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Egypt. J. Exp. Biol. (Bot.). 2013; 9(1): 81-86


STUDY THE EXPRESSION OF CHANNELRHODOPSIN-2 AS A DIRECTLY LIGHT-GATED CATION-SELECTIVE MEMBRANE CHANNEL IN HEK 293 CELLS

Ahmed El-Hussein Mohamed Kamel.

Abstract
Channelrhodopsins (ChRs) originated from Chlamydomonas genus has been considered as directly light-gated ion channels. In microalgae, their primary function is to direct the organism towards or away from the light stimulus as well to optimize the light conditions needed for photosynthesis. ChR2 originated from Chlamydomonas reinhardtii involved in generation of photocurrents in this green alga has been found to be 10 times better than ChR1 for the expression in most host cells like Xenopus oocytes. Despite of this fact and ChR2 can be easily targeted genetically; many of selective promoters cannot achieve sufficient expression levels of ChR2 for photostimulation. In the present work, Chop2T159C mCherry plasmid has been expressed in HEK 293 cells in one set of experiments, where cotransfection of Chop2T159C plasmid and mCherry plasmid in another set of experiments. The results have shown that ChR2 is a directly light-switched cation-selective ion channel upon stimulating the cells with 475 nm blue light. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. Furthermore the degree of expression of Chop2T159C mCherry plasmid in HEK 293 cells was better than that of Chop2T159C alone based on the resulted current in response of the incident light despite the initial thought of being mCherry genes would compete with the Chop2T159C genes.

Key words: HEK cells, ChR2, Optogenetics, patchclamping, Fluorescence, ion channel



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