Assay for Detection of Transient Intracellular CA2+

摘要

This invention relates to a simple end point assay for detection of transient intracellular Ca2+ with broad applicability to many Ca2+ channel proteins comprising, Generation of expression constructs for the fusion proteins having the Ca2+/calmodulin dependent protein kinase II (CaMKII) phosphorylation sites of NR2A or NR2B subunits of N-methyl-D-aspartate receptor (NMDAR) or the voltage gated potassium channel of Drosophila (Eag) or any protein sequence which binds to the T-site of CaMKII similar to NR2B, conjugated to mitochondrial localizing signal sequence, or mutants of these sequences as described herein. Generation of mammalian expression constructs of α-CaMK11 as a chimera with green fluorescent protein (GFP-α-CaMKII) or its mutants as described herein. Site-Directed mutagenesis, Transfection, Ca2+ stimulation, imaging and quantification of the number of cells with Ca2+-dependent signal, wherein, NMDA receptor activity assay, TRPVI receptor activity assay, GluR4 receptor activity assay are performed to detect the activity of Ca2+ channel proteins.

主权项

1. A stable calcium sensor cell line comprising:
a constitutively expressed protein carrying a sequence that can bind to the T-site of α-Ca2+/calmodulin dependent protein kinase II (CaMKII) or a fusion protein comprising any sequence that can bind to the T-site of CaMKII conjugated to a mitochondrial localizing sequence; and a chimeric protein, the chimeric protein comprising α-CaMKII or a mutant thereof and a fluorescent protein.