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

We describe in detail an automated and highly sensitive functional assay for 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 ([Ca(2+)](i)). The assay was originally established to investigate Galpha(q)-coupled prostanoid receptors, which are members of the G-protein-coupled receptor (GPCR) superfamily, signaling through elevation of [Ca(2+)](i), initially focusing on the human EP(1) 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 develop a clonal cell line which stably coexpresses hEP(1). This cell line was used to optimize assay parameters in order to maximize accuracy and throughput in an automated 96-well format with the result that each 96-well plate can 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 [Ca(2+)](i).