Optogenetics: a bright future for voltage gated ion 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 rectifi er hKir2.3 channel, was over-transfected with a ChR2 carrying a single amino acids mutation. This latter results 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 infl ux through Cav1.3 with the use of a calcium-sensitive fl uorescent dye (Fluo8) was tested in the Hamamatsu FDSSµcell. A well-known Cav1.3 blocker, Isradipine, tested in the resting and the partial inactivated Cav1.3 states, was used to confi rm the pharmacological profi le. Data obtained for the ChR2/hCav1.3 cell line by light stimulation have been also compared to the extracellular potassium stimulus and to patch-clamp to cross-check their reliability. Key-questions of the study Is the blue light produced by the FDSSµcell LED intense enough to activate ChR2? ➔ Can the light protocol be runned with the FDSSµcell? Is the activation of ChR2 suffi cient to induce cell membrane depola-rization? ➔ Can the ChR2 be used to depolarize cells avoiding the irreversible depolarization protocols using KCl injection? Is the resulting membrane depolarization suffi cient to activate the transfected voltage-gated channel Cav1.3? Can the cation fl ux through ChR2 disturbe the detction of Ca 2+ fl ux through the transfected target Cav1.3? Dihydropyridine Phenylalkylamine Benzothiazepin Membrane repolarization Time course of membrane repolarization after a fi rst blue light pulse (MPdye)