An automated aequorin luminescence-based functional calcium assay for G-protein-coupled receptors

We describe in detail an automated and highly sensitive functional assay fo r calcium-coupled receptors (those receptors whose activation results in an increase in intracellular calcium levels) utilizing coelenterazine-charged aequorin as a probe for intracellular calcium levels ([Ca2+](i)). The assa y was originally established to investigate G alpha(q)-coupled prostanoid r eceptors, which are members of the G-protein-coupled receptor (GPCR) superf amily, signaling through elevation of [Ca2+](i), initially focusing on the human EP1 prostanoid receptor (hEP(1)). The parental human embryonic kidney cell line 293-AEQ17, developed by Button and Brownstein (Cell Calcium 14, 663-671, 1993), constitutively expresses apoaequorin and was used to develo p a clonal cell line which stably coexpresses hEP(1). This cell Line was us ed to optimize assay parameters in order to maximize accuracy and throughpu t in an automated 96-well format with the result that each 96-well plate ca n be completed in 70 min. Use of this flexible system will greatly simplify the functional analysis of GPCRs and other receptors which when activated result in increases in [Ca2+](i). (C) 1999 Academic Press.