Improved trafficking and expression of luminopsins for more efficient optical and pharmacological control of neuronal activity.

Luminopsins (LMOs) are chimeric proteins consisting of a luciferase fused to an opsin that provide control of neuronal activity, allowing for less cumbersome and less invasive optogenetic manipulation. It was previously shown that both an external light source and the luciferase substrate, coelenterazine (CTZ), could modulate activity of LMO-expressing neurons, although the magnitudes of the photoresponses remained subpar. In this study, we created an enhanced iteration of the excitatory luminopsin LMO3, termed eLMO3, that has improved membrane targeting due to the insertion of a Golgi trafficking signal sequence. In cortical neurons in culture, the expression of eLMO3 resulted in significant reductions in the formation of intracellular aggregates, as well as in a significant increase in total photocurrents. Furthermore, we corroborated the findings with injections of adeno-associated viral vectors into the deep layers of the somatosensory cortex (the barrel cortex) of male mice. We observed greatly reduced numbers of intracellular puncta in eLMO3-expressing cortical neurons compared to those expressing the original LMO3. Finally, we quantified CTZ-driven behavior, namely whisker-touching behavior, in male mice with LMO3 expression in the barrel cortex. After CTZ administration, mice with eLMO3 displayed significantly longer whisker responses than mice with LMO3. In summary, we have engineered the superior LMO by resolving membrane trafficking defects, and we demonstrated improved membrane targeting, greater photocurrents, and greater functional responses to stimulate with CTZ.