title: Degradation of channelopsin-2 in the absence of retinal and degradation resistance in certain mutants.
abstract: Channelrhodopsin-2 is a light-gated cation channel from the green alga Chlamydomonas reinhardtii. It is functional in animal cells and therefore widely used for light-activated depolarization, especially in neurons. To achieve a fully functional protein, the chromophore all-trans-retinal is needed. It has not been investigated whether or not the apoprotein is stable without its cofactor until now. Here we show that channelopsin-2 (Chop2, protein without bound retinal) is much more prone to degradation than channelrhodopsin-2 (protein with retinal). Constructs of Chop2 fused to yellow fluorescent protein (Chop2::YFP) in the absence and presence of retinal confirm this observation by exhibiting strongly differing fluorescence. We present mutants of Chop2 with highly increased stability in the absence of retinal. Substitution of threonine 159 with aromatic amino acids causes enhanced resistance to degradation in the absence of retinal, which is confirmed by fluorescence intensity, the increase in photocurrents on the addition of retinal to previously expressed protein, and Western blot analysis. Exchanging threonine 159 with cysteine, however, increases photocurrents due to better binding of retinal, without obvious stabilization against degradation of the retinal-free opsin. We also show that the light-activated hyperpolarizing chloride pump halorhodopsin from Natronomonas pharaonis (NpHR) is not prone to retinal-dependent degradation.
authors.affiliation: University of Würzburg, Institute for Molecular Plant Physiology and Biophysics, Julius-von-Sachs Platz 2, D-97082 Würzburg, Germany.
authors.forename: Sybille
authors.initials: S
authors.lastname: Ullrich
authors.forename: Ronnie
authors.initials: R
authors.lastname: Gueta
authors.forename: Georg
authors.initials: G
authors.lastname: Nagel
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004351:Drug Resistance; D025602:Halorhodopsins / Q000378:metabolism / Q000528:radiation effects; D008027:Light; D009154:Mutation; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D012172:Retinaldehyde / Q000494:pharmacology; D014982:Xenopus laevis
chemical_list: D002352:Carrier Proteins; D025602:Halorhodopsins; C523009:channelopsin-2, Chlamydomonas reinhardtii; D012172:Retinaldehyde