NEAR-IR Resonance Raman Characterization of an Anion Channelrhodopsin from Guillardia Theta

Abstract

Optogenetics relies heavily on the expression of specific microbial rhodopsins in the neuronal plasma membrane. Most notably, this includes channelrhodopsins (ChRs), which normally function as light-gated cation channels, activating nerves upon illumination due to the flow of positive charge into the neuron and subsequent depolarization. Recently, a new class of ChRs, termed anion channelrhodopsins (ACRs), has been discovered (Govorunova et al. Science, 2015). These proteins function as true light-activated anion channels. They exclude the flow of protons and other cations and cause hyperpolarization of the membrane potential by allowing the inward flow of chloride ions. Compared to ion pumps previously used for neuronal silencing, ACRs have been shown to be effective at <1/1000th of the light intensity.In this study, near-infrared confocal resonance Raman spectroscopy (RRS) along with hydrogen/deuterium exchange, retinal analog substitution, and site-directed mutagenesis were used to study the retinal structure of the unphotolyzed state of an ACR from Guillardia theta (GtACR1) heterologously expressed and extracted from Pichia pastoris cells (Sineshchekov et al. PNAS, 2015, under review). These measurements reveal that: i) GtACR1 has an all-trans retinal structure similar to the structure of the red-shifted ChR1 from Chlamydomonas augustae (CaChR1); ii) varying the pH of GtACR1 from 3 to 11 or changing anion species did not alter the RRS, indicating that groups in the retinal binding pocket did not undergo changes in the protonation state over this pH range and that an exchangeable anion does not interact with the retinal under the conditions of the RRS measurement; iii) H/D exchange induced a downshift of the GtACR1 protonated Schiff base consistent with a weaker hydrogen bonding strength than other ChRs thus far characterized by RRS.