Abstract
Neurons integrate inputs over different time and space scales. Fast excitatory synapses at boutons (ms and μm), and slow modulation over entire dendritic arbors (seconds and mm) are all ultimately combined to produce behavior. Understanding the timing of signaling events mediated by G-protein-coupled receptors is necessary to elucidate the mechanism of action of therapeutics targeting the nervous system. Measuring signaling kinetics in live cells has been transformed by the adoption of fluorescent biosensors and dyes that convert biological signals into optical signals that are conveniently recorded by microscopic imaging or by fluorescence plate readers. Quantifying the timing of signaling has now become routine with the application of equations in familiar curve fitting software to estimate the rates of signaling from the waveform. Here we describe examples of the application of these methods, including (1) Kinetic analysis of opioid signaling dynamics and partial agonism measured using cAMP and arrestin biosensors; (2) Quantifying the signaling activity of illicit synthetic cannabinoid receptor agonists measured using a fluorescent membrane potential dye; (3) Demonstration of multiplicity of arrestin functions from analysis of biosensor waveforms and quantification of the rates of these processes. These examples show how temporal analysis provides additional dimensions to enhance the understanding of GPCR signaling and therapeutic mechanisms in the nervous system.
Highlights
The timing of molecular events is central to the orchestration of cellular activity that underlies the functions of the nervous system
The SteadyState value from the curve fit was corrected for the injection artifact. This was done by subtracting the mean Step value of the vehicle (4.4 %) from the fitted SteadyState value
The amount of sensor needs to be titrated to ensure sufficient signal but without signal saturation, as described previously (Hoare and Hughes, 2021)
Summary
The timing of molecular events is central to the orchestration of cellular activity that underlies the functions of the nervous system. Quantifying the extent to which MOR agonists activate the receptor is essential for understanding the liabilities of current mediations and in the optimization of safer new therapeutics targeting this receptor (Thompson et al, 2016; Schmid et al, 2017; Gillis et al, 2020c; Hill and Canals, 2021; Pineyro and Nagi, 2021) This quantification is central to the two approaches currently being advocated, which are: (1) Partial agonism, the development of ligands that only partially activate the receptor, sufficient to achieve analgesia but low enough to minimize side effects (Gillis et al, 2020a). This quantification is central to the two approaches currently being advocated, which are: (1) Partial agonism, the development of ligands that only partially activate the receptor, sufficient to achieve analgesia but low enough to minimize side effects (Gillis et al, 2020a). (2) Biased agonism, the development of ligands that are biased for activating G-protein vs. recruiting arrestin (Schmid et al, 2017)
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