Mosapride promotes internalization of a TREK1-HTR4 complex and exerts rapid antidepressant-like effects.

Major depressive disorder (MDD) remains a critical global health burden, and a substantial proportion of patients exhibit insufficient responses to conventional monoaminergic antidepressants. Selective inhibition of the two-pore domain potassium channel TREK1 has emerged as a promising antidepressant strategy; however, the regulatory mechanisms controlling TREK1 trafficking-particularly its functional coupling with G protein-coupled receptors (GPCRs)-remain poorly understood. Here, we established a cell-based screening platform using a biomolecular luminescence complementation (BiLC) assay to monitor agonist-induced changes in membrane-associated TREK1. Using this platform, we identified a TREK1-5-hydroxytryptamine receptor 4 (HTR4) complex in vitro and in native hippocampal tissues using biomolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), and proximity ligation assays (PLA). Live-cell imaging demonstrated that treatment with the HTR4 agonist mosapride induces redistribution/internalization of a plasma membrane-associated TREK1-HTR4 pool into intracellular compartments. Electrophysiological recordings further confirmed that mosapride reduces TREK1 channel activity in an HTR4-dependent manner. In vivo, oral administration of mosapride for five days ameliorated lipopolysaccharide (LPS)-induced depressive-like behaviors in mice and preserved markers associated with hippocampal neurogenesis. Notably, viral expression of the TREK1 C-terminal interaction domain (C1), which competitively disrupts the TREK1-HTR4 complex, attenuated the neurogenic and behavioral effects of mosapride, supporting a causal role of TREK1 regulation in mediating these antidepressant-like actions. Collectively, our findings reveal a previously unrecognized mechanism in which HTR4 dynamically regulates TREK1 trafficking and function, and they highlight GPCR-based modulation of TREK1 as a potential therapeutic strategy for depressive disorders.