Conversion of a light-driven proton pump into a light-gated ion channel

Area of Science:

• Microbial rhodopsins
• Optogenetics
• Algal biochemistry

Background:

• Microbial rhodopsins are light-activated proteins with ion-pumping capabilities.
• Optogenetics utilizes these pumps for cell hyperpolarization and voltage sensing.
• The arctic alga Coccomyxa subellipsoidea possesses novel opsin genes.

Purpose of the Study:

• To identify and characterize a novel microbial rhodopsin from Coccomyxa subellipsoidea.
• To analyze the functional roles of specific amino acid residues in proton pumping activity.
• To investigate the potential of CsR as a proton channel under altered conditions.

Main Methods:

• Gene identification and cloning from Coccomyxa subellipsoidea.
• Expression and photocurrent measurement in Xenopus oocytes.
• Site-directed mutagenesis of key residues (R83, Y57).
• Molecular dynamics simulations.

Main Results:

• Identified CsR opsin gene producing large photocurrents in Xenopus oocytes.
• Modification of R83 or Y57 significantly reduced proton pumping efficiency.
• Mutated CsR exhibited proton channel activity with rectification under moderate electrochemical load.
• R83, Y57, and water molecules form a proton shuttle essential for light-driven pumping.

Conclusions:

• CsR is a functional microbial rhodopsin with significant proton pumping activity.
• Residues R83 and Y57 are critical for the proton shuttle mechanism.
• CsR can be engineered into a light-gated proton channel by specific mutations.