Cao S et al. (2022), Protocol to identify ligands of odorant recepto...

XB-ART-58929

STAR Protoc 2022 Mar 15;32:101249. doi: 10.1016/j.xpro.2022.101249.

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Protocol to identify ligands of odorant receptors using two-electrode voltage clamp combined with the Xenopus oocytes heterologous expression system.

Cao S , Liu Y , Wang G .

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Two-electrode voltage clamp (TEVC) combined with the Xenopus laevis oocytes heterologous expression system is a powerful electrophysiological tool widely used to study the properties of many transmembrane proteins. Here, we describe a protocol using this combined approach to identify the ligands of odorant receptors that form ligand-gated ion channels. We detail the procedures for site-directed mutagenesis, oocyte microinjection, and TEVC recording. This protocol can also be used to identify the key residues and illustrate the structure-function relationships of these proteins. For complete details on the use and execution of this protocol, please refer to Cao et al. (2021).

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Species referenced: Xenopus laevis

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References [+] :

Briant, Transmembrane domain quality control systems operate at the endoplasmic reticulum and Golgi apparatus. 2017, Pubmed


	Figure 1. Schematic workflow of site-directed mutagenesis of target plasmid
	Figure 2. The main steps of oocytes microinjection and TEVC recording(A) Select mature and healthy oocytes.(B) Microinject oocytes under a stereomicroscope.(C) Culture injected oocytes into 24-well plates in an incubator at 18°C.(D) Perform TEVC recording experiment.
	Figure 3. The sequencing chromatograms of HvirOR6 and its mutant, HvirOR6-L321V
	Figure 4. The electrophysiological responses of HvirOR6 and HvirOR6-L321V to 10-4 M chemical solutions(A) Inward current responses of oocytes expressing HvirOR6 to Z9-14:Ald and Z9-16:Ald.(B) Inward current responses of oocytes expressing HvirOR6-L321V to Z9-14:Ald and Z9-16:Ald.(C) Response profiles of oocytes expressing HvirOR6 and its mutant to Z9-14:Ald and Z9-16:Ald. Error bars indicate SEM (n = 16–22).Figure adapted from Cao et al. (2021).
	Figure 5. The response sensitivity of HvirOR6 and HvirOR6-L321V to Z9-14:Ald and Z9-16:Ald(A) HvirOR6 expressing oocytes stimulated with a range of Z9-14:Ald concentrations.(B) Dose-response curve of HvirOR6 expressing oocytes treated with Z9-14:Ald. EC50 = 6.196 × 10-6 M (n = 10).(C) HvirOR6 and HvirOR6-L321V expressing oocytes stimulated with a range of Z9-16:Ald concentrations.(D) Dose-response curve of HvirOR6 and HvirOR6-L321V expressing oocytes treated with Z9-16:Ald. EC50 = 2.476 × 10-5 M and 4.083 × 10-5 M, respectively (n = 7).Figure adapted from Cao et al. (2021).