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Channelrhodopsin-2 (ChR2) is a light-activated channel that can conduct cations of multiple valencies down the electrochemical gradient. Under continuous light exposure, ChR2 transitions from a high-conducting open state (O1) to a low-conducting open state (O2) with differing ion selectivity. The molecular basis for the O1 → O2 transition and how ChR2 modulates selectivity between states is currently unresolved. To this end, we used steered molecular dynamics, electrophysiology, and kinetic modeling to identify residues that contribute to gating and selectivity in discrete open states. Analysis of steered molecular dynamics experiments identified three transmembrane residues (Val-86, Lys-93, and Asn-258) that form a putative barrier to ion translocation. Kinetic modeling of photocurrents generated from ChR2 proteins with conservative mutations at these positions demonstrated that these residues contribute to cation selectivity (Val-86 and Asn-258), the transition between the two open states (Val-86), open channel stability, and the hydrogen-bonding network (K93I and K93N). These results suggest that this approach can be used to identify residues that contribute to the open-state transitions and the discrete ion selectivity within these states. With the rise of ChR2 use in optogenetics, it will be critical to identify residues that contribute to O1 or O2 selectivity and gating to minimize undesirable effects., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Channelrhodopsin-2 is a light-activated cation channel. However, the mechanism of ion conductance is unresolved. Here, we performed cysteine scanning mutagenesis on transmembrane domain 7 followed by labeling with a methanethiosulfonate compound. Analysis of our results shows that residues that line the putative pore and interface with adjacent transmembrane domains 1 and 3, as proposed by our channelrhodopsin-2 homology model, affect ion conductance, decay kinetics, and/or off kinetics. Combined, these results suggest that negative charges at the extracellular side of transmembrane domain 7 funnel cations into the pore.

Channelrhodopsin-2 (ChR2) is a light-activated nonselective cation channel that is found in the eyespot of the unicellular green alga Chlamydomonas reinhardtii. Despite the wide employment of this protein to control the membrane potential of excitable membranes, the molecular determinants that define the unique ion conductance properties of this protein are not well understood. To elucidate the cation permeability pathway of ion conductance, we performed cysteine scanning mutagenesis of transmembrane domain three followed by labeling with methanethiosulfonate derivatives. An analysis of our experimental results as modeled onto the crystal structure of the C1C2 chimera demonstrate that the ion permeation pathway includes residues on one face of transmembrane domain three at the extracellular side of the channel that face the center of ChR2. Furthermore, we examined the role of a residue at the extracellular side of transmembrane domain three in ion conductance. We show that ion conductance is mediated, in part, by hydrogen bonding at the extracellular side of transmembrane domain three. These results provide a starting point for examining the cation permeability pathway for ChR2., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The light-gated cation channel channelrhodopsin-2 (ChR2) has rapidly become an important tool in neuroscience, and its use is being considered in therapeutic interventions. Although wild-type and known variant ChR2s are able to drive light-activated spike trains, their use in potential clinical applications is limited by either low light sensitivity or slow channel kinetics. We present a new variant, calcium translocating channelrhodopsin (CatCh), which mediates an accelerated response time and a voltage response that is ∼70-fold more light sensitive than that of wild-type ChR2. CatCh's superior properties stem from its enhanced Ca2+ permeability. An increase in [Ca2+]i elevates the internal surface potential, facilitating activation of voltage-gated Na+ channels and indirectly increasing light sensitivity. Repolarization following light-stimulation is markedly accelerated by Ca2+-dependent BK channel activation. Our results demonstrate a previously unknown principle: shifting permeability from monovalent to divalent cations to increase sensitivity without compromising fast kinetics of neuronal activation. This paves the way for clinical use of light-gated channels. [ABSTRACT FROM AUTHOR]