Presentation: Impedance measurements and Calcium imaging to investigate the role of store operated Ca2+ entry in GPCR-mediated endothelial barrier regulation.
The endothelium of our blood vessels is under the control of various G protein-coupled receptors (GPCRs), regulating endothelial barrier function, vascular tone, angiogenesis and inflammation. Some of these GPCRs signal via Gq,11 protein, which activates Ca2+-release from the IP3-sensitive internal stores of the endoplasmic reticulum (ER). Calcium store depletion can subsequently activate so-called store-operated Ca2+ entry (SOCE) via the ER-resident Ca2+-sensor stromal-interacting molecule 1 (STIM1) and Orai1 Ca2+ entry channels.
We used Impedance measurements and ratiometric Calcium imaging to investigate the role of calcium entry via Orai1 channels downstream of barrier-regulating GPCRs in response to their agonists Thrombin, Histamine and Sphingosine-1-Phosphate (S1P) in human endothelial cells.
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Presentation: Profiling the pharmacology of G-Protein coupled receptors (GPCR) in cell-based assays using label-free impedance analysis
Michael Skiba | PhD Student (University of Regensburg; Germany)
G-protein coupled receptors (GPCRs) are among the most heavily addressed drug targets in medicinal chemistry and pharmacology. It has been estimated that about 40 % of all prescription pharmaceuticals on the market address GPCRs in different target tissues. The screening for new agonists or antagonists has been largely based on assays studying genetically engineered cells for the (i) potential binding of the ligand to their receptors or (ii) the production of second messengers upon receptor activation. Both approaches require invasive experimental procedures. Thus, they need to be performed as endpoint assays that do not reveal the time course of the cell response or details about intrinsic signal amplification. In contrast to that, non-invasive and label-free impedance monitoring has been developed over the last decades providing the response of target cells to receptor activation in real time. The technique is referred to as electric cell-substrate impedance sensing or short ECIS. In ECIS the cells are grown on planar gold-film electrodes that are integrated into regular cell culture dishes. Most recently, these electrode bearing dishes have been made commercially available in standard 96well format. The impedance of the cell-covered electrodes is measured with non-invasive electrical signals and reports on the cell response with a time resolution that is adjustable from minutes to milliseconds. This article will highlight several different approaches how non-invasive impedance measurements are used to characterize the pharmacology of GPCRs in cell-based assays comprising agonist assays, antagonist assays, dose-response relationships, signal transduction profiling and it will introduce a new dosing scheme that increases the experimental throughput significantly.
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Presentation: Employing SSM electrophysiology to capture electrogenic fluxes in membrane proteins
Dr. Matthias Quick | Associate Professor of Neurobiology (Columbia University; NY, USA)
My lab has been focusing on the study of ion-dependent transporters with special emphasis on Na+ or H+-coupled symporters. Whereas flux studies with radiolabeled solutes using the target protein reconstituted in proteoliposomes provided a wealth of information, the determination of the thermodynamically-coupled solute transport-associated flux of H+ or Na+ has been challenging. This can be attributed in part to the low transport turnover numbers of these transporters and difficulties associated with their functional expression in suitable model systems that allow for their characterization with traditional electrophysiological methods (e.g., two-electrode voltage clamp or patch-clamp methods).
By using the SURFE2R N1 SSM platform, our team was able to quickly collect data of solute transport-associated flux of co-transported ions across the membrane of proteoliposomes containing different target proteins. With this technology it is possible to collect data for a full kinetic characterization of a target protein such as its dependence on substrate and ion concentrations, pH, and potential essential additives, as well as its substrate recognition profile. The SURFE2R system also enables the use of a wide range of substrates that are readily commercially available, avoiding the use of radiolabeled compounds.
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ePhys Trivia Session: Join us for Game Night ] so to speak [!
Here you will find a quiz - Top Total Cummulative Points To Be Awarded A Gift Basket. Our Annual User Meeting Beer Mugs are a hot commodity this time of year.
Jason Villagomez & Ciara Richards | Marketing Department (Nanion Technologies)