Design for Fast Optogenetic Screen In Mammalian Cells For Next Gen Ca 2+ Sensors

Blue light was used to activate an adenylyl cyclase enzyme from the soil bacterium (bPAC) that increases intracellular cAMP () as detected by the red sensor R-CaDDis. In turn the cAMP opened a cAMP gated channel (olfactory cyclic nucleotide gated channel, CNG, or the hyperpolarization-activated cyclic nucleotide gated channel, HCN2). This produced slow Ca transients as detected by R-GECO1.2. To speed these transients up, we added the inwardly rectifying potassium channel 2.1, Kir2.1, and the bacterial voltage gated sodium channel (NAVROSD). This is a modular system in which the kinds of channels, and their relative amounts, can be tuned to produce the cellular behavior crucial for screening a particular biosensor in an automated format.