Assays for L-type voltage gated calcium channels

Voltage gated calcium channels (VGCCs) are pursued as drug targets for neurodegenerative and cardiovascular diseases. High throughput drug screening targeting VGCCs depends on patch-clamp electrophysiology or fluorophore-based calcium imaging that requires powerful equipment and specialized expertise thus leading to cost escalation. Moreover, VGCC needs to be transfected into cell lines such as HEK-293. We report the presence of L-type VGCC (L-VGCC) subunit proteins, Cav1.2, alpha 2 delta and beta in HEK-293 cells and the application of simple methods for its assay. Endogenous expression of the channel in HEK-293 cells overcomes the need for transfection. L-VGCC in HEK-293 cells was activated either by the agonist, BayK8644 or by KCl-mediated depolarization. Activity was detected using the calcium sensing probe, GCaMP6m by live imaging. L-VGCC activity induced enhancement in GCaMP6m fluorescence returned to baseline corresponding to channel-closure. Activity was also shown using a methodology involving end-point detection of the calcium dependent interaction of alpha-CaMKII with NMDA receptor subunit GluN2B sequence. This methodology further simplifies the assay as it eliminates the need for real time imaging. Activation was blocked by the specific L-type VGCC antagonist, nifedipine. Finding the protein and activity of L-VGCC in HEK-293 cells offers commercially viable assays for drug screening.

Automated summary

This study reports the presence of L-type VGCC subunit proteins in HEK-293 cells and develops simple methods for its assay. The activity of L-VGCC in HEK-293 cells can be detected through endogenous expression, eliminating the need for transfection. Different experimental methods were used to detect the activity, and the antagonist nifedipine was found to block the activity. These findings offer commercially viable assays for drug screening.