An improved chloride-conducting channelrhodopsin for light-induced inhibition of neuronal activity in vivo

Abstract

Channelrhodopsins are light-gated cation channels that have been widely used for optogenetic stimulation of electrically excitable cells. Replacement of a glutamic acid in the central gate with a positively charged amino acid residue reverses the ion selectivity and produces chloride-conducting ChRs (ChloCs). Expressed in neurons, published ChloCs produced a strong shunting effect but also a small, yet significant depolarization from the resting potential. Depending on the state of the neuron, the net result of illumination might therefore be inhibitory or excitatory with respect to action potential generation. Here we report two additional amino acid substitutions that significantly shift the reversal potential of improved ChloC (iChloC) to the reversal potential of endogenous GABAA receptors. As a result, light-evoked membrane depolarization was strongly reduced and spike initiation after current injection or synaptic stimulation was reliably inhibited in iChloC-transfected neurons in vitro. In the primary visual cortex of anesthetized mice, activation of iChloC suppressed spiking activity evoked by visual stimulation. Due to its high operational light sensitivity, iChloC makes it possible to inhibit neurons in a large volume of brain tissue from a small, point-like light source.

The light‐driven sodium ion pump: A new player in rhodopsin research

“…In addition, as shown in recent successful examples [20,33,[69][70][71][72][73][74][75][76][77][78], the wealth of structural data available and advances in genomic technologies will lead to further engineering and discovery of functionally novel rhodopsins, and it is expected that the scope and impact of rhodopsin research will continue to expand.…”

“…Recently, the structural information on channelrhodopsin (ChR), a light-gated cation channel, has prompted the engineering of a light-gated anion channel [69][70][71][72], and soon after that, light-gated anion channels were discovered in a natural source [73,74]. Structurebased molecular engineering accomplished a 100 nm blue shift in the absorption spectrum of a proton pumping rhodopsin [75], and de novo transcriptome sequencing of green alga has led to the discovery of a novel ChR with a very red-shifted spectral peak (590 nm) [76].…”

Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity

“…Here we demonstrate the initial use of chloride-conducting channelrhodopsins in the behavior of freely moving animals performing tasks involving the VTA and LA. Beyond this generation of these optogenetic inhibitors that take advantage of physiological ion gradients rather than pumping ions unidirectionally, iC++ and related tools (8)(9)(10)33) also lay the groundwork for a deeper understanding of structure-function relationships involving the light-gated ion conductance pathway of channelrhodopsins. Along with prior work on chloride channel engineering (11,(34)(35)(36), this second round of predictive validation of the crystal structure-guided electrostatic model for the channelrhodopsin pore (7,8,11) provides basic insight into the operation of the microbial opsin channel proteins.…”

The form and function of channelrhodopsin

“…At the same time, different mutations (including negative-to-positive replacement of the central gate Glu 90 with Lys + or Arg + ) were used to create a Cl − -selective ChR2 (93), with the unifying theme of similar-direction effects on predicted internal pore electrostatics (93). Further mutagenesis of both variants [guided by the new model, in which complete elimination of proton conductance also required mutation of one or two putative proton wire pore glutamates (64, 94)] resulted in highly Cl − -selective ChRs [iChloC and iC++; Fig. 4G (64, 94)] now widely used for studies of animal behavior (conferring optogenetic inhibition in typical situations wherein internal Cl − concentration is low) (64, 95, 96).…”