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<p>OptoH<sub>3</sub>R is an artificial fusion protein that combines the photosensitive elements of rhodopsin with the signaling domain of the histamine H<sub>3</sub> receptor (H<sub>3</sub>R), enabling light-controlled activation of downstream H<sub>3</sub>R pathways. Although our previous study demonstrated that OptoH<sub>3</sub>R mimics the acute effects of H<sub>3</sub>R activation on neuronal activity, whether this tool can also recapitulate the long-term receptor desensitization and internalization processes associated with prolonged H<sub>3</sub>R activation remains unclear. In this study, primary cortical neurons and HeLa cells were employed to investigate the alterations in subcellular localization of OptoH<sub>3</sub>R upon sustained photoactivation, with histamine-stimulated wild-type (WT) H<sub>3</sub>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<sub>3</sub>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<sub>3</sub>R. Collectively, our findings demonstrate that OptoH<sub>3</sub>R recruits β-arrestin signaling upon sustained optical stimulation, thereby recapitulating H<sub>3</sub>R desensitization dynamics. This establishes OptoH<sub>3</sub>R as a useful tool for dissecting the spatiotemporally specific functions of H<sub>3</sub>R, including both its acute signaling and long-term β-arrestin-related mechanisms.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月20日

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  • Shuhui Deng,
  • Zhuchen Zhou,
  • Zhong Chen,
  • Yanrong Zheng,
  • Jing Zhou

摘要

OptoH3R is an artificial fusion protein that combines the photosensitive elements of rhodopsin with the signaling domain of the histamine H3 receptor (H3R), enabling light-controlled activation of downstream H3R pathways. Although our previous study demonstrated that OptoH3R mimics the acute effects of H3R activation on neuronal activity, whether this tool can also recapitulate the long-term receptor desensitization and internalization processes associated with prolonged H3R activation remains unclear. In this study, primary cortical neurons and HeLa cells were employed to investigate the alterations in subcellular localization of OptoH3R upon sustained photoactivation, with histamine-stimulated wild-type (WT) H3R 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 OptoH3R-positive vesicles within neuronal cells was attenuated by the β-arrestin inhibitor barbadin, a pattern consistent with the internalization observed in histamine-stimulated WT H3R. Collectively, our findings demonstrate that OptoH3R recruits β-arrestin signaling upon sustained optical stimulation, thereby recapitulating H3R desensitization dynamics. This establishes OptoH3R as a useful tool for dissecting the spatiotemporally specific functions of H3R, including both its acute signaling and long-term β-arrestin-related mechanisms.