Assessment of Cell Adhesion After Purinoceptor Activation.
Martínez-García JJ1, Pelegrín P2.
Author information
1
Biomedical Research Institute of Murcia (IMIB), Clinical University Hospital Virgen de la Arrixaca, Murcia, Spain.
2
Biomedical Research Institute of Murcia (IMIB), Clinical University Hospital Virgen de la Arrixaca, Murcia, Spain. pablo.pelegrin@imib.es.
Abstract
Cell adhesion is a characteristic feature of phagocytic myeloid cells, which is important for several inflammatory processes, such as migration, invasion, and proliferation. Purinergic signaling in macrophages plays an important role in cell adhesion of this cell type to different extracellular substrates. This protocol describes the use of two different detection methods to quantify cell adhesion upon P2X7 receptor activation by extracellular ATP.

KEYWORDS:
Cell adhesion; Extracellular ATP; Extracellular matrix; MTT; Macrophage; P2X7 receptor; Yo-Pro-1

PMID: 31646503 DOI: 10.1007/978-1-4939-9717-6_27
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2.
Methods Mol Biol. 2020;2041:345-349. doi: 10.1007/978-1-4939-9717-6_26.
Measuring Leukocyte Migration to Nucleotides.
Moon TJ1,2, Elliott MR3,4.
Author information
1
David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, NY, USA.
2
Department of Microbiology and Immunology, University of Rochester, Rochester, NY, USA.
3
David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, NY, USA. Michael_Elliott@urmc.rochester.edu.
4
Department of Microbiology and Immunology, University of Rochester, Rochester, NY, USA. Michael_Elliott@urmc.rochester.edu.
Abstract
Extracellular nucleotides are potent damage-associated molecular patterns that shape the immune response to cell stress and tissue damage. These nucleotides are sensed by purinergic receptors and mediate a wide range of cellular effects. Among the best characterized of these effects is cellular migration. While the motility responses of leukocytes to nucleotides can be achieved by microscopic live-cell imaging approaches, such systems are time-consuming and require costly equipment and analysis tools not readily available to all researchers. Transwell migration chambers are a widely used alternative to microscopy due to their relatively low cost and moderate through-put capacity. However, extracellular nucleotides are labile and rapidly degraded in serum-containing cell cultures due to the presence of phosphohydrolases. Thus, evaluating leukocyte migration to nucleotides presents a number of challenges not seen with more stable classes of chemoattractants like proteins and lipids. Here we describe a method to measure leukocyte migration to nucleotides that is cost-effective, rapid and produces robust and reproducible migration of leukocytes using transwell migration chambers.

KEYWORDS:
ATP; Boyden chamber; Cell migration; Chemotaxis; Leukocytes; Nucleotides; Purinergic signaling; Transwell; UTP

PMID: 31646502 DOI: 10.1007/978-1-4939-9717-6_26
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3.
Methods Mol Biol. 2020;2041:335-343. doi: 10.1007/978-1-4939-9717-6_25.
Detection of Inflammasome Activation by P2X7 Purinoceptor Activation by Determining ASC Oligomerization.
Martínez-Banaclocha H1, Pelegrín P2.
Author information
1
Biomedical Research Institute of Murcia (IMIB), Clinical University Hospital Virgen de la Arrixaca, Murcia, Spain. heliosmar@live.com.
2
Biomedical Research Institute of Murcia (IMIB), Clinical University Hospital Virgen de la Arrixaca, Murcia, Spain.
Abstract
The purinoceptor P2X7 is highly expressed in cells of the innate immune system, including monocytes and macrophages. Its activation is a potent signal to activate the NLRP3 inflammasome and induce the release of proinflammatory cytokines of the IL-1 family. In this chapter, we present a method to monitor NLRP3 inflammasome activation in human monocytes upon P2X7 receptor stimulation by detecting intracellular oligomers of ASC by flow cytometry. This method could be used to evaluate the degree of inflammasome activation in blood samples from patients suffering different chronic inflammatory diseases.

