Novel Fluorescence-Based High-Throughput FLIPR Assay Utilizing Membrane-Tethered Genetic Calcium Sensors to Identify T-Type Calcium Channel Modulators

[Image: see text] T-type voltage-gated Ca(2+) channels have been implicated in many human disorders, and there has been increasing interest in developing highly selective and potent T-type Ca(2+) channel modulators for potential clinical use. However, the unique biophysical properties of T-type Ca(2+) channels are not conducive for developing high-throughput screening (HTS) assays to identify modulators, particularly potentiators. To illustrate, T-type Ca(2+) channels are largely inactivated and unable to open to allow Ca(2+) 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 K(ir)2.3 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 Ca(2+) 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 Ca(2+) 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.