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2 0 0 0 OA Gate-keeper of ion transport ―a highly conserved helix-3 tryptophan in a channelrhodopsin chimera, C1C2/ChRWR

著者
Yujiro Nagasaka Shoko Hososhima Naoko Kubo Takashi Nagata Hideki Kandori Keiichi Inoue Hiromu Yawo
出版者
The Biophysical Society of Japan
雑誌
Biophysics and Physicobiology (ISSN:21894779)
巻号頁・発行日
pp.BSJ-2020007, (Released:2020-06-09)
被引用文献数
5

1 0 0 0 OA Gate-keeper of ion transport—a highly conserved helix-3 tryptophan in a channelrhodopsin chimera, C1C2/ChRWR

著者
Yujiro Nagasaka Shoko Hososhima Naoko Kubo Takashi Nagata Hideki Kandori Keiichi Inoue Hiromu Yawo
出版者
The Biophysical Society of Japan
雑誌
Biophysics and Physicobiology (ISSN:21894779)
巻号頁・発行日
vol.17, pp.59-70, 2020 (Released:2020-07-22)
参考文献数
51
被引用文献数
5
Microbial rhodopsin is a large family of membrane proteins having seven transmembrane helices (TM1-7) with an all-trans retinal (ATR) chromophore that is covalently bound to Lys in the TM7. The Trp residue in the middle of TM3, which is homologous to W86 of bacteriorhodopsin (BR), is highly conserved among microbial rhodopsins with various light-driven functions. However, the significance of this Trp for the ion transport function of microbial rhodopsins has long remained unknown. Here, we replaced the W163 (BR W86 counterpart) of a channelrhodopsin (ChR), C1C2/ChRWR, which is a chimera between ChR1 and 2, with a smaller aromatic residue, Phe to verify its role in the ion transport. Under whole-cell patch clamp recordings from the ND7/23 cells that were transfected with the DNA plasmid coding human codon optimized C1C2/ChRWR (hWR) or its W163F mutant (hWR-W163F), the photocurrents were evoked by a pulsatile light at 475 nm. The ion-transporting activity of hWR was strongly altered by the W163F mutation in 3 points: (1) the H+ leak at positive membrane potential (Vm) and its light-adaptation, (2) the attenuation of cation channel activity and (3) the manifestation of outward H+ pump activity. All of these results strongly suggest that W163 has a role in stabilizing the structure involved in the gating-on and -off of the cation channel, the role of “gate keeper”. We can attribute the attenuation of cation channel activity to the incomplete gating-on and the H+ leak to the incomplete gating-off.