Crystal structure of the natural anion-conducting channelrhodopsin GtACR1

Abstract

The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta, exhibits large light-gated anion conductance and high anion selectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9 Å resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools.

Molecular mechanism of light-driven sodium pumping

“…15). Indeed, the CGs of the native cation channelrhodopsin-2 (CrChR2) 50 , chimeric cation channelrhodopsin (C1C2) 51 and also natural anion channelrhodopsin (GtACR1) 52,53 are composed of the S63-N258-E90, S102-N297-E129 and S43-N239-E68 triads ( Supplementary Fig. 15).…”

Viral channelrhodopsins: calcium-dependent Na+/K+selective light-gated channels

“…Microbial and animal rhodopsins (type-1 and 2 rhodopsins, respectively) comprise a superfamily of heptahelical (7-TM) transmembrane proteins covalently linked to a retinal chromophore (Ernst et al, 2014;Gushchin and Gordeliy, 2018). Type-1 rhodopsins are the most abundant light-harvesting proteins that have diverse functions, such as ion pumping, ion channeling, sensory and enzymatic activities (Gordeliy et al, 2002;Govorunova et al, 2015;Gushchin et al, 2013Gushchin et al, , 2015Kato et al, 2015;Kim et al, 2018;Mukherjee et al, 2019;Shevchenko et al, 2017;Volkov et al, 2017). The discovery, in 2000, of the light-driven pump proteorhodopsin (PR) in marine microbes triggered extensive search of metagenomes for light-activated proteins (Béjà et al, 2000).…”

“…The study of microbial rhodopsins culminated in the development of optogenetics, the revolutionary method for controlling cell behavior in vivo using light-gated channels and light-driven pumps (Deisseroth, 2015). Currently, major efforts are being undertaken to identify rhodopsins with novel functions and properties that could be harnessed to enhance optogenetic applications (Berndt and Deisseroth, 2015;Berndt et al, 2014;Govorunova et al, 2015;Kato et al, 2018;Kim et al, 2018;Sineshchekov et al, 2017a).…”

The photoactive site modulates current rectification and channel closing in the natural anion channelrhodopsin GtACR1

“…Anion channelrhodopsins from the cryptophyte alga Guillardia theta ( Gt ACRs) are the most potent optogenetic inhibitors of neuronal firing and regulators of animal behavior currently available (Govorunova et al 2015; Mahn et al 2018; Messier et al 2018; Mohammad et al 2017; Wilson et al 2018). Recently we and others have obtained high-resolution X-ray structures of the dark (closed) state of G. theta anion channelrhodopsin 1 ( Gt ACR1) (Kim et al 2018; Li et al 2019). In contrast to available structures of cation channelrhodopsins (CCRs) from green algae (Kato et al 2012; Oda et al 2018; Volkov et al 2017), Gt ACR1 exhibits a narrow continuous intramolecular tunnel formed by helices 1-3 and 7 that connects the cytoplasmic and extracellular aqueous phases and presumably expands upon illumination to pass anions (Li et al 2019).…”

“…However, no open channel structure is yet available, and many questions regarding its architecture remain unanswered. In particular, a possible role of the second extracellular vestibule (Kim et al 2018) in anion conductance has not so far been tested experimentally. Also, the functional significance of the many structural differences between ACRs and CCRs is not yet clear.…”