Search for: All records

Abstract Although molecular tools for controlling neuronal activity by light have vastly expanded, there are still unmet needs which require development and refinement. For example, light delivery into the brain is still a major practical challenge that hinders potential translation of optogenetics in human patients. In addition, it would be advantageous to manipulate neuronal activity acutely and precisely as well as chronically and non‐invasively, using the same genetic construct in animal models. We have previously addressed these challenges by employing bioluminescence and have created a new line of opto‐chemogenetic probes termed luminopsins by fusing light‐sensing opsins with light‐emitting luciferases. In this report, we incorporatedChlamydomonaschannelrhodopsin 2 with step‐function mutations as the opsin moiety in the new luminopsin fusion protein termed step‐function luminopsin (SFLMO). Bioluminescence‐induced photocurrent lasted longer than the bioluminescence signal due to very slow deactivation of the mutated channel. In addition, bioluminescence was able to activate most of the channels on the cell surface due to the extremely high light sensitivity of the channel. This efficient channel activation was partly mediated by radiationless bioluminescence resonance energy transfer due to the proximity of luciferase and opsin. To test the utility of SFLMOs in vivo, we transduced the substantia nigra unilaterally via a viral vector in male rats. Injection of the luciferase substrate as well as conventional photostimulation via fiber optics elicited circling behaviors. Thus, SFLMOs expand the current approaches for manipulation of neuronal activity in the brain and add more versatility and practicality to optogenetics in freely behaving animals. 

Abstract Multi‐scale calcium (Ca²⁺) dynamics, exhibiting wide‐ranging temporal kinetics, constitutes a ubiquitous mode of signal transduction. We report a novel endoplasmic‐reticulum (ER)‐targeted Ca²⁺indicator, R‐CatchER, which showed superior kinetics in vitro (k_off≥2×10³ s⁻¹,k_on≥7×10⁶ M⁻¹ s⁻¹) and in multiple cell types. R‐CatchER captured spatiotemporal ER Ca²⁺dynamics in neurons and hotspots at dendritic branchpoints, enabled the first report of ER Ca²⁺oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca²⁺‐based functional cooperativity of CaSR. We elucidate the mechanism of R‐CatchER and propose a principle to rationally design genetically encoded Ca²⁺indicators with a single Ca²⁺‐binding site and fast kinetics by tuning rapid fluorescent‐protein dynamics and the electrostatic potential around the chromophore. The design principle is supported by the development of G‐CatchER2, an upgrade of our previous (G‐)CatchER with improved dynamic range. Our work may facilitate protein design, visualizing Ca²⁺dynamics, and drug discovery.