Channelrhodopsin-1 Phosphorylation Changes with Phototactic Behavior and Responds to Physiological Stimuli in Chlamydomonas

The unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) exhibits oriented movement responses (phototaxis) to light over more than three log units of intensity. Phototaxis thus depends on the cell's ability to adjust the sensitivity of its photoreceptors to ambient light conditions. In Chlamydomonas, the photoreceptors for phototaxis are the channelrhodopsins (ChR)1 and ChR2; these light-gated cation channels are located in the plasma membrane. Although ChRs are widely used in optogenetic studies, little is known about ChR signaling in algae. We characterized the in vivo phosphorylation of ChR1. Its reversible phosphorylation occurred within seconds as a graded response to changes in the light intensity and ionic composition of the medium and depended on an elevated cytosolic Ca2+ concentration. Changes in the phototactic sign were accompanied by alterations in the phosphorylation status of ChR1. Furthermore, compared with the wild type, a permanently negative phototactic mutant required higher light intensities to evoke ChR1 phosphorylation. C-terminal truncation of ChR1 disturbed its reversible phosphorylation, whereas it was normal in ChR2-knockout and eyespot-assembly mutants. The identification of phosphosites in regions important for ChR1 function points to their potential regulatory role(s). We propose that multiple ChR1 phosphorylation, regulated via a Ca2+-based feedback loop, is an important component in the adaptation of phototactic sensitivity in Chlamydomonas.