Functional characterisation of the human α1 glycine receptor in a fluorescence-based membrane potential assay

Abstract

In the present study, we have created a stable HEK293 cell line expressing the human homomeric α1 glycine receptor (GlyR) and characterised its functional pharmacology in a conventional patch-clamp assay and in the FLIPR ® Membrane Potential (FMP) assay, a fluorescence-based high throughput screening assay. In the patch-clamp assay, the α1 GlyR exhibited the properties expected from a strychnine-sensitive glycine-gated chloride channel. In the FMP assay exposure of the cell line to GlyR agonists elicited a concentration-dependent increase in fluorescent intensity, a signal that could be suppressed by pre-incubation with GlyR antagonists. Agonists and antagonists displayed EC 50 and K i values in good agreement with previously reported values from studies of recombinant α1 GlyRs and native α1β GlyRs. The rank orders of potencies was glycine > β-alanine > taurine for the agonists and RU 5135>strychnine>brucine>PMBA=picrotoxin>atropine for the antagonists. The actions of three allosteric modulators at the α1 GlyR cell line were also characterised in the FMP assay. Micromolar concentrations of Zn 2+ inhibited α1 GlyR signalling but in contrast to previous reports the metal ion did not appear to potentiate GlyR function at lower concentrations. Analogously, whereas pregnenolone sulphate inhibited α1 GlyR function, the potentiation of α1 GlyR by pregnenolone in electrophysiological studies could not be reproduced in the assay. In conclusion, the FMP assay may not be suited for sophisticated studies of GlyR pharmacology and kinetics. However, the assay offers several advantages in studies of ligand–receptor interactions. Furthermore, the assay could be highly useful in the search for structurally novel ligands acting at GlyRs.