Structural mechanisms of selectivity and gating in anion channelrhodopsins

Abstract

Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behaviour studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anion channels, but also to create next-generation optogenetic tools. Here we report crystal structures of the dACR iC++, along with spectroscopic, electrophysiological and computational analyses that provide unexpected insights into pH dependence, substrate recognition, channel gating and ion selectivity of both dACRs and nACRs. These results enabled us to create an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.

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

“…Individual glutamate replacement of nearly all of these residues (Pro91, Met95, Ser214, Ser 215, Thr216 and Ser226; Figure 3 – figure supplement 1) did not affect rectification, which suggests that the second extracellular vestibule plays only a minor, if any, role in anion conduction in Gt ACR1. Thus our functional data provide empirical confirmation of the stability of hydrogen bonding between Tyr81, Arg94 and Glu223 deduced from all-atom molecular dynamic simulations of Gt ACR1 (Kato et al 2018).…”

“…An intramolecular cavity opened to the extracellular space (a vestibule) was found in the Gt ACR1 dark structure in addition to the continuous tunnel (Figure 3 – figure supplement 1). The crystal structures of the hybrid CCR known as C1C2 (Kato et al 2012) and its Cl - -conducting mutant iC++ (Kato et al 2018) also show a vestibule in the corresponding position (EV2), which is thought to represent the main extracellular entry into the channel pore in these molecules. In the dark Gt ACR1 structure EV2 is separated from the intramolecular tunnel by hydrogen-bonded Tyr81, Arg94 and Glu223 (Kim et al 2018; Li et al 2019).…”

Genomic stability of Self-inactivating Rabies

“…The development of innovative technologies to record and manipulate the activity of large populations of neurons (Jun et al, 2017;Lin and Schnitzer, 2016;Stirman et al, 2016;Yizhar et al, 2011) has had a transformative impact on systems neuroscience leading to a deeper understanding of how specific networks control essential aspects of animal behaviour (Fadok et al, 2017;Kohl et al, 2018;Stuber and Wise, 2016). In particular, the latest generation of molecular sensors and actuators allow researchers to visualize (Abdelfattah et al, 2019;Dana et al, 2019) and perturb (Kato et al, 2018;Shemesh et al, 2017) the activity of individual neurons with unprecedented genetic, spatial and temporal resolution. However, strategies to express these tools in any desired neuron within a neural network structure remain scarce.…”

Protein structures guide the design of a much-needed tool for neuroscience

“…Kato et al now report the latest development: FLASH. This anion-conducting channel rhodopsin was designed using information gleaned from the two new crystal structures 1,2 , and supplants the previous bestin-class protein for inhibitory optogenetics 17 , ZipACR. FLASH can suppress individual action potentials in trains produced at frequencies of up to 40 hertz, with fewer offtarget effects than ZipACR.…”