Abstract
Channelrhodopsins are light-gated ion channels of green algae used for the precise temporal and spatial control of transmembrane ion fluxes. The channelrhodopsin Chrimson from Chlamydomonas noctigama allows unprecedented deep tissue penetration due to peak absorption at 590 nm. We demonstrate by electrophysiological recordings and imaging techniques that Chrimson is highly proton selective causing intracellular acidification in HEK cells that is responsible for slow photocurrent decline during prolonged illumination. We localized molecular determinants of both high proton selectivity and red light activation to the extracellular pore. Whereas exchange of Glu143 only drops proton conductance and generates an operational Na-channel with 590 nm activation, exchange of Glu139 in addition increased the open state lifetime and shifted the absorption hypsochromic by 70 nm. In conjunction with Glu300 in the center and Glu124 and Glu125 at the intracellular end of the pore, Glu139 contributes to a delocalized activation gate and stabilizes by long-range interaction counterion configuration involving protonation of Glu165 that we identified as a key determinant of the large opsin shift in Chrimson.
Highlights
Understanding the complex interplay of molecular processes in living systems requires the ability to control the activity of distinct molecular components in a non-invasive manner
We demonstrate that under physiological conditions, Chrimson photocurrents are primarily carried by protons, which causes significant intracellular acidification in human embryonic kidney (HEK) cells during prolonged illumination
We identified an altered gate structure in Chrimson compared to other channelrhodopsins featuring an additional putative pore constriction around E4’, that determines proton selectivity, photocurrent kinetics, and is essential for the large opsin shift in Chrimson
Summary
Understanding the complex interplay of molecular processes in living systems requires the ability to control the activity of distinct molecular components in a non-invasive manner. Recent studies have likewise emphasized the impact of microbial rhodopsin stimulation on local sodium, calcium, chloride, or proton concentrations leading to numerous secondary effects at the subcellular level[8]. These effects include the active import of calcium ions into astrocytes by a Na+-Ca2+-exchanger[9], the pH-induced influx of calcium in synaptic terminals[10], shifts in the gamma-aminobutyric acid (GABA) receptor reversal potential in synaptic terminals[11], or the activation of acid-sensing ion channels (ASIC) in proton microdomains on the extracellular surface[12] or near astrocytes expressing proton pumps[13]. Replacing E3’ with a polar glutamine or a neutral alanine residue reduces proton conductance stepwise[25, 26], whereas replacing E3’ with a positively charged arginine abolishes cation conductance, transforming CrChR2 into an anion-selective ChR6
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
