Engineered Sensor Enables Probing of PAC1 Receptor Activation Across Species

Core Concepts

A novel genetically-encoded sensor, PAClight1P78A, based on the human PAC1 receptor enables highly sensitive and selective optical detection of PAC1 receptor activation by the endogenous ligand PACAP across different species.

Abstract

The content describes the development and characterization of a novel genetically-encoded sensor, PAClight1P78A, based on the human PAC1 receptor. The sensor was engineered to have a large dynamic range (up to 1100% ΔF/F0), high ligand selectivity, and rapid activation kinetics.
Key highlights:

PAClight1P78A can be used to optically detect PAC1 receptor activation by the endogenous ligand PACAP in various cell types, including HEK293T cells and primary rat neurons, with high sensitivity.
The sensor is selective for PACAP and does not respond to other neuropeptides like VIP, CRF, or enkephalin.
PAClight1P78A can be used to study ligand binding and diffusion in acute mouse brain slices and in vivo in behaving mice.
The sensor also functions well in zebrafish larvae, enabling two-photon imaging of PACAP-induced fluorescence changes in the olfactory region.
The authors also developed a non-responsive control sensor, PAClight1P78A-ctrl, by mutating key residues involved in PACAP binding.
Stable cell lines expressing PAClight1P78A show improved homogeneity and reproducibility compared to transient transfections.
Overall, the PAClight1P78A sensor provides a powerful tool for studying PAC1 receptor signaling in a wide range of in vitro and in vivo applications across different model organisms.

Stats

PACAP1-38 induced a 1109% ΔF/F0 response in HEK293T cells expressing PAClight1P78A.
PAClight1P78A showed a 729% ΔF/F0 response to PACAP1-38 in rat primary neurons.
The EC50 of PAClight1P78A for PACAP1-38 was 29.91 nM in stable HEK293 cell lines.
PAClight1P78A showed no response to up to 50 μM VIP in stable HEK293 cell lines.
In acute mouse brain slices, PAClight1P78A showed a 17.5% ΔF/F0 response to 3000 nM PACAP1-38.
In vivo in behaving mice, microinfusion of 300 μM PACAP1-38 induced a 126% ΔF/F0 response in PAClight1P78A-expressing cortex.

Quotes

"PAClight1P78A displays an average fluorescent response of 1109% ΔF/F0 in HEK293T cells to bath application of 10μM PACAP1-38."
"PAClight1P78A clearly increased its fluorescence in response to 3 μM PACAP1-38 (+17.5% peak ΔF/F0), but not to VIP (-5.5% peak ΔF/F0), CRF (-4.3% peak ΔF/F0), or ENK (+1.4% peak ΔF/F0)."
"Microinfusion of PACAP1-38 (300 μM, 200 nl) led to peak fluorescence increases of 126% ΔF/F0 in PAClight1P78A-expressing mice."

Key Insights Distilled From

Probing PAC1 receptor activation across species with an engineered sensor

by Cola,R. B., ... at www.biorxiv.org 02-07-2024

https://www.biorxiv.org/content/10.1101/2024.02.06.579048v1

Deeper Inquiries

How could the PAClight1P78A sensor be used to study the spatiotemporal dynamics of PACAP signaling in the brain and other tissues?

The PAClight1P78A sensor can be utilized to investigate the spatiotemporal dynamics of PACAP signaling in various tissues, including the brain. By expressing the sensor in specific cell types or regions of interest, researchers can monitor the real-time activation of the PAC1 receptor in response to PACAP ligands. This allows for the visualization of where and when PACAP signaling occurs, providing insights into the distribution and timing of receptor activation in different physiological and pathological conditions. In the brain, for example, the sensor can be used to study the role of PACAP signaling in neuronal circuits, synaptic transmission, and neuroplasticity. By combining the sensor with imaging techniques such as two-photon microscopy, researchers can observe the activation of the PAC1 receptor at the cellular and subcellular levels, providing detailed information on the dynamics of PACAP signaling in neuronal networks.

What are some potential limitations or caveats of using genetically-encoded GPCR sensors like PAClight1P78A compared to other techniques for studying receptor activation?

While genetically-encoded GPCR sensors like PAClight1P78A offer numerous advantages for studying receptor activation, there are also some limitations and caveats to consider. One limitation is the potential for overexpression artifacts, where the high levels of sensor expression may lead to non-physiological responses or interfere with endogenous signaling pathways. Careful optimization of sensor expression levels and controls are necessary to mitigate this issue. Another limitation is the specificity of the sensor to the ligand of interest, as cross-reactivity with other molecules could lead to false-positive results. Validation of the sensor's selectivity and sensitivity is crucial to ensure accurate interpretation of the data. Additionally, the kinetics of the sensor response may not always reflect the kinetics of endogenous receptor activation, as the sensor's design and properties can influence the speed and magnitude of the fluorescent signal. Finally, the use of genetically-encoded sensors requires genetic manipulation of cells or organisms, which may introduce experimental variability and complexity compared to other techniques for studying receptor activation.

Given the widespread distribution of PACAP and its receptors, how might the PAClight1P78A sensor be leveraged to uncover novel physiological functions of the PACAP/PAC1R system across different organ systems?

The PAClight1P78A sensor can be leveraged to uncover novel physiological functions of the PACAP/PAC1R system across different organ systems by enabling the visualization and quantification of PACAP signaling in various tissues. In the central nervous system, the sensor can be used to study the role of PACAP in modulating neuronal activity, synaptic plasticity, and neurodevelopment. In peripheral tissues, such as the immune system, endocrine glands, and reproductive organs, the sensor can provide insights into the effects of PACAP signaling on immune responses, hormone secretion, and reproductive functions. By expressing the sensor in specific cell types or tissues of interest, researchers can investigate the spatiotemporal dynamics of PACAP signaling and its impact on physiological processes. This approach can lead to the discovery of novel functions of the PACAP/PAC1R system in different organ systems and provide valuable information for drug development and therapeutic interventions targeting this signaling pathway.