In vitro Activity of a Purified Natural Anion Channelrhodopsin

Abstract

Natural anion channelrhodopsins (ACRs) recently discovered in cryptophyte algae are the most active rhodopsin channels known. They are of interest both because of their unique natural function of light‐gated chloride conductance, and their unprecedented efficiency of membrane hyperpolarization for optogenetic neuron silencing. Light‐induced currents of ACRs have been studied in HEK cells and neurons, but light‐gated channel conductance of ACRs in vitro has not been demonstrated. Here we report light‐induced chloride channel activity of a purified ACR protein reconstituted in large unilamellar vesicles (LUVs). Electron paramagnetic resonance (EPR) measurements establish that the channels are inserted uniformly ‘inside‐out’ with their cytoplasmic surface facing the medium of the LUV suspension. We show by time‐resolved flash spectroscopy that the photochemical reaction cycle of a functional purified ACR from Guillardia theta (GtACR1) in LUVs exhibits similar spectral shifts indicating similar photocycle intermediates as GtACR1 in detergent micelles. Furthermore, the photocycle rate is dependent on electric potential generated by chloride gradients in the LUVs in the same manner as in voltage‐clamped animal cells. We confirm with this system that, opposite to cation‐conducting channelrhodopsins, opening of the channel occurs prior to deprotonation of the Schiff‐base. However, the photointermediate transitions in the LUVs exhibit faster kinetics. The ACR‐incorporated LUVs provides a purified defined system amenable to EPR, optical and vibrational spectroscopy, and fluorescence resonance energy transfer measurements of structural changes of ACRs with the molecules in a demonstrably functional state.Support or Funding InformationThis work was supported by NIH grant R01GM027750 and Endowed Chair AU‐0009 from the Robert A. Welch Foundation.