In channelrhodopsin-2 Glu-90 is crucial for ion selectivity and is deprotonated during the photocycle.
The light-activated microbial ion channel channelrhodopsin-2 (ChR2) is a powerful tool to study cellular processes with high spatiotemporal resolution in the emerging field of optogenetics. To customize the channel properties for optogenetic experiments, a detailed understanding of its molecular reaction mechanism is essential. Here, Glu-90, a key residue involved in the gating and selectivity mechanism of the ion channel is characterized in detail. The deprotonation of Glu-90 during the photocycle is elucidated by time-resolved FTIR spectroscopy, which seems to be part of the opening mechanism of the conductive pore. Furthermore, Glu-90 is crucial to ion selectivity as also revealed by mutation of this residue combined with voltage clamp experiments. By dynamic homology modeling, we further hypothesized that the conductive pore is flanked by Glu-90 and located between helices A, B, C, and G.
Department of Biophysics, Ruhr-University Bochum, Universitätsstrasse 150, Bochum 44801, Germany.
Kirstin
K
Eisenhauer
Jens
J
Kuhne
Eglof
E
Ritter
André
A
Berndt
Steffen
S
Wolf
Erik
E
Freier
Franz
F
Bartl
Peter
P
Hegemann
Klaus
K
Gerwert
D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000502:physiology*; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D018698:Glutamic Acid / Q000737:chemistry*; D007834:Lasers; D008027:Light; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared; D014867:Water / Q000737:chemistry; D014981:Xenopus
D002352:Carrier Proteins; D011522:Protons; C523009:channelopsin-2, Chlamydomonas reinhardtii; D014867:Water; D018698:Glutamic Acid