Cytoplasmic extensions of the channelrhodopsins 1 and 2 interacts in Chlamydomonas reinhardtii

Abstract

Channelrhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2) are directly light-gated ion channels that are being extensively utilized in the field of optogenetics. These proteins are present in the eyespot of a phototactic alga called Chlamydomonas reinhardtii and mediate its motility behavior (phototaxis and photophobic responses). Both ChR1 and ChR2 are two seven transmembrane proteins with N-terminal rhodopsin domain and C-terminal cytoplasmic extensions of unidentified function. Earlier studies have suggested the interaction between ChR1 and ChR2. However, the evidences of physical interaction between ChRs are still lacking. Here, we have dissected the possibility of the direct interaction between the cytoplasmic C-terminus extensions of the ChR1 and ChR2. The immunoprecipitation, co-precipitation, colocalization and the proximity ligation assay showed the interaction between ChR1 and ChR2. In addition, the in vitro GST pull down assay showed that ChR1 and ChR2 interact directly with their C-terminal extensions. Our study leads to the model where C-terminal cytoplasmic extensions of ChR1 and ChR2 could directly interact with each other in Chlamydomonas. In vivo, this interaction could serve to directly regulate the activity or control binding to downstream effectors (Figure 1).