AGONIST-STIMULATED FREE CALCIUM IN SUBCELLULAR COMPARTMENTS - DELIVERY OF RECOMBINANT AEQUORIN TO ORGANELLES USING A REPLICATION DEFICIENT ADENOVIRUS VECTOR

Changes in the concentration of calcium ions ([Ca2+]) within cellular organelles play a central role in controlling cellular function. We ha ve engineered the Ca2+ sensitive photoprotein aequorin to monitor sele ctively [Ca2+] within defined subcellular compartments, namely the cyt osol, nucleus and endoplasmic reticulum. DNA encoding the engineered a equorins have been inserted into a replication deficient adenovirus (A d) type 5 E1(-) vector, under control of the cytomegalovirus (CMV) maj or immediate early promoter. The Ad vector provides a simple and effic ient method to express the photoproteins in a wide variety of mammalia n cell types. Efficient targeting of the photoproteins to the appropri ate cellular compartment was established immunocytochemically in COS7 cells, where it was expressed in up to 100% of the target population. Levels of expression could be controlled by virus dose and chemical ag ents which affect the activity of the CMV promoter. In HeLa cells expr essing nuclear targeted aequorin or cytosolic aequorin, ATP or histami ne induced immediate biphasic elevations of both nuclear and cytosolic [Ca2+]; subsequent challenge with agonist evoked similar responses. I n addition to epithelial type adherent cell lines (COS7 and HeLa), aeq uorin expression was also readily detected in non-adherent cells of my eloid lineage (K562 and HL60) and non-adherent primary cells polymorph onuclear leucocytes (neutrophils). The Ad vectors can, therefore, be u sed to express targeted aequorin in a range of different cell types an d represents a novel method to monitor changes in free [Ca2+] in cellu lar organelles.