Novel Fluorescence-Based High-Throughput FLIPR Assay Utilizing Membrane-Tethered Genetic Calcium Sensors to Identify
Yan-Ling Zhang1, Sean P Moran1, Andrew Allen1
1Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States.
Abstract:
T-type voltage-gated Ca2+ channels have been implicated in many human disorders, and there has been increasing interest in developing highly selective and potent T-type Ca2+ channel modulators for potential clinical use. However, the unique biophysical properties of T-type Ca2+ channels are not conducive for developing high-throughput screening (HTS) assays to identify modulators, particularly potentiators. To illustrate, T-type Ca2+ channels are largely inactivated and unable to open to allow Ca2+ influx at -25 mV, the typical resting membrane potential of the cell lines commonly used in cellular screening assays. To address this issue, we developed cell lines that express Kir2.3 channels to hyperpolarize the membrane potential to -70 mV, thus allowing T-type channels to return to their resting state where they can be subsequently activated by membrane depolarization in the presence of extracellular KCl. Furthermore, to simplify the HTS assay and to reduce reagent cost, we stably expressed a membrane-tethered genetic calcium sensor, GCaMP6s-CAAX, that displays superior signal to the background compared to the untethered GCaMP6s or the synthetic Ca2+ sensor Fluo-4AM. Here, we describe a novel GCaMP6s-CAAX-based calcium assay utilizing a high-throughput fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA) format that can identify both activators and inhibitors of T-type Ca2+ channels. Lastly, we demonstrate the utility of this novel fluorescence-based assay to evaluate the activities of two distinct G-protein-coupled receptors, thus expanding the use of GCaMP6s-CAAX to a wide range of applications relevant for developing cellular assays in drug discovery.
Insights
Researchers developed a novel assay for T-type calcium channels, crucial for drug discovery. This high-throughput screening method uses a genetic calcium sensor and engineered cell lines to identify potential modulators for various human disorders.
Area of Science:
- Pharmacology
- Neuroscience
- Biophysics
Background:
- T-type voltage-gated Ca2+ channels are implicated in human disorders, driving interest in selective modulators.
- Existing high-throughput screening (HTS) assays face challenges due to T-type Ca2+ channel biophysical properties, hindering modulator identification, especially potentiators.
- T-type channels are typically inactivated at common cellular screening potentials, preventing Ca2+ influx and assay functionality.
Purpose of the Study:
- To develop a robust and cost-effective HTS assay for identifying T-type Ca2+ channel modulators.
- To overcome the limitations of T-type Ca2+ channel biophysics in cellular screening assays.
- To validate the assay's utility for drug discovery and broader cellular assay applications.
Main Methods:
- Engineered cell lines expressing K ir2.3 channels to hyperpolarize membrane potential to -70 mV, restoring T-type channel resting states.
- Stable expression of a membrane-tethered genetic calcium sensor, GCaMP6s-CAAX, for enhanced signal-to-background ratio.
- Utilized a high-throughput fluorometric imaging plate reader for calcium influx detection.
Main Results:
- Successfully developed a novel GCaMP6s-CAAX-based fluorescence assay for T-type Ca2+ channels.
- The assay effectively identifies both activators and inhibitors of T-type Ca2+ channels.
- Demonstrated the assay's versatility by evaluating G-protein-coupled receptor activities.
Conclusions:
- The developed assay overcomes previous limitations in screening T-type Ca2+ channel modulators.
- This novel fluorescence-based HTS assay offers a valuable tool for drug discovery.
- The GCaMP6s-CAAX system is adaptable for various cellular assay applications beyond T-type Ca2+ channels.
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