Optogenetics: a bright future for voltage gated ion channels

Channels Channelrhodopsin-2 (ChR2) is a light-activated microbial cation channel which can be used to depolarize neurons through the incidence of blue light (470 nm). The possibility to optically control the plasma membrane voltage opens new and interesting perspectives for the characterization of voltage-gated ion channels and the search for modulators. Proof of principle studies have been performed to verify the applicability of this tool for the development of cell based assays in High Throughput Screening (HTS) platforms as microplate readers.An HEK-293 cell line, which stably co-expresses the human voltage-gated calcium channel hCav1.3 and the inward rectifier hKir2.3 channel, was over-transfected with a ChR2 carrying a single amino acids mutation. This latter result in a prolonged lifetime of the conducting state of ChR2 and in a reduced light power requirement for its activation. A protocol of light stimulation of ChR2 and record of calcium ion influx through Cav1.3 with the use of a calcium-sensitive fluorescent dye (Fluo8) was tested in the Hamamatsu FDSS?cell. A well-known Cav1.3 blocker, Isradipine, tested in the resting and the partial inactivated Cav1.3 states, was used to confirm the pharmacological profile.Data obtained for the ChR2/hCav1.3 cell line by light stimulation have been also compared to the extracellular potassium stimulus and to patchclamp to cross-check their reliability