Elusive equilibrium: the challenge of interpreting receptor pharmacology using calcium assays

Calcium is a key intracellular signal that controls manifold cellular processes over a wide temporal range. The development of calcium-sensitive fluorescent dyes and proteins revolutionized our ability to visualize this important second messenger and its complex signalling characteristics. The subsequent advent of high throughput plate-based fluorescence readers has resulted in the calcium assay becoming the most widely utilized assay system for the characterization of novel receptor ligands. In this review we discuss common approaches to calcium assays, paying particular attention to the potential issues associated with interpretation of receptor pharmacology using this system. Topics covered include dye saturation and forced-coupling of receptors to the calcium pathway, but special consideration is given to the influence of non-equilibrium conditions in this rapid signalling system. Modelling the calcium transient in a kinetic mode allows the influence of ligand kinetics, receptor reserve and read time to be explored. This demonstrates that observed ligand pharmacology at very early time points can be quite different to that determined after longer incubations, even resulting in reversal of agonist potency orders that may be misinterpreted as agonist biased signalling. It also shows that estimates of antagonist affinity, whether by Schild analysis or inhibition curves, are similarly affected by hemi-equilibrium conditions. Finally we end with a discussion on practical approaches to accurately estimate the affinity of insurmountable antagonists using calcium assays.