Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis

• Published in: The Journal of biological chemistry 2015-05, Vol.290 (20), p.12719-12730
• Main Authors: Ogren, John I., Yi, Adrian, Mamaev, Sergey, Li, Hai, Spudich, John L., Rothschild, Kenneth J.
• Format: Article
• Language: English
• Subjects: Chlamydomonas reinhardtii - chemistry; Chlamydomonas reinhardtii - genetics; Chlamydomonas reinhardtii - metabolism; Fourier Transform IR (FTIR); Ion Channel; Membrane Protein; Molecular Biophysics; Mutagenesis, Site-Directed; Optogenetics; Plant Proteins - genetics; Plant Proteins - metabolism; Protons; Rhodopsin; Rhodopsin - genetics; Rhodopsin - metabolism; Spectroscopy, Fourier Transform Infrared
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.634840

Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2380 state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2380 formation. The unusual charge neutrality of both Schiff base counterions in the P2380 conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs. Channelrhodopsin-1 is a red-shifted light-gated cation channel. Proton transfers and H-bonding changes involving the Schiff base counterion residues Asp-169 and Glu-299 were detected. A two-step proton transfer relay mechanism consistent with our results and earlier photoinduced channel current and pH titration measurements is proposed. Understanding the mechanism of channelrhodopsin can lead to improved optogenetic control of neurons.