Arunkumar Renganathan, Chandrika and Mathew, Steephan and RajeevkumarRaveendran , Nair and Suma PriyaSudarsana , Devi and Mantosh, Kumar and Soumya, Paul and Mayadevi, Madhavan and R V., Omkumar (2018) A simple end-point assay for calcium channel activity. Cell calcium, 74. 73-85. ISSN 1532-1991

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Abstract

Cellular calcium signaling events are transient. Hence they are observed in real time using fluorescence imaging or electrophysiological methods that require sophisticated instrumentation and specialized skills. For high throughput assays simple and inexpensive techniques are desirable. Many calcium channels that serve as drug targets have subtypes arising from diverse subunit combinations. These need to be targeted selectively for achieving efficacy and for avoiding side effects in therapies. This in turn increases the number of calcium channels that act as drug targets. We report a novel method for intracellular calcium sensing that utilizes the calcium dependent stable interaction between CaM kinase II (CaMKII) and its ligands such as the NMDA receptor subunit GluN2B. The CaMKII-GluN2B complex formed persists as a memory of the transient increase in calcium. In a cell-based assay system GFP-α-CaMKII expressed in the cytosol responds to calcium by translocating towards GluN2B sequence motif exogenously expressed on mitochondria or endoplasmic reticulum. The resulting punctate fluorescence pattern serves as the signal for intracellular calcium release. The pattern is stable, unaffected by sample processing and is observable without real time imaging. The activities of calcium channel proteins heterologously expressed in HEK-293 cells were detected with specificity using this technique. A calcium sensor vector and a calcium sensor cell line were developed as tools to perform this technique. This technique being simple and less expensive could significantly facilitate high throughput screening in calcium channel drug discovery.

Item Type:	Article
Uncontrolled Keywords:	Calcium channel Calcium imaging Drug screening Purinergic receptor Transient receptor potential channels (TRP channels) End-point measurement Calcium sensing Calcium channel assay CaMKII NMDA receptor
Subjects:	Molecular Neurobiology
Depositing User:	Central Library RGCB
Date Deposited:	14 Sep 2018 08:25
Last Modified:	14 Sep 2018 08:25
URI:	http://rgcb.sciencecentral.in/id/eprint/687

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