title: Functional characterisation of the human alpha1 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 alpha1 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 alpha1 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 EC50 and Ki values in good agreement with previously reported values from studies of recombinant alpha1 GlyRs and native alpha1beta GlyRs. The rank orders of potencies was glycine > beta-alanine > taurine for the agonists and RU 5135>strychnine>brucine>PMBA=picrotoxin>atropine for the antagonists. The actions of three allosteric modulators at the alpha1 GlyR cell line were also characterised in the FMP assay. Micromolar concentrations of Zn2+ inhibited alpha1 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 alpha1 GlyR function, the potentiation of alpha1 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.
authors.affiliation: Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, Copenhagen DK-2100, Denmark. aaj@dfuni.dk
authors.forename: Anders A
authors.initials: AA
authors.lastname: Jensen
authors.forename: Uffe
authors.initials: U
authors.lastname: Kristiansen
mesh_terms: D002460:Cell Line; D005453:Fluorescence; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D018009:Receptors, Glycine / Q000502:physiology*
chemical_list: D018009:Receptors, Glycine