Label-Free Whole Cell Biosensing for High-Throughput Discovery of Activators and Inhibitors Targeting G Protein-Activated Inwardly Rectifying Potassium Channels

Abstract

Dynamic mass redistribution (DMR) and cellular dielectric spectroscopy (CDS) are label-free biosensor technologies that capture real-time integrated cellular responses upon exposure to extra- and intracellular stimuli. They register signaling routes that are accompanied by cell shape changes and/or molecular movement of cells proximal to the biosensor to which they are attached. Here, we report the unexpected observation that robust DMR and CDS signatures are also elicited upon direct stimulation of G protein-activated inwardly rectifying potassium (GIRK) channels, which are involved in the regulation of excitability in the heart and brain. Using ML297, a small-molecule GIRK activator, along with channel blockers and cytoskeletal network inhibitors, we found that GIRK activation exerts its effects on cell shape by a mechanism which depends on actin but not the microtubule network. Because label-free real-time biosensing (i) quantitatively determines concentration dependency of GIRK activators, (ii) accurately assesses the impact of GIRK channel blockers, (iii) is high throughput-compatible, and (iv) visualizes previously unknown cellular consequences downstream of direct GIRK activation, we do not only provide a novel experimental strategy for identification of GIRK ligands but also an entirely new angle to probe GIRK (ligand) biology. We envision that DMR and CDS may add to the repertoire of technologies for systematic exploitation of ion channel function and, in turn, to the identification of novel GIRK ligands in order to treat cardiovascular and neurological disorders.

The atypical chemokine receptor ACKR3/CXCR7 is a broad-spectrum scavenger for opioid peptides

“…Label-free dynamic mass redistribution (DMR) assay. Dynamic mass redistribution (DMR) experiments were conducted using the Corning Epic (Corning) biosensor system 43,[94][95][96][97] . In brief, 6 × 10 5 U87 cells were seeded in 6-cm dishes.…”

An experimental strategy to probe Gq contribution to signal transduction in living cells

“…DMR measurements were performed as described previously ( 98 , 99 , 134 ) using the Epic biosensor (Corning) together with the CyBi-SELMA semiautomated electronic pipetting system (Analytik Jena AG). In brief, 24 h after transfection with the indicated G protein α subunits, HEK-ΔGq/11 cells were counted and seeded at a density of 17,000 cells per well on 384-well fibronectin-coated biosensor plates.…”

Heterotrimeric G Protein Subunit Gαq Is a Master Switch for Gβγ-Mediated Calcium Mobilization by Gi-Coupled GPCRs

“…Activation of GIRK channels was measured using a Thallos AM dye-based thallium flux assay kit and recorded with the FlexStation 3 MultiMode Bench Top reader (Molecular Devices, Sunnyvale, CA, USA) according to the previously published protocol (Krebs et al, 2018), which is a modified version of Wydeven et al (2014). Briefly, the day before the experiment, 20,000 cells/well were seeded in 384 well plates.…”