Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity

被引：159

作者：

Berndt, Andre ^{[1
]}

Lee, Soo Yeun ^{[1
]}

Wietek, Jonas ^{[2
]}

Ramakrishnan, Charu ^{[1
]}

Steinberg, Elizabeth E. ^{[3
,4
]}

Rashid, Asim J. ^{[5
]}

Kim, Hoseok ^{[6
]}

Park, Sungmo ^{[5
]}

Santoro, Adam ^{[5
]}

Frankland, Paul W. ^{[5
]}

Iyer, Shrivats M. ^{[1
]}

Pak, Sally ^{[1
]}

Ahrlund-Richter, Sofie ^{[6
]}

Delp, Scott L. ^{[1
]}

Malenka, Robert C. ^{[3
,4
]}

Josselyn, Sheena A. ^{[5
]}

Carlen, Marie ^{[6
]}

Hegemann, Peter ^{[2
]}

Deisseroth, Karl ^{[1
,3
,7
]}

机构：

[1] Stanford Univ, Dept Bioengn, Stanford, CA 94305 USA

[2] Humboldt Univ, Inst Biol, Expt Biophys, D-10115 Berlin, Germany

[3] Stanford Univ, Dept Psychiat & Behav Sci, Stanford, CA 94305 USA

[4] Stanford Univ, Nancy Pritzker Lab, Stanford, CA 94305 USA

[5] Univ Toronto, Hosp Sick Children, Program Neurosci & Mental Hlth, Toronto, ON M5G 1X8, Canada

[6] Karolinska Inst, Dept Neurosci, SE-17177 Stockholm, Sweden

[7] Stanford Univ, Howard Hughes Med Inst, Stanford, CA 94305 USA

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 2016年 / 113卷 / 04期

基金：

欧洲研究理事会; 美国国家卫生研究院; 加拿大健康研究院;

关键词：

optogenetics; channelrhodopsin; structure; chloride; neuronal inhibition; CHLORIDE CHANNELS; CATION CHANNEL; FEAR MEMORY; NEURONS; INHIBITION; AMYGDALA; EXCITABILITY; CONVERSION; AVERSION; RECEPTOR;

D O I：

10.1073/pnas.1523341113

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near -65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another similar to 15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor-based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structurefunction relationships of the light-gated pore.

引用

页码：822 / 829

页数：8

共 37 条

[1] Expanding the optogenetics toolkit [J].