| abstract |
This invention relates to a simple end point assay for detection of transient intracellular Ca 2+ with broad applicability to many Ca 2+ channel proteins comprising, Generation of expression constructs for the fusion proteins having the Ca 2+/ 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-CaMKll as a chimera with green fluorescent protein (GFP-α-CaMKII) or its mutants as described herein. Site-Directed mutagenesis, Transfection, Ca 2+ stimulation, imaging and quantification of the number of cells with Ca 2+ -dependent signal, wherein, NMDA receptor activity assay, TRPVI receptor activity assay, GluR4 receptor activity assay are performed to detect the activity Of Ca 2+ channel proteins. |