Validation of High Throughput Screening Assays Against Three Subtypes of Ca v 3 T-Type Channels Using Molecular and Pharmacologic Approaches

T-type Ca²⁺ channels encoded by voltage-gated Ca²⁺ channel (Ca_v) 3.1, 3.2, and 3.3 genes play important physiological roles and serve as therapeutic targets for neurological and cardiovascular disorders. Currently there is no selective T-channel blocker. To screen for such a blocker, we developed three stable cell lines expressing human recombinant Ca_v3.1, 3.2, or 3.3 channels and then examined their usefulness in high throughput screens. All three cell lines displayed an increase in intracellular Ca²⁺ in response to changes in extracellular Ca²⁺ as detected with Ca²⁺-sensitive dyes using a fluorometric imaging plate reader (FLIPR^® [Molecular Devices, Sunnyvale, CA] or FlexStation^® [Molecular Devices]). The signal-to-noise ratio was 2–4. Co-expression of Ca_v3.2 with a mouse leak K⁺ channel, which by virtue of being open at rest hyperpolarizes the cell membrane, blocked the fluorescent signal. Co-addition of KCl to these cells induced a Ca²⁺ signal that was similar to that observed in the cell line expressing Ca_v3.2 alone. These results confirm that the detection of intracellular Ca²⁺ increase in cells expressing Ca_v3.2 alone results from Ca²⁺ entry through channels that are open at the resting membrane potential of each cell line (i.e., window currents). Testing known drugs on Ca_v3 channels showed that block could be reliably detected using the FlexStation assay, FLIPR assay, or voltage clamp recordings using the IonWorks^® HT system (Molecular Devices). These results support the use of the FLIPR window current assay for primary drug screening and high throughput patch recordings for secondary screening of novel T-channel blockers.