Functional characterization of 5-HT and 5-HT serotonin receptor signaling through G-protein-activated inwardly rectifying K channels in a fluorescence-based membrane potential assay.

The 5-HT and 5-HT serotonin receptors are abundantly expressed in the CNS and constitute validated as well as putative drug targets in a variety of psychiatric and cognitive disorders, alcoholism/addiction, pain and migraine. In the present study we have characterized the functional properties of human 5-HT and 5-HT stably co-expressed with the human G-protein-activated inwardly rectifying K channel 2 (GIRK2) in HEK293 cells in the fluorescence-based FLIPR® Membrane Potential Blue (FMP) assay. Serotonin and other agonists induced robust decreases in fluorescence levels in the 5-HT/GIRK2- and 5-HT/GIRK2-HEK293 cells in a concentration-dependent manner in the assay, and these responses could be inhibited by selective 5-HT/5-HT antagonists and by the Gα-protein inhibitor pertussis toxin (PTX). Five additional stable HEK293 cell lines co-expressing 5-HT or 5-HT with GIRK2 and one of the PTX-insensitive Gα-subunit mutants Gα, Gα and Gα were constructed, and 5-HT/5-HT-mediated responses through these specific Gα-subunits were measured in these cells pretreated with PTX in the FMP assay. The functional properties of 16 reference 5-HT agonists were characterized at the seven cell lines, which constitutes the most detailed pharmacological profiling and comparison of 5-HT and 5-HT receptor signaling in the same assay published to date. We propose that this approach to assay 5-HT-mediated signaling through endogenous Gα-proteins in HEK293 cells or through specific Gα-subunits in a fairly high-throughput manner holds some advantages to other functional assays for Gα-coupled receptors. The assay will facilitate detailed profiling of the Gα- and Gβγ-mediated signaling of 5-HT and 5-HT at the molecular level, and it could also be used to identify novel modulators for the receptors.