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Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor
摘要： Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 fluorescence probe at the end of trans-membrane helix 6 and reconstituted in phospholipid nanodiscs tethered to a microscope slide. Individual receptor molecules are then monitored over time by single-molecule total internal reflection fluorescence microscopy, revealing spontaneous transitions between inactive and active-like conformations. The assay provides information on the equilibrium distribution of inactive and active receptor conformations and the rate constants for conformational exchange. The experiments can be performed in the absence of ligands, revealing the spontaneous conformational transitions responsible for basal signaling activity, or in the presence of agonist or inverse agonist ligands, revealing how the ligands alter the dynamics of the receptor to either stimulate or repress signaling activity. The resulting mechanistic information is useful for the design of improved GPCR-targeting drugs. The single-molecule assay is described in the context of the β2 adrenergic receptor, but can be extended to a variety of GPCRs.
关键词： Phospholipid nanodiscs,G-protein coupled receptors,Conformational dynamics,β2 adrenergic receptor,Single-molecule fluorescence
更新于2025-11-21 11:24:58
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Pharmacological interference of adrenergic receptor signaling preserve photoreceptors after retinal detachment through inhibition of oxidative stress and inflammation
摘要： Background and Purpose: The current strategy is not sufficient to halt progression of photoreceptor death and subsequent visual impairment related to retinal detachment (RD) which is observed in various retinal disorders. This study investigated the neuroprotective effects of adrenergic receptor (AR)-targeting pharmaceuticals, the α1-AR antagonist doxazosin or the α2-AR agonist guanabenz, against photoreceptor cell death in RD. Experimental Approach: Brown-Norway rats were created with experimental RD by subretinal injection of sodium hyaluronate. Oxidative stress biomarkers and cytokine production were quantified with ELISA. Protein expression levels and immunofluorescent labelling were determined in rats with RD and controls for mechanistic elucidation. The effects of systemic administration of doxazosin, guanabenz on photoreceptor apoptosis, retinal histology and electroretinography were evaluated in rats with RD compared to vehicle controls. Key Results: Photoreceptors were the major source of RD-induced ROS overproduction in the rat retina through the regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Systemic administration of doxazosin or guanabenz significantly alleviated the RD-induced production of ROS and proinflammatory cytokines, including IL-1β and MCP-1, and suppressed retinal gliosis, resulting in attenuation of photoreceptor death and preservation of retinal structures and functions in RD. Conclusions and Implications: Our findings point to adrenergic receptors as novel therapeutic targets for photoreceptor protection and suggest that both doxazosin and guanabenz, two FDA-approved drugs, could be further explored to treat retinal diseases.
关键词： G protein-coupled receptors,adrenergic receptors,retinal detachment,photoreceptor neuroprotection,NADPH oxidase
更新于2025-09-23 15:23:52
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New light on neurotransmitter-gated receptors: Optical approaches for controlling physiological function
摘要： Neurotransmitter-gated receptors contribute to synaptic transmission and modulation in many ways. Considering glutamate receptors as an example, it becomes clear that these receptor families are highly diverse and that it is experimentally challenging to disentangle the different functional contributions of closely related receptor subtypes. Pharmacological and genetic methods are now complemented by optogenetic approaches, which allow for controlling receptor signaling with light. Using glutamate receptors as an example, I summarize how tethered photoswitchable ligands can be used to control individual receptor subtypes with high spatial and temporal precision, and in specific cells. These, and similarly exciting approaches, offer new possibilities for probing the function of individual receptors in the nervous system.
关键词： optogenetics,ligand-gated ion channels,optopharmacology,G protein-coupled receptors,glutamate receptors
更新于2025-09-23 15:23:52