The functional characterization of mammalian olfactory receptors (ORs) remains a significant challenge due to their complex expression and signaling mechanisms. While in vitro systems have enabled the identification of agonists and antagonists for several ORs, including Olfr73, these findings often fail to translate to native conditions. This study directly compares the response dynamics of ectopically expressed mouse Olfr73 in two distinct environments: in vivo within native olfactory sensory neurons (OSNs) and in vitro in HEK293T cells. The results demonstrate that ligand interactions are not static but are dynamically modulated by both the cellular context and the temporal pattern of stimulation.
In vivo experiments utilized rAAV2/5-mediated gene delivery to express full-length Olfr73 fused with GCaMP3 in mature OSNs of adult mice and rats. Using high-resolution confocal imaging, we monitored calcium responses in dendritic knobs and cilia following brief (5-second) odorant pulses. The data revealed that eugenol, a known agonist, evoked robust, concentration-dependent activation in both species, with mouse OSNs exhibiting significantly higher sensitivity than rat OSNs—EC50 values of 4.8 ± 1.1 µM and 41 ± 12 µM, respectively. Notably, methylisoeugenol (MIEG), previously reported as an antagonist, elicited a measurable calcium response in mouse OSNs with an EC50 of 430 ± 130 µM, indicating partial agonism rather than inhibition. Similarly, nootkatone, identified as a potent agonist in vitro, acted only as a weak agonist in native OSNs, producing a response at just 50% of eugenol’s amplitude. Isosafrole, another putative antagonist, failed to activate Olfr73 even at high concentrations, while dimerized forms of eugenol and isoeugenol were inactive altogether.
To assess binary interactions, we applied mixtures of eugenol and MIEG, or other putative antagonists, and found no evidence of inhibition. Even when MIEG was pre-incubated for 30 seconds before eugenol application, no suppression of the response was observed. Co-application of eugenol and MIEG resulted in additive responses, confirming that antagonism was absent under physiological stimulus conditions. These findings mirror those from recent electrophysiological studies using intact OSNs and confirm that in the native cellular environment, MIEG behaves as a true agonist.
In contrast, in vitro assays using HEK293T cells expressing Olfr73 with G-proteins such as G15, Golf, or Gq11/β1/γ13 consistently demonstrated antagonism between eugenol and MIEG when tested with short pulses. However, when prolonged stimulation (10–30 minutes) was used, the same ligands produced sustained cAMP accumulation, and mixtures of eugenol and MIEG showed no inhibition—only additive responses. This indicates that the functional outcome is highly dependent on stimulation duration. Furthermore, the use of different G-protein pathways did not alter this result, suggesting that the absence of antagonism is not pathway-specific but rather a consequence of the cellular milieu.KMT2C Antibody Autophagy
These results collectively show that the functional profile of Olfr73 is profoundly influenced by its cellular environment.182498-32-4 References The native OSN environment, rich in endogenous chaperones, lipid microdomains, and signaling complexes, appears to reconfigure receptor-ligand interactions in ways that cannot be replicated in heterologous systems.PMID:34920791 Moreover, the temporal dynamics of odor exposure—short versus prolonged—can switch the outcome from antagonism to agonism, highlighting the importance of mimicking natural stimulus patterns in pharmacological studies.
This work underscores the necessity of validating in vitro findings in native tissues. Future efforts to define the molecular receptive range (MRR) of ORs should prioritize in vivo functional assessment in intact OSNs, particularly through viral vector-mediated expression and real-time imaging. Such approaches will provide a more accurate picture of how odorants interact with ORs in the living organism. Ultimately, understanding the context-dependent nature of OR signaling will be critical for advancing our knowledge of olfactory coding and for developing targeted therapeutics based on chemosensory receptors.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com