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

Optogenetics uses light-sensitive proteins to precisely manipulate specific neurons [1]. However, its limitations in elucidating the precise contributions of individual protein molecules to physiological and behavioral outcomes have spurred the emergence of optopharmacology, which employs photosensitive molecules to directly modulate endogenous proteins (e.g., ion channels, GPCRs) [2, 3]. Among these, OptoXRs, engineered from rhodopsin, enable optical control of intracellular signaling, even in behaving animals [4].

The histamine H₃R plays a critical role in the central nervous system (CNS) by modulating the release of histamine and other neurotransmitters [5]. Emerging evidence indicates that histamine receptor functions are highly cell-type specific, and their downstream signaling encompasses both G protein-dependent pathways and β-arrestin-mediated regulatory mechanisms [6]. OptoXRs technology offers a powerful means to investigate such cell-type-specific signaling by enabling precise spatiotemporal control of receptor activation [7]. We previously demonstrated that OptoH₃R (a photosensitive histamine H₃R fusion protein) successfully engages downstream G protein pathways and mimics the acute effects of H₃R activation on neuronal activity [8]; however, its role in β-arrestin-mediated signaling and long-term regulation—including receptor internalization and subsequent desensitization—remains unexplored and requires further investigation.

In this study, we assessed whether sustained OptoH₃R activation recruits β-arrestin and induces internalization, thereby further validating its utility as a precision tool for receptor research. The OptoH₃R was constructed by fusing the domain fusion of bovine rhodopsin and human H₃R. Specifically, the extracellular and transmembrane domains of rhodopsin are retained for photosensitivity, while its intracellular loops and C-terminus are replaced with corresponding functional regions of H₃R (based on Uniprot Q9Y5N1 annotations). A short rhodopsin-derived sequence (TETSQVAPA) is added to the C-terminus to ensure proper folding and localization. In addition, the constructed OptoH₃R is expressed via a Cre-dependent adeno-associated virus (AAV-CAG-FLEX-OptoH₃R-EGFP, kindly provided by Professor Xuhua Wang from Zhejiang University). This OptoH₃R chimeric receptor integrates rhodopsin’s light-sensing properties with the intracellular signaling capabilities of the H₃R, enabling optical control (488 nm laser stimulation) of H₃R-mediated downstream signaling pathways. The photosensitive module consists of the rhodopsin (Bos Taurus, NM_001014890) transmembrane domain, which undergoes conformational changes upon illumination at specific wavelengths. The intracellular domain of the histamine H₃R (Homo sapiens, NM_007232.3) serves as the signal transduction element and is fused directly to the rhodopsin transmembrane region. Specifically, OptoH₃R retains the extracellular and transmembrane domains of rhodopsin (Fig. 1a), which provides light sensitivity and removes native H₃R ligand-binding pockets, while incorporating the intracellular loops of H₃R to preserve specific downstream signaling functionality. This configuration allows light-induced activation of H₃R signaling pathways without the need for endogenous histamine binding.

To investigate whether the OptoH₃R can mimic endogenous histamine H₃R activation of its downstream β-arrestin signaling pathways, primary mouse neuronal cells were transfected with AAV-FLEX-OptoH₃R-EGFP and AAV-hSyn-Cre-IRES-mCherry. After 72 h transfection, cells were illuminated with a 488 nm laser (Olympus IX83-FV3000 confocal, Coherent OBIS 488 nm LS 20 mW CW, 80% laser power) for 10 min per imaging region and observed using the confocal microscope, in contrast to the acute 30‑s stimulation typically used for rapid signaling events, to allow sufficient time for receptor internalization to occur. Using histamine-stimulated WT H₃R as a control, OptoH₃R-positive vesicles exhibited internalization patterns similar to that of WT H₃R, including comparable puncta number per cell (7.333 ± 0.8819 for light-stimulated OptoH₃R vs. 8.4 ± 0.5583for histamine-stimulated WT H₃R after 1 h) and subcellular distribution (Fig. 1d and f), which were both attenuated by barbadin, a β-arrestin inhibitor (Fig. 1b-e, p < 0.001). Furthermore, the time-lapse imaging was conducted in living HeLa cells expressing OptoH3R-EGFP to monitor the dynamic changes of these vesicles within subcellular structures over 1 h (with imaging every 10 min), and an increase in the number of puncta over time was observed (Fig. 1f-g). Subsequently, these cells were co-stained with an early endosome marker, rabbit anti-EEA1 (1:100; Cell Signaling, 3288T, USA), which revealed a high degree of colocalization (75.5%) between the vesicular puncta and endosomes (Fig. 1h). This finding indicates that the light-induced increase in OptoH₃R puncta represents receptor endocytic vesicles. Taken together, these results demonstrate that OptoH₃R can mimic the activation of H₃R and its downstream β-arrestin signaling, including acute G protein-mediated effects and β-arrestin-dependent regulatory processes.

As a key CNS receptor involved in fear memory, cognition, and other physiological processes, the functional interpretation of the H₃R has long been constrained by the inherent limitations of traditional research methods, leading to substantial contradictions across different studies [6, 9]. These apparently paradoxical findings underscore the critical importance of spatial specificity in H₃R function: receptor activity in different brain regions, even within distinct neuronal populations in the same region, may produce opposing behavioral outcomes. Beyond spatial complexity, the temporal dynamics of H₃R signaling add another layer of intricacy [10]. Our previous study demonstrated that OptoH₃R faithfully mimics H₃R-mediated G protein signaling and the acute effects on neuronal activity [8]. The present findings extend this observation by demonstrating that OptoH₃R undergoes internalization in response to sustained light stimulation. The blockade of this phenomenon by a β-arrestin inhibitor indicates the specific involvement of the β-arrestin pathway in this internalization process. The ability of OptoH₃R to replicate both arms of H₃R signaling—G protein-mediated effects [8] and β-arrestin-dependent internalization—highlights its utility as a precise optogenetic tool for dissecting spatiotemporally specific functions of H₃R. Moreover, the successful use of OptoH₃R in primary mouse neurons expressing Cre recombinase (via AAV-FLEX and AAV-hSyn-Cre) underscores the versatility of this approach for cell-type-specific manipulations.

However, it is worth noting that the present study employed global illumination of entire imaging regions; future experiments incorporating spatially restricted light patterns could reveal whether localized OptoH₃R activation is sufficient to drive endocytosis or whether widespread receptor engagement is required. Another limitation is the use of relatively high laser power (80%), which raises the possibility of phototoxicity or off-heating effects. Additionally, this study was conducted exclusively in vitro, whether optogenetic activation of OptoH₃R in vivo produces long-term effects on animal behavior or neuronal population activity remains unexplored. Given that H₃R primarily functions as a presynaptic autoreceptor regulating histamine and other neurotransmitter release, future validation in living animals combined with behavioral paradigms and fiber photometry would be of greater translational value. Collectively, our findings establish OptoH₃R as a mimic of endogenous H₃R β-arrestin signaling. This optogenetic tool provides a valuable platform for interrogating the physiological and pathophysiological roles of H₃R with high spatiotemporal resolution, particularly in complex neuronal circuits where histamine release patterns are highly dynamic.