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Neurotransmission is the primary mechanism by which neurons communicate with each other to achieve fast and dynamic operation of the nervous system. Elucidating the physiological function of neurotransmission requires technologies that can manipulate signal release or detection with high spatial, temporal, and targeting precision. Optogenetics and photopharmacology offer powerful approaches to achieve this purpose. However, existing tools are often limited by their low efficacy and/or poor target specificity. This study presents two novel tools to advance the repertoire of neurotransmission control: a high-performance kalium channelrhodopsin for long-range optogenetic neuropathway inhibition, and a photoswitchable agonist for subtype-selective activation of GABAA receptors.
Dissecting neurocircuits underlying a physiological process requires reliable and targeted inhibition of specific neuropathways. HcKCR1, a light-gated potassium channel (i.e., kalium channelrhodopsin) with large conductance and superb photosensitivity, is a promising tool for this approach. However, its application for neuropathway inhibition is constrained by suboptimal gating kinetics and inefficient axonal trafficking. To address the functional issues, we conducted structure-guided mutagenesis to engineer a performance-improved HcKCR1 (piKCR) that offers sustained and reliable inhibition. piKCR activation strongly reduces neuronal gain, enabling effective suppression of action potential firing using very low intensities of green or red light. To achieve projection silencing, we developed an axon-targeted variant (piKCR.AT) by fusing the intracellular domain of neurexin to the C-terminus of piKCR. Confocal imaging and electrophysiology confirmed robust cross-hemispheric trafficking and long-range presynaptic inhibition by piKCR.AT in the mouse hippocampus. Furthermore, piKCR.AT was expressed in the muscle afferents, achieving non-invasive, targeted optical antinociception in a chronic pain mouse model.
Inhibitory neurotransmission is largely mediated by GABAARs, a family of heteropentameric ligand-gated chloride-permeable channels. Unravelling the function and distribution of each GABAAR subtype is essential for a holistic understanding of GABAergic inhibition in health and disease. Photopharmacology, a technique that utilizes light-switchable drugs, can manipulate endogenous proteins with high spatiotemporal precision. Within this receptor family, the delta subunit-containing GABAARs (δ-GABAARs) represent high-value therapeutic targets due to their distinct pharmacology and critical role in regulating neuronal excitability. To achieve subtype-selective photopharmacological control of δ-GABAARs, we developed Az-IGU, a photoswitchable agonist targeting α4/6β3δ GABAARs. Under UV light, cis Az-IGU triggered robust photoagonism, which was reversibly deactivated by green light. Mechanistically, cis Az-IGU targets the orthosteric GABA binding site, with its efficacy correlating directly with the intrinsic spontaneous activity of δ-GABAARs. Furthermore, Az-IGU achieved robust photocontrol of GABAergic activity and action potential firing, offering a powerful strategy to precisely manipulate neuronal excitability.
Together, these findings establish novel optogenetic and photopharmacological technologies for precise manipulation of neurotransmission. By developing reliable tools to interrogate neuropathways and specific neurotransmitter receptor subtypes, this work provides new avenues to explore uncharacterized mechanisms of neurotransmission. Ultimately, the information obtained through these technologies offer insights for developing novel therapeutic approaches for health and disease.