A Family of Photoswitchable Metabotropic Glutamate Receptors for High‐Efficiency Optical Interrogation of Specific Receptor Populations in vivo

Abstract

G protein‐coupled receptors (GPCRs) form a large family of membrane bound receptors that initiate intracellular responses which control cellular signaling cascades to contribute to a wide range of biological functions. The metabotropic glutamate receptors (mGluRs) consist of an eight‐member family of class C GPCRs that respond to the excitatory neurotransmitter glutamate and serve as promising pharmacological targets for a range of neurological and psychiatric disorders. mGluRs are localized in a variety of cell types throughout the central nervous system where they can modulate neuronal activity in both normal and disease‐related states. Despite the prevalence of these receptors, the underlying molecular, synaptic and circuit mechanisms of mGluR‐mediated neuromodulation are still poorly understood due to the limitations of classical pharmacological and genetic approaches. Here we report an improved chemical optogenetic toolkit that permits the reversible and repeatable activation of specific mGluRs with genetic targeting, subtype‐specificity and spatiotemporal precision. Using an updated family of azobenzene‐based photoswitchable glutamate compounds that covalently attach to protein tags, we have dramatically enhanced the efficiency and applicability of these tools. A branched SNAP‐targeting compound termed “doubleBGAG” enables near‐complete photoagonism of mGluR2 when compared to saturating glutamate. The improved efficiency of SNAP‐mGluR2 activation by doubleBGAG permits superior optical inhibition of cortical neurons. Strikingly, doubleBGAG also drastically improves the efficiency of photoagonism of SNAP‐mGluR3 from <10% to ~85% compared to saturating glutamate, allowing investigation of group II mGluR signaling with enhanced subtype specificity. In addition, development of a chimera‐based approach allows for photoactivation of mGluR5 signaling with sub‐cellular targeting. Branched photoswitches targeting CLIP or Halo‐tags along with spectral variants allow multiplexing optical control of multiple receptors. Toward the ultimate goal of targeted delivery of photoswitches to native mGluRs, doubleBGAG also enhances photoswitch efficiency when conjugated to GFP‐targeting nanobodies. Finally, we report the development and testing of fluorophore‐conjugated photoswitches, which allow dual photoactivation and detection of mGluR2 in rodent medial prefrontal cortex in acute slice preparations and in vivo. Together this optogenetic toolkit allows for the dissection of mGluR subtype‐specific contributions to physiological and behavioral changes in mouse models of psychiatric disorders.Support or Funding InformationNIH 2018 R35 GM: 1R35GM124731‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.