KEYWORDS:
ASC; ATP; Flow cytometry; Inflammasome; Monocyte; NLRP3; P2X7

PMID: 31646501 DOI: 10.1007/978-1-4939-9717-6_25
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4.
Methods Mol Biol. 2020;2041:323-334. doi: 10.1007/978-1-4939-9717-6_24.
Assays to Measure Purinoceptor Pore Dilation.
Gu BJ1,2, Avula P1, Wiley JS3.
Author information
1
The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC, Australia.
2
National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University, Shanghai, China.
3
The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC, Australia. jwiley@unimelb.edu.au.
Abstract
The P2X7 receptor is a classic purinoceptor/ion channel. After activated by ATP, it opens a cation selective channel, which dilates to a large pore over tens of seconds, allowing the entry of big molecules. This unique feature is often used to evaluate this receptor's function with DNA-binding dyes (MW 300-400 Da), such as ethidium bromide and Yo-Pro-1. Here we describe two-color flow cytometry based protocols for measuring P2X7 pore dilation. One is ATP-induced ethidium uptake by real-time multicolor flow cytometry for standardized and accurate quantitation, and the other is a quick whole blood assay which is particularly useful for ex vivo study.

KEYWORDS:
Area under curve; Ethidium; Excel formula; FlowJo; Pore dilation; Real-time flow cytometry; WinMDI

PMID: 31646500 DOI: 10.1007/978-1-4939-9717-6_24
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5.
Methods Mol Biol. 2020;2041:311-321. doi: 10.1007/978-1-4939-9717-6_23.
Intracellular Calcium Recording After Purinoceptor Activation Using a Video-Microscopy Equipment.
Miras-Portugal MT1,2,3, Ortega F1,2,3, Gualix J1,2,3, Perez-Sen R1,2,3, Delicado EG1,2,3, Gomez-Villafuertes R4,5,6.
Author information
1
Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain.
2
Instituto Universitario de Investigación en Neuroquímica (IUIN), Madrid, Spain.
3
Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain.
4
Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain. marosa@ucm.es.
5
Instituto Universitario de Investigación en Neuroquímica (IUIN), Madrid, Spain. marosa@ucm.es.
6
Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain. marosa@ucm.es.
Abstract
Calcium is one of the most important intracellular messengers, triggering a wide range of cellular responses. Changes in intracellular free calcium concentration can be measured using calcium sensitive fluorescent dyes, which are either EGTA- or BAPTA-based organic molecules that change their spectral properties in response to Ca2+ binding. One of the most common calcium indicators is the ratiometric dye Fura-2. The main advantage of using ratiometric dyes is that the ratio signal is independent of the illumination intensity, dye concentration, photobleaching, and focus changes among others, allowing for the concentration of intracellular calcium to be determined independently of these artifacts. In this protocol, we describe the use of Fura-2 to measure intracellular calcium elevations in single cultured cells after purinoceptor activation using a video-microscopy equipment. This method, usually known as calcium imaging, allows for real-time quantification of intracellular calcium dynamics and can be adapted to measure agonist mediated intracellular calcium responses due to the activation of different purinergic receptors in several cellular models using the appropriate growth conditions.

KEYWORDS:
Calcium imaging; Calcium responses; Fura-2; Intracellular free calcium concentration; Purinergic receptor; Ratiometric calcium dye; Video microscopy

