(344) A Photoswitchable GPCR-Based Opsin for Synaptic Inhibition

Abstract

The development of diverse molecular tools to manipulate the activity of defined cell types has greatly accelerated our understanding of the neural circuits underlying complex behaviors. The most commonly used opsins are light-gated ion channels or pumps to control activity on millisecond timescales. While optogenetic activation can be precisely tuned across a range of firing frequencies, neuronal inhibition has proven to be more problematic. The most commonly used inhibitory opsins are chloride or proton pumps requiring constant illumination, and biophysical constraints due to different subcellular ion gradients can trigger unwanted excitation. The development of opto-GPCR chimeras grants spatial and temporal control of distinct G-protein coupled intracellular signaling cascades, and in contrast to binary on/off manipulations, modulating endogenous activity patterns may more accurately reflect circuit dynamics. However, these rhodopsin-based approaches possess several limitations, including high photosensitivity and irreversible activation. Here we have identified a novel photoswitchable GPCR-based opsin that engages endogenous inhibitory signaling cascades to silence synaptic transmission. This UV/blue light-sensitive opsin couples to G-proteins to reversibly inhibit neuronal voltage-gated calcium channel function, with similar efficacy to endogenous GABABRs. Long-term optical inhibition can be achieved with pulsed light, does not desensitize, and most importantly, inhibition is rapidly reversed by illumination with amber light. This opsin can also be stimulated by 2-photon excitation, permitting subcellular activation of G-protein subunits, which may allow for precise patterns of inhibition deep within tissues. Lastly, we found that optical stimulation in vivo inhibits dopamine neurons to reduce motivated behaviors. We are now adapting this GPCR-based opsin to serve as a novel scaffold for next-generation opto-XR chimeras with photoswitchable control of GPCR signaling cascades to better understand the roles of these important therapeutic targets in different pain circuits.