Abstract
Channelrhodopsins are light-activated ion channels that mediate cation permeation across cell membranes upon light absorption. Red-light-activated channelrhodopsins are of particular interest, because red light penetrates deeper into biological tissues and also enables dual-color experiments in combination with blue-light-activated optogenetic tools. Here we report the crystal structure of the most red-shifted channelrhodopsin from the algae Chlamydomonas noctigama, Chrimson, at 2.6 Å resolution. Chrimson resembles prokaryotic proton pumps in the retinal binding pocket, while sharing similarity with other channelrhodopsins in the ion-conducting pore. Concomitant mutation analysis identified the structural features that are responsible for Chrimson’s red light sensitivity; namely, the protonation of the counterion for the retinal Schiff base, and the polar residue distribution and rigidity of the retinal binding pocket. Based on these mechanistic insights, we engineered ChrimsonSA, a mutant with a maximum activation wavelength red-shifted beyond 605 nm and accelerated closing kinetics.
Highlights
Channelrhodopsins are light-activated ion channels that mediate cation permeation across cell membranes upon light absorption
The photocurrent properties of Chrimson were minimally affected by the different N-terminus, and red light activation, high proton selectivity, and fast pHdependent photocurrent kinetics were preserved in all three Chrimson variants (Supplementary Figure 4a–e)
Chrimson is widely used in optogenetic experiments, since its red-shifted action spectrum allows deep tissue penetration of the excitation light, activation in a spectral range beyond the natural sensitivity of native photoreceptors in many animal species and combination with blue-light-activated tools for dual-color experiments
Summary
Channelrhodopsins are light-activated ion channels that mediate cation permeation across cell membranes upon light absorption. We present the crystal structure of Chrimson at 2.6 Å resolution, which reveals (i) the presence of an outer gate occluding the retinal Schiff base from the extracellular solvent, (ii) unique protonation state of the counterion residue destabilizing the protonated retinal Schiff base in the ground state, (iii) highly biased polar residue distribution toward the β-ionone ring, and (iv) tight association with the retinal chromophore to improve the retinal planarity, which appears essential for fast photocycle kinetics Based on these molecular insights, we have generated ChrimsonSA (S169A, Super red-shifted and Accelerated) with more than 20 nm further red-shifted absorption, significantly reduced blue-light-sensitivity, and accelerated closing kinetics, compared to wild-type Chrimson. ChrimsonSA expressed well in hippocampal neurons and allowed spiking with red light while the prevalence of blue-light-evoked action potentials was strongly reduced, making it a useful tool for dualcolor applications
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