Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

Summary

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

Abstract

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Introduction

The sense of smell plays an important role in animals' survival as they rely on their olfactory abilities to obtain food, avoid predators and danger, distinguish species, and select mate^1,2. The realization of these functions depends on the odorant receptors (ORs), which are individually expressed at the ciliary surface of olfactory sensory neurons (OSNs) located in the olfactory epithelium (OE). ORs constitute the largest family of the G-protein coupled receptor (GPCR) superfamily with approximately 400 and 1200 diverse OR genes in human and mouse, respectively^3,

Protocol

1. Culturing and Maintenance of Hana3A Cells

1. Maintain cells in 10 mL of minimum essential medium (MEM) with 10% fetal bovine serum (FBS), 100 µg/mL penicillin-streptomycin, and 1.25 µg/mL amphotericin B in a 100-mm cell culture dish in a 37 °C cell culture incubator with 5% CO₂. With every other passage, add 1 µg/mL puromycin to maintain stable transfection of plasmids (See Introduction).
   NOTE: Perform all steps involving cell culture in a class II biological safety cabinet to ensure sterile environment.
2. Subculture at a ratio of 10-20% in 100-mm dishes every 2-3 days.

Results

Muscone is the main aromatic component from natural musk. Recent studies identified OR5AN1 as a human receptor for muscone and other macrocyclic musk compounds based on homology to the mouse OR, MOR215-1, cloned from muscone-responsive glomeruli in behaving mice^37,38. By screening the human OR repertoire, our group and the Touhara group also identified OR5AN1 as a major receptor for two macrocyclic musk compounds, cyclopentadecano.......

Discussion

Accurately measuring an OR's activation upon exposure to a certain odorant is the first step in deciphering the coding of olfactory information. The experiments shown in this study represent an example of how one can identify, using an in vitro OR expression system, responsive ORs among the human OR repertoire for the odorous chemical of interest and subsequently characterizing the receptor pharmacology using various concentrations of the chemical. Our results confirm OR5AN1 as a bona fide receptor .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amphotericin B	Sigma	A2942
DMSO	Sigma	D2650
FBS	Gibco	10099-141
GloSensor cAMP reagent	Promega	E1290
pGloSensor-20F cAMP plasmid	Promega	E1171
Hana3A cells			available from authors upon request
HBSS, without calcium or magnesium	GIBCO	14175095
HEPES	Hyclone	SH30237
Lipofectamine2000	Invitrogen	11668-019
M3R plasmid			cloned into a mammalian expression vector such as pCI
MEM, with EBSS and L-glutamine	Hyclone	SH30024
Muscone	Santa Cruz	sc-200528
Musk tibetene	Sigma-Aldrich	S359165
OR plasmids			cloned with a Rho-tag into a mammalian expression vector such as pCI
PBS, without calcium or magnesium	Cellgro	21-040-CV
Penicillin-streptomycin	Hyclone	SV30010
Plasmid miniprep kit	Tiangen	DP103-03
Puromycin	Sigma	P8833
RTP1S plasmid			cloned into a mammalian expression vector such as pCI
Trypsin-EDTA	Hyclone	SH30236
0.2-mL PCR tube	Axygen	PCR-02-C
1.5-mL Eppendorf tube	Eppendorf
15-mL 17 mm x 120 mm conical tube	BD Falcon	352096
8-well and/or 12-well multichannel pipetman	Eppendorf
96-well flat-bottomed white cell culture plate	Greiner	655098
100 mm x 20 mm cell culture dish	BD Falcon	353003
Class II biological safety cabinet with laminar flow
Cell culture incubator, with 5% CO2
Centrifuge, with swinging bucket rotor for 15-ml conical tubes
Infinite F200 plate reader	Tecan
Phase-contrast microscope with x10 and x20 objectives
Spectrophotometer
Sterile reagent reservoirs for multichannel distribution
Sterile paper towel