PMID: 31646499 DOI: 10.1007/978-1-4939-9717-6_23
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6.
Methods Mol Biol. 2020;2041:301-309. doi: 10.1007/978-1-4939-9717-6_22.
Controlling Engineered P2X Receptors with Light.
Atkinson BN1,2,3, Chudasama V1, Browne LE4,5.
Author information
1
Department of Chemistry, University College London, London, UK.
2
Wolfson Institute for Biomedical Research, University College London, London, UK.
3
Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK.
4
Wolfson Institute for Biomedical Research, University College London, London, UK. liam.browne@ucl.ac.uk.
5
Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK. liam.browne@ucl.ac.uk.
Abstract
This chapter details methods to express and modify ATP-gated P2X receptor channels so that they can be controlled using light. Following expression in cells, a photoswitchable tool compound can be used to covalently modify mutant P2X receptors, as previously demonstrated for homomeric P2X2 and P2X3 receptors, and heteromeric P2X2/3 receptors. Engineered P2X receptors can be rapidly and reversibly opened and closed by different wavelengths of light. Light-activated P2X receptors can be mutated further to impart ATP-insensitivity if required. This method offers control of specific P2X receptor channels with high spatiotemporal precision to study their roles in physiology and pathophysiology.

KEYWORDS:
ATP; Azobenzene; Ion channel; Optochemicals; P2X; Photoswitchable; Synthetic optogenetics

PMID: 31646498 DOI: 10.1007/978-1-4939-9717-6_22
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7.
Methods Mol Biol. 2020;2041:285-300. doi: 10.1007/978-1-4939-9717-6_21.
Automated Planar Patch-Clamp Recording of P2X Receptors.
Milligan CJ1, Jiang LH2.
Author information
1
The Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, Parkville, VIC, Australia. carol.milligan@florey.edu.au.
2
School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
Abstract
P2X receptors are a structurally and functionally distinctive family of ligand-gated ion channels that play important roles in mediating extracellular adenosine 5'-triphosphate (ATP) signaling in diverse physiological and pathophysiological processes. For several decades, the "manual" patch-clamp technique was regarded as the gold standard assay for investigating ion channel properties. More recently, breakthroughs in the development of automated patch-clamp technologies are enabling the study of ion channels, with much greater throughput capacities. These automated platforms, of which there are many, generate consistent, reliable, high-fidelity data. This chapter demonstrates the versatility of one of these technologies for ligand-gated ion channels, with a particular emphasis on protocols that address some of the issues of receptor desensitization that are commonly associated with P2X receptor-mediated currents.

KEYWORDS:
Automated electrophysiology; Ligand-gated ion channels; Microfluidics; P2X receptors; Planar chip; Planar patch-clamp; Stacked solution application; Voltage-clamp

PMID: 31646497 DOI: 10.1007/978-1-4939-9717-6_21
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8.
Methods Mol Biol. 2020;2041:275-283. doi: 10.1007/978-1-4939-9717-6_20.
Recording P2X Receptors Using Whole-Cell Patch Clamp from Native Monocytes and Macrophages.
Stokes L1.
Author information
1
School of Pharmacy, University of East Anglia, Norwich, UK. L.Stokes@uea.ac.uk.
Abstract
Investigating ion channels in their native cell type is important when striving to understand their regulation and function, but this comes with added complexities due to the plethora of channels and receptors present. Details of recording ATP-gated ion channels in macrophages are presented together with information on how to prepare the primary cells for electrophysiological analysis.

KEYWORDS:
ATP; EPC10; Macrophage; Monocyte; P2X receptor; P2X4; P2X7; Whole-cell patch clamp

PMID: 31646496 DOI: 10.1007/978-1-4939-9717-6_20
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9.
Methods Mol Biol. 2020;2041:261-273. doi: 10.1007/978-1-4939-9717-6_19.
Heterologous Expression and Patch-Clamp Recording of P2X Receptors in HEK293 Cells.
Jiang LH1, Roger S2.
Author information
1
School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK. l.h.jiang@leeds.ac.uk.
2
EA4245, Transplantation, Immunology, Inflammation, Faculty of Medicine, University of Tours, Tours, France.
Abstract
P2X receptors (P2XRs) are ligand-gated ion channels gated by extracellular adenosine 5'-triphosphate (ATP) and play a critical role in mediating ATP-induced purinergic signaling in physiological and pathological processes. Heterologous expression of P2XR in human embryonic kidney 293 (HEK293) cells and measurement of P2XR-mediated currents using patch-clamp recording technique have been widely used to study the biophysical and pharmacological properties of these receptors. Combination of electrophysiology with site-directed mutagenesis and structural information has shed light on the molecular basis for receptor activation and mechanisms of actions by receptor antagonists and modulators. It is anticipated that such methodologies will continue helping us to provide more mechanistic understanding of P2XRs and to test novel receptor antagonists and allosteric modulators for therapeutical purposes. In this chapter, we describe protocols of transiently or stably expressing the P2XR in HEK293 cells and measuring P2XR-mediated currents by using whole-cell recording.

