Characterization of engineered channelrhodopsin variants with improved properties and kinetics.
Channelrhodopsin 2 (ChR2), a light-activated nonselective cationic channel from Chlamydomonas reinhardtii, has become a useful tool to excite neurons into which it is transfected. The other ChR from Chlamydomonas, ChR1, has attracted less attention because of its proton-selective permeability. By making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis, we developed a ChR variant, named ChEF, that exhibits significantly less inactivation during persistent light stimulation. ChEF undergoes only 33% inactivation, compared with 77% for ChR2. Point mutation of Ile(170) of ChEF to Val (yielding "ChIEF") accelerates the rate of channel closure while retaining reduced inactivation, leading to more consistent responses when stimulated above 25 Hz in both HEK293 cells and cultured hippocampal neurons. In addition, these variants have altered spectral responses, light sensitivity, and channel selectivity. ChEF and ChIEF allow more precise temporal control of depolarization, and can induce action potential trains that more closely resemble natural spiking patterns.
Lin
John Y
JY
Department of Pharmacology, University of California, San Diego, California, USA. j8lin@ucsd.edu
Lin
Michael Z
MZ
Steinbach
Paul
P
Tsien
Roger Y
RY
D020418:Algal Proteins / Q000737:chemistry* / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D016825:Chlamydomonas reinhardtii; D004558:Electric Stimulation; D006624:Hippocampus / Q000502:physiology; D006801:Humans; D016018:Least-Squares Analysis; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D009474:Neurons / Q000502:physiology; D018408:Patch-Clamp Techniques; D010775:Photic Stimulation; D015202:Protein Engineering; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D020418:Algal Proteins; D011993:Recombinant Fusion Proteins; D012243:Rhodopsin