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

Steven J. Charlton, Georges Vauquelin

Research output: Contribution to journalArticlepeer-review

83 Citations (Scopus)

Abstract

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 revolutionised our ability to visualise 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 utilised assay system for the characterisation 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 possible practical approaches to accurately estimate the affinity of insurmountable antagonists using calcium assays.
Original languageEnglish
Pages (from-to)1250-1265
Number of pages16
JournalBritish Journal of Pharmacology
Volume161
Issue number6
Publication statusPublished - Nov 2010

Keywords

  • Calcium assay
  • Equilibrium
  • Hemi-equilibrium
  • Antagonist
  • Agonist
  • Modelling and simulation
  • Receptor theory
  • Drug discovery

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