Format

Send to

Choose Destination
J Biol Chem. 2019 Mar 15;294(11):3806-3821. doi: 10.1074/jbc.RA118.006996. Epub 2019 Jan 4.

Multidimensional screening yields channelrhodopsin variants having improved photocurrent and order-of-magnitude reductions in calcium and proton currents.

Author information

1
From the MIT Media Lab, McGovern Institute, and Koch Institute, Departments of Biological Engineering and Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139.
2
From the MIT Media Lab, McGovern Institute, and Koch Institute, Departments of Biological Engineering and Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139 esb@media.mit.edu.

Abstract

Channelrhodopsins (ChRs) are light-gated ion channels in widespread use in neuroscience for mediating the genetically targetable optical control of neurons (optogenetics). ChRs pass multiple kinds of ions, and although nonspecific ChR-mediated conductance is not an issue in many neuroscience studies, conductance of calcium and protons, which can mediate diverse cellular signals, may be undesirable in some instances. Here, we turned our attention to the creation of ChRs that have high cation photocurrent but pass fewer calcium ions and protons. We developed an automated, time-resolved screening method capable of rapidly phenotyping channelrhodopsin-2 (ChR2) variants. We found substitution mutations throughout ChR2 that could boost current while altering ion selectivity and observed that the mutations that reduced calcium or proton conductance have additive effects. By combining four mutations, we obtained a ChR, ChromeQ, with improved photocurrent that possesses order-of-magnitude reductions in calcium and proton conductance and high fidelity in driving repetitive action potentials in neurons. The approach presented here offers a viable pathway toward customization of complex physiological properties of optogenetic tools. We propose that our screening method not only enables elucidation of new ChR variants that affect microbial opsin performance but may also reveal new principles of optogenetic protein engineering.

KEYWORDS:

calcium; channelrhodopsin; neurobiology; optogenetics; physiology; protein engineering; proton; screening

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center