Homology Cloning, Heterologous Expression and Characterization of A New Channelrhodopsin

<p>Channelrhodopsins are phototaxis receptors in the plasma membranes of motile unicellular algae. They function as light-gated cation channels and this channel activity has been exploited to trigger action potentials in neurons with light to control neural circuits (“optogenetics"). Four channelrhodopsins were identified in two algal species, <em>Chlamydomonas reinhardtii</em> and <em>Volvox carteri</em>, with known genome sequences; each species contains 2 channelrhodopsins, one absorbing at longer wavelengths and one at shorter wavelengths, named <em>Cr</em>ChR1 and <em>Cr</em>ChR2, respectively. Our goals are to expand knowledge of channelrhodopsin mechanisms and also to identify new channelrhodopsins from various algal species with improved properties for optogenetic use. For these aims we are targeting algae from extreme environments to establish the natural diversity of their properties. We cloned a new channelrhodopsin from the psychrophilic (cold-loving) alga, <em>Chlamydomonas augustae</em>, with degenerate primers based on the 4 known homologs. The new protein is 48% and 52% identical to <em>Cr</em>ChR1 and <em>Cr</em>ChR2, respectively. We expressed the channelrhodopsin in HEK293 cells and measured light-induced currents to assess their kinetics and action spectrum. Based on the primary structure, kinetics of light-induced photocurrents in HEK293 cells, and action spectrum maximum of 520 nm near that of the two previously found <em>Cr</em>ChR1, we named the new channelrhodopsin <em>Ca</em>ChR1. The properties of robust channel activity at physiological pH, fast on-and-off kinetics, and greatly red-shifted action spectrum maximum from that of <em>Cr</em>ChR2, make <em>Ca</em>ChR1 advantageous as an optogenetic tool. To know this new channelrhodopsin better, we expressed His-tagged <em>Ca</em>ChR1 in <em>Pichia pastoris</em> and the yield is about 6 mg/L. The purified His-tagged <em>Ca</em>ChR1 exhibited an absorption spectrum identical to the action spectrum of <em>Ca</em>ChR1-generated photocurrents. The future work will be measurement of the photocycles of <em>Ca</em>ChR1 by flash photolysis, crystallization of <em>Ca</em>ChR1 for the structure and mutagenesis of <em>Ca</em>ChR1 to find the critical amino acids accounting for red-shifted spectra, slow inactivation and rapid on-and-off kinetics.</p> <p>Seven new channelrhodopsins including <em>Ca</em>ChR1 from different algal species have been cloned in our lab at this time, bringing the total known to 13. The work of cloning of these new channelrhodopsins along with the expression of <em>Ca</em>ChR1 was published in Photochemistry and Photobiology in January 2012</p>

Name thesis:degree_name

Masters of Science (MS)

Level thesis:degree_level

Thesis (MS)

Year dc:date.available

2012

Author dc:creator

• Hou, Sing-Yi

Contributors dc:contributor

• John Spudich
• Vasanthi Jayaraman
• Lei Zheng

• channelrhodopsin
• optogenetics
• algae

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/326

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1361