Molecular properties of a DTD channelrhodopsin from .

Microbial rhodopsins are membrane proteins found widely in archaea, eubacteria and eukaryotes (fungal and algal species). They have various functions, such as light-driven ion pumps, light-gated ion channels, light sensors and light-activated enzymes. A light-driven proton pump bacteriorhodopsin (BR) contains a DTD motif at positions 85, 89, and 96, which is unique to archaeal proton pumps. Recently, channelrhodopsins (ChRs) containing the DTD motif, whose sequential identity is ~20% similar to BR and to cation ChRs in (CCRs), were found. While extensive studies on ChRs have been performed with CCR2, the molecular properties of DTD ChRs remain an intrigue. In this paper, we studied a DTD rhodopsin from (CCR4) using electrophysiological measurements, flash photolysis, and low-temperature difference FTIR spectroscopy. Electrophysiological measurements clearly showed that CCR4 functions as a light-gated cation channel, similar to other DTD ChRs (CCR1-3). Light-driven proton pump activity was also suggested for CCR4. Both electrophysiological and flash photolysis experiments showed that channel closing occurs upon reprotonation of the Schiff base, suggesting that the dynamics of retinal and channels are tightly coupled in CCR4. From Fourier transform infrared (FTIR) spectroscopy at 77 K, we found that the primary reaction is an all- to a 13- photoisomerization, like other microbial rhodopsins, although perturbations in the secondary structure were much smaller in CCR4 than in CCR2.