Assays for L-type voltage gated calcium channels.

Autor: Archana GM; Molecular Neurobiology Division, Rajiv Gandhi Centre for Biotechnology, Thycaud, P. O., Thiruvananthapuram, 695014, India; University of Kerala, India., Arunkumar RC; Molecular Neurobiology Division, Rajiv Gandhi Centre for Biotechnology, Thycaud, P. O., Thiruvananthapuram, 695014, India; University of Kerala, India., Omkumar RV; Molecular Neurobiology Division, Rajiv Gandhi Centre for Biotechnology, Thycaud, P. O., Thiruvananthapuram, 695014, India. Electronic address: omkumar@rgcb.res.in.
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 2022 Nov 01; Vol. 656, pp. 114827. Date of Electronic Publication: 2022 Aug 12.
DOI: 10.1016/j.ab.2022.114827
Abstrakt: 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.
Competing Interests: Declaration of competing interest The authors declare no conflict of interest.
(Copyright © 2022 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
Externí odkaz: