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 (V_m) 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 cellular V_m in advance of T-type Ca²⁺ channel activation. Using selected ionic conditions in the presence of gramicidin, T-type Ca²⁺ channels are converted from the unavailable, inactivated state to the available, resting state, where they can be subsequently activated by application of extracellular K⁺. The fidelity of the assay has been pharmacologically characterized with sample T-type Ca²⁺ channel blockers whose potency has been determined by conventional manual patch-clamp techniques. This method has the potential for applications in high-throughput fluorometric imaging plate reader (FLIPR®, Molecular Devices, Sunnyvale, CA) formats with cell lines expressing either recombinant or endogenous T-type Ca²⁺ channels.