KEYWORDS:
HEK293 cells; Heterologous expression; P2X receptors; Patch-clamp recording

PMID: 31646495 DOI: 10.1007/978-1-4939-9717-6_19
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10.
Methods Mol Biol. 2020;2041:243-259. doi: 10.1007/978-1-4939-9717-6_18.
P2X Electrophysiology and Surface Trafficking in Xenopus Oocytes.
Bertin E1, Martínez A1, Boué-Grabot E2.
Author information
1
Institut des Maladies Neurodégénératives, CNRS UMR 5293, Université de Bordeaux, Bordeaux, France.
2
Institut des Maladies Neurodégénératives, CNRS UMR 5293, Université de Bordeaux, Bordeaux, France. eric.boue-grabot@u-bordeaux.fr.
Abstract
Xenopus oocytes serve as a standard heterologous expression system for the study of various ligand-gated ion channels including ATP P2X receptors. Here we describe the whole-cell two-electrode voltage clamp and biotinylation/Western blotting techniques to investigate the functional properties and surface trafficking from P2X-expressing oocytes.

KEYWORDS:
ATP-gated channels; Double-electrode patch clamp; Electrophysiology; Oocytes; P2X; Receptor function; Surface trafficking; Xenopus

PMID: 31646494 DOI: 10.1007/978-1-4939-9717-6_18
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11.
Methods Mol Biol. 2020;2041:233-241. doi: 10.1007/978-1-4939-9717-6_17.
ATP Measurement in Cerebrospinal Fluid Using a Microplate Reader.
de Diego-García L1,2,3,4, Sebastián-Serrano Á1,2,5, Bianchi C1,2, Di Lauro C1,2, Díaz-Hernández M6,7.
Author information
1
Department of Biochemistry and Molecular Biology, Veterinary School, Complutense University of Madrid, Madrid, Spain.
2
Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain.
3
Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland.
4
FutureNeuro Research Center, Dublin, Ireland.
5
Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain.
6
Department of Biochemistry and Molecular Biology, Veterinary School, Complutense University of Madrid, Madrid, Spain. migueldiaz@ucm.es.
7
Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain. migueldiaz@ucm.es.
Abstract
Imbalance in extracellular ATP levels in brain tissue has been suggested as a triggering factor for several neurological disorders. Here, we describe the most sensitive and reliable technique for monitoring the ATP levels in mice cerebrospinal samples collected by cisterna magna puncture technique and quantified using a microplate reader.

KEYWORDS:
ATP; Cerebrospinal fluid; In vivo; Luciferase; Luciferin; Microplate reader; Neurological disorders

PMID: 31646493 DOI: 10.1007/978-1-4939-9717-6_17
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12.
Methods Mol Biol. 2020;2041:223-231. doi: 10.1007/978-1-4939-9717-6_16.
Using FRET-Based Fluorescent Sensors to Monitor Cytosolic and Membrane-Proximal Extracellular ATP Levels.
Kaschubowski KE1, Kraft AE2, Nikolaev VO2, Haag F3.
Author information
1
Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
2
Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
3
Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. haag@uke.de.
Abstract
The assessment of local concentrations of extracellular ATP (eATP) at the site of receptor binding remains a challenge in the field of purinergic signaling. In many cases, biosensors exploiting the principle of Förster resonance energy transfer (FRET) have provided useful tools to visualize local concentrations of metabolites. A series of FRET-based biosensors based on the epsilon subunits of bacterial ATP synthases have been described for the visualisation of ATP. These sensors carry ATP-sensing units with different affinities for ATP, permitting imaging of ATP under the widely different concentration conditions found in subcellular locations such as the cytoplasm and the membrane-proximal extracellular space.

