Abstract
Abstract
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.