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. 2022 Mar 15;3(2):101249.
doi: 10.1016/j.xpro.2022.101249. eCollection 2022 Jun 17.

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

Song Cao  1   2 , Yang Liu  1 , Guirong Wang  1   2
Affiliations

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

Song Cao et al. STAR Protoc. .

Abstract

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).

Keywords: Gene Expression; Model Organisms; Molecular Biology; Neuroscience.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Schematic workflow of site-directed mutagenesis of target plasmid
Figure 2
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
Figure 3
The sequencing chromatograms of HvirOR6 and its mutant, HvirOR6-L321V
Figure 4
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
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).
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