KEYWORDS:
Biosensor; Extracellular ATP; FACS; FRET; Live-cell imaging; Microscopy

PMID: 31646492 DOI: 10.1007/978-1-4939-9717-6_16
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13.
Methods Mol Biol. 2020;2041:209-221. doi: 10.1007/978-1-4939-9717-6_15.
Fluorescent Labeling and Quantification of Vesicular ATP Release Using Live Cell Imaging.
Vessey KA1, Ho T2, Jobling AI2, Wang AY2, Fletcher EL2.
Author information
1
Visual Neuroscience Laboratory, Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, VIC, Australia. k.vessey@unimelb.edu.au.
2
Visual Neuroscience Laboratory, Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, VIC, Australia.
Abstract
Adenosine triphosphate (ATP) is actively transported into vesicles for purinergic neurotransmission by the vesicular nucleotide transporter, VNUT, encoded by the gene, solute carrier 17, member 9 (SLC17A9). In this chapter, methods are described for fluorescent labeling of VNUT positive cells and quantification of vesicular ATP release using live cell imaging. Directions for preparation of viable dissociated neurons and cellular labeling with an antibody against VNUT and for ATP containing synaptic vesicles with fluorescent ATP markers, quinacrine or MANT-ATP, are detailed. Using confocal microscope live cell imaging, cells positive for VNUT can be observed colocalized with fluorescent ATP vesicular markers, which occur as discrete puncta near the cell membrane. Vesicular release, stimulated with a depolarizing, high potassium physiological saline solution induces ATP marker fluorescence reduction at the cell membrane and this can be quantified over time to assess ATP release. Pretreatment with the voltage gated calcium channel blocker, cadmium, blocks depolarization-induced membrane fluorescence changes, suggesting that VNUT-positive neurons release ATP via calcium-dependent exocytosis. This technique may be applied for quantifying vesicular ATP release across the peripheral and central nervous system and is useful for unveiling the intricacies of purinergic neurotransmission.

KEYWORDS:
Adenosine; Dopamine; Dopaminergic; P2X7; Purine; Purinergic; Retina; Solute carrier 17, member 9 (SLC17A9); VNUT

PMID: 31646491 DOI: 10.1007/978-1-4939-9717-6_15
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14.
Methods Mol Biol. 2020;2041:197-207. doi: 10.1007/978-1-4939-9717-6_14.
Using Amperometric, Enzyme-Based Biosensors for Performing Longitudinal Measurements of Extracellular Adenosine 5-Triphosphate in the Mouse.
Beamer E1, Engel T2.
Author information
1
Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland. edwardbeamer@rcsi.com.
2
Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland.
Abstract
Adenosine 5-triphosphate (ATP) functions in the central nervous system as an extracellular signaling molecule. While much progress has been made in understanding the circumstances under which it is released, from in vitro preparations, in vivo has proven more challenging. Microdialysis followed by high-performance liquid chromatography has been employed to demonstrate a spike in extracellular concentrations under some pathological conditions including seizures, but this method lacks the sensitivity to detect extracellular ATP at concentrations present under normal physiological conditions. An alternative approach, the use of amperometric, enzyme-based microelectrode biosensors for measuring extracellular ATP in vivo have been employed in the rabbit. Here, we describe a protocol for measuring ATP concentrations using these biosensors in the mouse, simultaneously with electroencephalogram recordings. This approach is ideal for investigating the relationship between ATP release and seizures.

