Light-driven Anion-Pumping Rhodopsin with Unique Cytoplasmic Anion-release Mechanism.

Microbial rhodopsins are photoreceptive membrane proteins found in microorganisms with an all-trans-retinal chromophore. The function of many microbial rhodopsins is determined by three residues in the third transmembrane helix called motif residues. Here, we report a group of microbial rhodopsins with a novel Thr-Thr-Gly (TTG) motif. The ion-transport assay revealed that they function as light-driven inward anion pumps similar to halorhodopsins previously found in archaea and bacteria. Based on the characteristic glycine residue in their motif and light-driven anion-pumping function, these new rhodopsins are called glycylhalorhodopsins (GHRs). X-ray crystallographic analysis found large cavities on the cytoplasmic side, which are produced by the small side-chain volume of the glycine residue in the motif. The opened structure of GHR on the cytoplasmic side is related to the anion releasing process to the cytoplasm during the photoreaction compared to canonical halorhodopsin from Natronomonas pharaonis (NpHR). GHR also transports SO42- and the extracellular glutamate residue plays an essential role in extracellular SO42- uptake. In summary, we have identified TTG motif-containing microbial rhodopsins that display an anion-releasing mechanism.

Twisting and Protonation of Retinal Chromophore Regulate Channel Gating of Channelrhodopsin C1C2.

Channelrhodopsins (ChRs) are light-gated ion channels and central optogenetic tools that can control neuronal activity with high temporal resolution at the single-cell level. Although their application in optogenetics has rapidly progressed, it is unsolved how their channels open and close. ChRs transport ions through a series of interlocking elementary processes that occur over a broad time scale of subpicoseconds to seconds. During these processes, the retinal chromophore functions as a channel regulatory domain and transfers the optical input as local structural changes to the channel operating domain, the helices, leading to channel gating. Thus, the core question on channel gating dynamics is how the retinal chromophore structure changes throughout the photocycle and what rate-limits the kinetics. Here, we investigated the structural changes in the retinal chromophore of canonical ChR, C1C2, in all photointermediates using time-resolved resonance Raman spectroscopy. Moreover, to reveal the rate-limiting factors of the photocycle and channel gating, we measured the kinetic isotope effect of all photoreaction processes using laser flash photolysis and laser patch clamp, respectively. Spectroscopic and electrophysiological results provided the following understanding of the channel gating: the retinal chromophore highly twists upon the retinal Schiff base (RSB) deprotonation, causing the surrounding helices to move and open the channel. The ion-conducting pathway includes the RSB, where inflowing water mediates the proton to the deprotonated RSB. The twisting of the retinal chromophore relaxes upon the RSB reprotonation, which closes the channel. The RSB reprotonation rate-limits the channel closing.