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Expression of olfactory receptors in Xenopus oocytes
N Dahmen1, H L Wang, F L Margolis
1Department of Neurosciences, Roche Institute of Molecular Biology, Nutley, New Jersey 07110.
Abstract:
The rat olfactory epithelium and the amino acid-sensitive catfish olfactory system have been used as models to study the molecular mechanisms of olfactory transduction. Here we report the functional expression of rat and catfish olfactory receptors in Xenopus oocytes injected with mRNA isolated from the respective tissues. Application of odor ligands to injected oocytes, monitored by two-electrode voltage clamp, activates stimulus-dependent transmembrane currents that reverse direction at about the chloride equilibrium potential. The currents show characteristic secondary oscillations that are presumed to reflect underlying Ca2+ oscillations. Similar ligand-activated membrane currents induced in oocytes after injection of other mRNAs have been shown to be due to activation of endogenous Ca(2+)-activated chloride channels. In summary, our results demonstrate the usefulness of the Xenopus oocyte expression system for cloning and characterization of olfactory receptors in both fish and mammalian species.
Insights
Researchers expressed rat and catfish olfactory receptors in Xenopus oocytes. This study demonstrates the Xenopus oocyte system is effective for characterizing olfactory receptors in diverse species.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- The rat olfactory epithelium and catfish olfactory system are key models for studying olfactory transduction.
- Understanding the molecular mechanisms of smell is crucial for various biological and medical applications.
Purpose of the Study:
- To functionally express rat and catfish olfactory receptors in a heterologous system.
- To validate the Xenopus oocyte as a versatile platform for olfactory receptor research.
Main Methods:
- Isolation of mRNA from rat olfactory epithelium and catfish olfactory tissue.
- Injection of isolated mRNA into Xenopus oocytes.
- Two-electrode voltage clamp electrophysiology to measure ligand-activated currents.
- Analysis of transmembrane currents and associated oscillations.
Main Results:
- Successful functional expression of both rat and catfish olfactory receptors in Xenopus oocytes.
- Odorant application induced stimulus-dependent transmembrane currents.
- Observed currents reversed at the chloride equilibrium potential and exhibited secondary oscillations, indicative of Ca2+ signaling.
- Demonstrated the utility of Ca(2+)-activated chloride channels in the oocyte system.
Conclusions:
- The Xenopus oocyte expression system is a powerful tool for cloning and characterizing olfactory receptors.
- This system is applicable to both fish and mammalian olfactory receptor research.
- Provides a foundation for future studies on olfactory receptor function and drug discovery.