KEYWORDS:
Adenosine-5-triphosphate; Amperometric detection; Enzyme-based; In vivo electrochemistry; Microelectrode biosensors

PMID: 31646490 DOI: 10.1007/978-1-4939-9717-6_14
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15.
Methods Mol Biol. 2020;2041:183-195. doi: 10.1007/978-1-4939-9717-6_13.
Detection of Extracellular ATP in the Tumor Microenvironment, Using the pmeLUC Biosensor.
De Marchi E1, Orioli E1, Pegoraro A1, Adinolfi E1, Di Virgilio F2.
Author information
1
Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy.
2
Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy. fdv@unife.it.
Abstract
ATP is one of the main components of the tumor microenvironment, where it affects cell growth, tumor progression and antitumor immune response. The development of the pmeLUC probe, a luciferase engineered to be expressed on the outer facet of the plasma membrane, allowed real-time measurement of extracellular ATP in vitro and in vivo systems, among which the tumor microenvironment. Here we describe the experimental procedures to measure extracellular ATP levels in the tumor microenvironment of three different cancer models generated by the implant of pmeLUC-expressing tumor cells into the appropriate mice strain: ACN human neuroblastoma (nude/nude mice host), WEHI-3B murine leukemia (BALB/c host), and B16F10 murine melanoma (C57Bl/6 host). The procedure to obtain stable expression of pmeLUC in different cell types and methods for the measurement of extracellular ATP with pmeLUC in vitro are also described.

KEYWORDS:
Extracellular ATP; Luciferin; Luminescence; Luminometry; Plasma membrane luciferase

PMID: 31646489 DOI: 10.1007/978-1-4939-9717-6_13
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16.
Methods Mol Biol. 2020;2041:163-181. doi: 10.1007/978-1-4939-9717-6_12.
Application of Fluorescent Purinoceptor Antagonists for Bioluminescence Resonance Energy Transfer Assays and Fluorescent Microscopy.
Soave M1,2, Goulding J1,2, Markus R3, Hill SJ1,2, Stoddart LA4,5.
Author information
1
Cell Signalling and Pharmacology Research Group, Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, UK.
2
Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, UK.
3
School of Life Sciences Imaging (SLIM), School of Life Sciences, University of Nottingham, Nottingham, UK.
4
Cell Signalling and Pharmacology Research Group, Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, UK. leigh.stoddart@nottingham.ac.uk.
5
Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, UK. leigh.stoddart@nottingham.ac.uk.
Abstract
Fluorescent antagonists offer the ability to interrogate G protein-coupled receptor pharmacology. With resonance energy transfer techniques, fluorescent antagonists can be implemented to monitor receptor-ligand interactions using assays originally designed for radiolabeled probes. The fluorescent nature of these antagonists also enables the localization and distribution of the receptors to be visualized in living cells. Here, we describe the generation of modified purinergic receptors with the NanoLuc luciferase or SNAP-tag, using the P1 adenosine A3 receptor as an example. We also describe the procedure of characterizing a novel fluorescent purinergic antagonist using ligand-mediated bioluminescence resonance energy transfer assays and confocal microscopy.

KEYWORDS:
Antagonist; BRET; Confocal microscopy; Fluorescence; Ligand binding; Purinergic receptor

PMID: 31646488 DOI: 10.1007/978-1-4939-9717-6_12
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17.
Methods Mol Biol. 2020;2041:155-162. doi: 10.1007/978-1-4939-9717-6_11.
Multimeric Purinoceptor Detection by Bioluminescence Resonance Energy Transfer.
Compan V1,2, Rassendren F3,4.
Author information
1
IGF, University of Montpellier, CNRS, INSERM, Montpellier, France. vincent.compan@igf.cnrs.fr.
2
Labex ICST, Montpellier, France. vincent.compan@igf.cnrs.fr.
3
IGF, University of Montpellier, CNRS, INSERM, Montpellier, France.
4
Labex ICST, Montpellier, France.
Abstract
Assays based on bioluminescence resonance energy transfer (BRET) provide a sensitive and simple method to study protein-protein interactions in live cells. Here we describe a protocol using BRET technique to investigate potential interactions between P2X subunits. This approach combined with bimolecular fluorescence complementation (BiFC) can also be employed to determine the stoichiometry of heteromeric P2X receptors.

