Phytomedicine Plus (Aug 2022)
St. John's wort extract STW3-VI affects the binding behaviour, signal transduction and lateral mobility of 5-HT2A receptors observed by single molecule detection and the use of a novel fluorescence-labeled ligand
Abstract
Background: 5-HT2A receptors play an important role in the development and therapy of depression. St. John's wort (SJW) extracts are used in the treatment of depression. The effects of pre-treatment with SJW extract on the behaviour of 5-HT2A receptors in the plasma membrane have not been well described at the molecular level. Methods: In this work, the influence of the SJW extract STW3-VI on 5-HT2A receptor internalization and calcium response under stimulating conditions are determined using different reporter cell lines. Influences on 5-HT2A receptor binding as well as on the lateral diffusion behaviour of receptor-ligand complexes (RLC) in the plasma membrane are investigated by fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT) in real time on living cells. Therefore, a selective fluorescently labeled ligand was first developed. Results: STW3-VI pre-treatment inhibited both the 5-HT2A receptor internalization and the calcium response after agonistic stimulation with (±)-TCB-2, which suggests an inhibitory effect. Receptor binding studies using FCS surprisingly showed a dose-dependent increase in 5-HT2A receptor binding under STW3-VI. However, it is noticeable that there was a clear redistribution towards faster diffusing RLC, who possibly contribute less to signal transduction. The diffusive states of 5-HT2A receptors identified by SPT changed also under STW3-VI. Remarkably, there was a redistribution from faster diffusing to immobile RLC, while at the same time the transition probabilities for a receptor to change the diffusive state decreased clearly compared to control cells. This particular lateral diffusion behaviour likely characterizes 5-HT2A receptors with impaired signal transduction. Conclusion: The present data indicate a correlation between receptor status and lateral mobility, with STW3-VI apparently having an inhibitory effect on 5-HT2A receptor-mediated signaling in the cell models used here. Single-molecule techniques such as FCS and SPT provide important data that contribute to a better understanding of therapeutically relevant drug-target interactions and downstream regulatory processes.
