons among the examined tools, which include channelrhodopsins, enzymerhodopsins (in this study), and animal G protein-coupled bistable rhodopsins (in the accompanying paper). Truncation of Kn ChR prolongs the channel open lifetime by more than 10-fold ( Tashiro et al, 2021). Kn ChR conducts various monovalent and bivalent cations, including H + , Na + , and Ca 2+ , while Kn ChR has a higher permeability to Na + and Ca 2+ and a higher permeability ratio of Ca 2+ to Na + than Cr ChR2 ( Tashiro et al, 2021 ).…”

“…Kn ChR is another cation channelrhodopsin derived from the filamentous terrestrial alga Klebsormidium nitens (Tashiro et al, 2021 ). Truncation of the carboxyterminal of Kn ChR prolonged the channel open lifetime by more than 10-fold, providing strong light-induced channel activity ( Tashiro et al, 2021 ). These findings imply that Gt CCR4 and truncated variants of Kn ChR are alternative optogenetic tools that can compensate for the shortcomings of Cr ChRs or display stronger photo-inducing activity than Cr ChRs ( Hososhima et al, 2020, Tashiro et al, 2021 ).…”

Emerging Diversity of Channelrhodopsins and Their Structure-Function Relationships

“…ChR sequences were also found in the other three classes of the “core” chlorophytes, i.e., Chlorodendrophyceae, Trebouxiophyceae and Ulvophyceae, in Pedinomonas minor (class Pedinophyceae), and in the three classes of the polyphyletic group known as “prasinophytes” (Mamiellophyceae, Nephroselmidophyceae and Pyramimonadophyceae; Klapoetke et al, 2014 ; Rozenberg et al, 2020 ). ChR homologs have also been found in the several representatives of the phylum Streptophyta: the flagellate alga Mesostigma viride (Govorunova et al, 2011 ), included in Streptophyta as an early diverging lineage, the filamentous alga Klebsormidium nitens (Klebsormidiophyceae; Awasthi et al, 2020 ; Tashiro et al, 2021 ) and thallus-forming alga Coleochaete irregularis (Coleochaetophyceae; Rozenberg et al, 2020 ), the latter of which is considered as the closest relative of land plants (Leliaert et al, 2012 ). However, the sequenced genomes of land plants do not seem to encode ChRs (nor rhodopsins of any kind).…”