Assay for Detection of Transient Intracellular CA2+

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-CaMK11 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.

What is claimed is: 
     
         1 . A stable calcium sensor cell line comprising:
 a constitutively expressed protein carrying a sequence that can bind to the T-site of α-Ca 2+ /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.   
     
     
         2 . The stable calcium sensor cell line as claimed in  claim 1 , further comprising at least one expression construct comprising a calcium channel protein. 
     
     
         3 . The stable calcium sensor cell line as claim in  claim 1 , wherein the cell line is produced by:
 i) generating a construct encoding a protein carrying any sequence that can bind to the T-site of 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-Ca 2+ /calmodulin dependent protein kinase (CaMKII) or one of its mutants and a fluorescent protein; and an IRES sequence between the above mentioned chimeric protein and protein or fusion protein having any sequence that can bind to the T-site of CaMKII;   ii) generating an expression vector with a unique antibiotic selection marker for the construct mentioned in item (i) by subcloning the construct mentioned in item (i) having a protein carrying any sequence that can bind to the T-site of 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-Ca 2+ /calmodulin dependent protein kinase (CaMKII) or a mutant thereof and a fluorescent protein; and an IRES sequence between the above mentioned chimeric protein and protein or fusion protein having any sequence that can bind to the T-site of CaMKII;   iii) transfecting cells with the expression vector generated as mentioned in item (ii);   iv) subjecting the cells to antibiotic selection pressure using the antibiotic referred in item (ii);   v) sorting all the cells expressing the fluorescent protein mentioned in item (i) by fluorescence activated cell sorter (FACS) technology and subjecting the sorted cells again to antibiotic selection pressure as mentioned in item (iv);   vi) repeating cycles comprising the procedures mentioned in item (iv) followed by item (v) until almost all cells are positive for the fluorescent protein; and   
       subjecting the cells obtained in item (vi) that stably express the fluorescent protein to clonal selection by seeding at low density and separating colonies formed from single cells by colony picking method. 
     
     
         4 . An assay for quantitation of intracellular calcium wherein for the activity assay of any calcium channel protein, at least one expression construct of the calcium channel protein is transfected to the calcium sensor cell line of  claim 1 .