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eNeuro. 2018 Jul 10;5(3). pii: ENEURO.0174-18.2018. doi: 10.1523/ENEURO.0174-18.2018. eCollection 2018 May-Jun.

Extending the Time Domain of Neuronal Silencing with Cryptophyte Anion Channelrhodopsins.

Author information

1
Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, McGovern Medical School, Houston, TX 77030.
2
Department of Neurobiology and Anatomy, The University of Texas Health Science Center at Houston, McGovern Medical School, Houston, TX 77030.
3
Institute of Botany, Cologne Biocenter, University of Cologne, Cologne D-50674, Germany.
4
Departments of Biological Sciences and of Medicine, University of Alberta, Edmonton, AB T6G 2E1, Canada.
5
Beijing Genomics Institute-Shenzhen, Shenzhen 518083, China.

Abstract

Optogenetic inhibition of specific neuronal types in the brain enables analysis of neural circuitry and is promising for the treatment of a number of neurological disorders. Anion channelrhodopsins (ACRs) from the cryptophyte alga Guillardia theta generate larger photocurrents than other available inhibitory optogenetic tools, but more rapid channels are needed for temporally precise inhibition, such as single-spike suppression, of high-frequency firing neurons. Faster ACRs have been reported, but their potential advantages for time-resolved inhibitory optogenetics have not so far been verified in neurons. We report RapACR, nicknamed so for "rapid," an ACR from Rhodomonas salina, that exhibits channel half-closing times below 10 ms and achieves equivalent inhibition at 50-fold lower light intensity in lentivirally transduced cultured mouse hippocampal neurons as the second-generation engineered Cl--conducting channelrhodopsin iC++. The upper limit of the time resolution of neuronal silencing with RapACR determined by measuring the dependence of spiking recovery after photoinhibition on the light intensity was calculated to be 100 Hz, whereas that with the faster of the two G. theta ACRs was 13 Hz. Further acceleration of RapACR channel kinetics was achieved by site-directed mutagenesis of a single residue in transmembrane helix 3 (Thr111 to Cys). We also show that mutation of another ACR (Cys to Ala at the same position) with a greatly extended lifetime of the channel open state acts as a bistable photochromic tool in mammalian neurons. These molecules extend the time domain of optogenetic neuronal silencing while retaining the high light sensitivity of Guillardia ACRs.

KEYWORDS:

channelrhodopsins; chloride ion channels; neuronal inhibition; optogenetics

PMID:
30027111
PMCID:
PMC6051594
DOI:
10.1523/ENEURO.0174-18.2018
[Indexed for MEDLINE]
Free PMC Article

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