A BRET Ca ²⁺ sensor enables high-throughput screening in the presence of background fluorescence

Abstract

The intrinsic fluorescence of samples confounds the use of fluorescence-based sensors. This is of particular concern in high-throughput screening (HTS) applications using large chemical libraries containing intrinsically fluorescent compounds. To overcome this problem, we developed a bioluminescence resonance energy transfer (BRET) Ca 2+ sensor, CalfluxCTN. We demonstrated that it reliably reported changes in intracellular Ca 2+ concentrations evoked by an agonist and an antagonist of the human muscarinic acetylcholine receptor M 1 (hM 1 R) even in the presence of the fluorescent compound fluorescein, which interfered with a standard fluorescent HTS sensor (Fluo-8). In an HTS using a chemical library containing fluorescent compounds, CalfluxCTN accurately identified agonists and antagonists that were missed or miscategorized using Fluo-8. Moreover, we showed that a luciferase substrate that becomes activated only when inside cells generated long-lasting BRET signals in HTS, enabling results to be reliably compared among replicate samples for hours. Thus, the use of a self-luminescent sensor instead of a fluorescent sensor could facilitate the complete screening of chemical libraries in a high-throughput context and enable analysis of autofluorescent samples in many different applications.

Detecting and measuring of GPCR signaling – comparison of human induced pluripotent stem cells and immortal cell lines

“…However, a paired substrate is required, which can be costly if used in massive quantities, necessitating economic considerations. BRET has been used to develop luminescence-based biosensors for monitoring the downstream effects of GPCRs, including Ca 2+ , cAMP, ERK activity, ATP concentration, membrane voltage, and ubiquitination ( 49 , 65 , 71 , 86 , 87 ). In addition, BRET assays have been utilized to study GPCR activation, the assembly of GPCR signaling elements on endosomal surfaces, and the conformational dynamics of the receptor-G protein complex; however, the intensity of these sensors has been suboptimal.…”