Characterization of a rhodopsin-phosphodiesterase from Choanoeca flexa to be combined with rhodopsin-cyclases for bidirectional optogenetic cGMP control

Rhodopsin phosphodiesterases (RhPDEs) were first discovered in the choanoflagellate Salpingoeca rosetta, but their physiological role remained unknown. Their light-dependent modulation was found to be low, limiting optogenetic application. However, recent in vivo studies in the choanoflagellate Choanoeca flexa revealed a strong linkage of RhPDE to the actomyosin-mediated contraction and colony sheet inversion and identified downstream cGMP effectors. Through screening various RhPDE variants from C. flexa, we identified four photomodulated PDEs of which C. flexa RhPDE1 (CfRhPDE1) revealed the highest cGMP affinity and the most pronounced light regulation with K_m values of 1.9 and 4.4 μM in light and darkness. By coexpressing CfRhPDE1 with the rhodopsin-guanylyl-cyclase from the fungus Catenaria anguillulae and a cyclic nucleotide-gated ion channel from olfactory neurons in ND7/23 cells, we demonstrate bidirectional dual-color modulation of cGMP levels and ion channel conductance. Together with spectroscopic characterization, our fast functional recordings suggest that the M-state of the photocycle initiates functional changes in the phosphodiesterase domain via rapid rhodopsin-PDE coupling. With efficient expression and 3.5 s lifetime of the active state, this protein provides high photosensitivity to the host cells. This demonstrates that RhPDEs can regulate cGMP signaling in mammalian cells on a subsecond timescale, closing a present gap in optogenetics and assisting researchers in setting up multicomponent optogenetic systems for bidirectional control of cyclic nucleotides.