A Fluorescence-Based High-Throughput Screening Assay for the Identification of T-Type Calcium Channel Blockers

T-type voltage-gated Ca2+ channels have been implicated in contributing to a broad variety of human disorders, including pain, epilepsy, sleep disturbances, cardiac arrhythmias, and certain types of cancer. However, potent and selective T-type Ca2+ channel modulators are not yet available for clinical use. This may in part be due to their unique biophysical properties that have delayed the development of high-throughput screening (HTS) assays for identifying blockers. One notable challenge is that at the normal resting membrane potential (Vm) of cell lines commonly utilized for drug screening purposes, T-type Ca2+ channels are largely inactivated and thus cannot be supported by typical formats of functional HTS assays to both evoke and quantify the Ca2+ channel signal. Here we describe a simple method that can successfully support a fluorescence-based functional assay for compounds that modulate T-type Ca2+channels. The assay functions by exploiting the pore-forming properties of gramicidin to control the...