OptoH3R fusion protein mimics β-arrestin-mediated membrane endocytosis of histamine H3 receptor in vitro

OptoH 3 R is an artificial fusion protein that combines the photosensitive elements of rhodopsin with the signaling domain of the histamine H 3 receptor (H 3 R), enabling light-controlled activation of downstream H 3 R pathways. Although our previous study demonstrated that OptoH 3 R mimics the acute effects of H 3 R activation on neuronal activity, whether this tool can also recapitulate the long-term receptor desensitization and internalization processes associated with prolonged H 3 R activation remains unclear. In this study, primary cortical neurons and HeLa cells were employed to investigate the alterations in subcellular localization of OptoH 3 R upon sustained photoactivation, with histamine-stimulated wild-type (WT) H 3 R serving as a control. Furthermore, the role of β-arrestin in this process was explored. Time-lapse fluorescence imaging revealed that the number of puncta progressively increased over time following laser stimulation. Subsequent co-staining experiments with endosome marker EEA1 showed that 75.5% of light‑induced puncta were EEA1‑positive. Notably, the increase in OptoH 3 R-positive vesicles within neuronal cells was attenuated by the β-arrestin inhibitor barbadin, a pattern consistent with the internalization observed in histamine-stimulated WT H 3 R. Collectively, our findings demonstrate that OptoH 3 R recruits β-arrestin signaling upon sustained optical stimulation, thereby recapitulating H 3 R desensitization dynamics. This establishes OptoH 3 R as a useful tool for dissecting the spatiotemporally specific functions of H 3 R, including both its acute signaling and long-term β-arrestin-related mechanisms.