Molecular Determinants of Photocurrent Kinetics of the Red Light Activable Channelrhodopsin Chrimson

Abstract

Channelrhodopsin (ChR) are light gated ion channels which expressed in neurons enable the temporal and spacial precise regulation of membrane voltage and action potential firing by light [1]. Recently an extended screen of 61 potential ChR genes for photocurrents in human kidney cells discovered a far-red activable ChR from the freshwater algae Chlamydomonas noctigama CnChR1 also named Chrimson [2]. Chrimson shows peak photocurrents at 590 nm more than 50 nm further red shifted than previous ChRs and is therefor best suited for optogenetic experiments requiring deep tissue penetration or dual color activation with blue and red light. Photocurrent kinetics are key properties of ChRs determining their potential optogenetic application. Whereas fast photocurrent kinetics enable the temporally precise triggering of high frequency action potential trains, slow photocurrent kinetics increase the operational range of ChRs to lower light intensities and enable the prolonged manipulation of membrane voltage by single low intensity light pulses. In the well-characterized CrChR2 from Chlamydomonas reinhardtii the residue D156 has been identified as a key determinant of photocurrent kinetics possibly participating in the reprotonation of the retinal Schiff base [3]. In Chrimson this aspartate is not conserved. Interestingly photocurrent kinetics are still fast indicating different kinetic determinants in Chrimson than in CrChR2. By sight-directed mutagenesis we investigated amino acid substitution in Chrimson compared to CrChR2 in the inner gate, central gate and in the retinal binding pocket and functionally characterized kinetic properties of these mutants in whole cell patch clamp measurements. Whereas specific substitutions in all three regions of the protein were affecting channel kinetics, especially the retinal binding pocket proved essential for fast photocurrent kinetics.