期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 108, 期 18, 页码 7595-7600出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1017210108
关键词
hippocampus; optogenetics; photocycle; pyramidal cell; spike frequency
资金
- Leibniz Graduate School for Molecular Biophysics
- European Molecular Biology Organization
- Novartis Research Foundation
- Gebert Ruf Foundation
- German Research foundation, DFG [HE 3824/9-3, 18-1]
Channelrhodopsin-2 (ChR2) has become an indispensable tool in neuroscience, allowing precise induction of action potentials with short light pulses. A limiting factor for many optophysiological experiments is the relatively small photocurrent induced by ChR2. We screened a large number of ChR2 point mutants and discovered a dramatic increase in photocurrent amplitude after threonine-to-cysteine substitution at position 159. When we tested the T159C mutant in hippocampal pyramidal neurons, action potentials could be induced at very low light intensities, where currently available channelrhodopsins were unable to drive spiking. Biophysical characterization revealed that the kinetics of most ChR2 variants slows down considerably at depolarized membrane potentials. We show that the recently published E123T substitution abolishes this voltage sensitivity and speeds up channel kinetics. When we combined T159C with E123T, the resulting double mutant delivered fast photocurrents with large amplitudes and increased the precision of single action potential induction over a broad range of frequencies, suggesting it may become the standard for light-controlled activation of neurons.
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