Delay of intracellular calcium transients using a calcium chelator: application to high-throughput screening of the capsaicin receptor ion channel and G-protein-coupled receptors.

Autor: Grant SK; Department of High Throughput Screening and Automation, Merck Research Laboratories, Rahway, New Jersey 07065, USA., Bansal A, Mitra A, Feighner SD, Dai G, Kaczorowski GJ, Middleton RE
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 2001 Jul 01; Vol. 294 (1), pp. 27-35.
DOI: 10.1006/abio.2001.5157
Abstrakt: Whole-cell functional assays are often used for high-throughput screening (HTS) of molecular targets such as ion channels and G-protein-coupled receptors. A common method for assaying the activity of these membrane proteins is to measure the change in intracellular calcium concentration upon receptor stimulation. These changes in calcium concentration are typically transient and therefore not readily adapted to high-density plate formats used in HTS instruments. We have demonstrated that an intracellular calcium chelator, BAPTA, was able to delay by 5- to 20-fold and extend for several minutes the observed calcium signals initiated by extracellular calcium influx or release of calcium from intracellular stores. As examples, we used cells expressing a calcium-permeable ion channel, vanilloid receptor type 1 (the capsaicin receptor), and two G-protein-coupled receptors. These receptor-mediated increases in intracellular calcium concentration were measured by both fluorescence-based and luminescence-based detection methods. The use of an intracellular calcium chelator to delay calcium signaling should have wide application since it allows the measurement of the functional activity of any cellular receptor that signals through calcium. With this procedure, calcium fluorescence and luminescence whole-cell functional assays may be performed with standard laboratory pipetting and detection systems.
(Copyright 2001 Academic Press.)
Databáze: MEDLINE
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