KEYWORDS:
BRET; BiFC; Bioluminescence; Heteromer; P2X; Stoichiometry

PMID: 31646487 DOI: 10.1007/978-1-4939-9717-6_11
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18.
Methods Mol Biol. 2020;2041:147-153. doi: 10.1007/978-1-4939-9717-6_10.
Multimeric Ionotropic Purinoceptor Detection by Protein Cross-Linking.
Compan V1,2, Rassendren F3,4.
Author information
1
IGF, University of Montpellier, CNRS, INSERM, Montpellier, France. vincent.compan@igf.cnrs.fr.
2
Labex ICST, Montpellier, France. vincent.compan@igf.cnrs.fr.
3
IGF, University of Montpellier, CNRS, INSERM, Montpellier, France.
4
Labex ICST, Montpellier, France.
Abstract
P2X receptor subunits (P2X1 to P2X7) assemble to form trimeric homomers or heteromers. Here, we describe the use of protein cross-linking to study the composition of P2X receptor complexes. This simple protocol is useful for determining the stoichiometry of P2X heteromeric receptors as well as for assessing the effect of point mutation, truncation, or concatenation on the quaternary architecture of these receptors.

KEYWORDS:
Cross-linking; Heteromer; Homomer; P2X; Quaternary structure; Trimer

PMID: 31646486 DOI: 10.1007/978-1-4939-9717-6_10
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19.
Methods Mol Biol. 2020;2041:137-146. doi: 10.1007/978-1-4939-9717-6_9.
Studying Purinoceptor Cell-Surface Expression by Protein Biotinylation.
Young MT1.
Author information
1
School of Biosciences, Cardiff University, Cardiff, UK. youngmt@cardiff.ac.uk.
Abstract
Covalent labeling of protein with biotin (biotinylation) is a versatile technique which enables the capture and analysis of labeled protein with streptavidin-coated beads. It is particularly useful for detecting and quantifying the cell-surface expression of membrane proteins and widely used to analyze protein trafficking and the effect of mutations (particularly those which render the protein nonfunctional) on cell-surface expression. Here I describe the procedure for biotinylation and capture of cell-surface rat P2X2 receptors expressed in mammalian cells, and outline the steps in data analysis required to measure the proportion of cell-surface expressed protein of single point mutants relative to a wild-type control.

KEYWORDS:
Biotinylation; Cell-surface; Crosslinking; Membrane protein; P2X; Protein expression; Western blot

PMID: 31646485 DOI: 10.1007/978-1-4939-9717-6_9
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20.
Methods Mol Biol. 2020;2041:117-136. doi: 10.1007/978-1-4939-9717-6_8.
Flow Cytometry of Membrane Purinoreceptors.
Schwarz N1, Junge M1, Haag F1, Koch-Nolte F2.
Author information
1
Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
2
Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. nolte@uke.de.
Abstract
Mammalian purinoreceptors respond to extracellular nucleotides and their metabolites, for example, following the release of ATP or NAD+ from cells and their hydrolysis by ectonucleotidases. Membrane purinoreceptors are expressed as ionotropic ligand-gated ion channels designated P2X receptors, or as metabotropic G-protein coupled receptors designated P1 or P2Y receptors, on the cell surface of different cell types. In this chapter, we provide protocols to monitor the expression and activity of purinoreceptors on the cell membrane of living cells by flow cytometry.

KEYWORDS:
Ca2+-influx; Ectodomain shedding; Externalization of phosphatidylserine; Flow cytometry; Inflammasome; P2X7; Purinergic signaling; Purinoreceptors

PMID: 31646484 DOI: 10.1007/978-1-4939-9717-6_8
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