Assays for L-type voltage gated calcium channels
G M Archana1, R C Arunkumar1, R V Omkumar2
1Molecular Neurobiology Division, Rajiv Gandhi Centre for Biotechnology, Thycaud, P. O., Thiruvananthapuram, 695014, India; University of Kerala, India.
Abstract:
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, α2δ and β 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 α-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.
Insights
Researchers found L-type voltage-gated calcium channels (L-VGCC) in HEK-293 cells, enabling simpler, cost-effective drug screening assays. This discovery avoids cell transfection and streamlines high-throughput screening for neurodegenerative and cardiovascular diseases.
Area of Science:
- Pharmacology
- Neuroscience
- Cell Biology
Background:
- Voltage-gated calcium channels (VGCCs) are key drug targets for neurological and cardiovascular conditions.
- Current high-throughput screening methods (electrophysiology, calcium imaging) are costly and complex, often requiring cell transfection.
- There is a need for more accessible and cost-effective assays for VGCC drug discovery.
Purpose of the Study:
- To identify and characterize L-type VGCC (L-VGCC) expression in HEK-293 cells.
- To develop simplified, cost-effective assay methods for L-VGCC activity.
- To validate these assays for drug screening applications.
Main Methods:
- Detection of endogenous L-VGCC subunit proteins (Cav1.2, α2δ, β) in HEK-293 cells.
- Activation of L-VGCC using BayK8644 or KCl depolarization.
- Monitoring intracellular calcium changes with GCaMP6m live imaging.
- Utilizing an end-point assay based on calcium-dependent α-CaMKII/GluN2B interaction.
Main Results:
- Endogenous L-VGCC proteins were confirmed in HEK-293 cells, eliminating the need for transfection.
- L-VGCC activation led to measurable increases in intracellular calcium.
- Both live imaging and end-point detection methods successfully demonstrated L-VGCC activity.
- The L-VGCC antagonist nifedipine effectively blocked channel activity.
Conclusions:
- HEK-293 cells endogenously express functional L-VGCCs.
- Simple, cost-effective assays for L-VGCC activity can be established using these cells.
- These findings facilitate commercially viable drug screening for diseases involving L-VGCCs.
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