High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Summary

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Abstract

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors^1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors—an essential step toward a model of odor coding in the mammalian olfactory system.

Introduction

The mammalian olfactory system has the ability to respond to a vast number of odorous stimuli, allowing for the detection and discrimination of thousands of odorants. Olfactory receptors (ORs) are the molecular sensors expressed by the olfactory sensory neurons in the olfactory epithelium¹². Mammalian odor recognition occurs through differential activation of ORs by odorants, and the OR gene family is extensive, with roughly 1,000 murine and 400 human receptors^12-16. Previous functional analyses of ORs in olfactory neurons and in heterologous cells have shown that different odorants are recognized by unique, but overlapping ensembles of ORs^1....

Protocol

1. Culture of Hana3A Cells

1. Prepare M10 media by supplementing minimum essential medium (MEM) with 10% (v/v) FBS.
2. Culture Maintenance
   1. Maintain cells in M10 media. NOTE: The expression vectors for RTP1L, RTP2, REEP1, and G_αolf confer puromycin resistance to Hana3A cells, but maintaining the cells with this antibiotic does not significantly affect assay results.
   2. Subculture at a ratio of 1:8 in 10 cm dishes every 2-3 days.
   3. Incubate at 37 °C with 5% CO₂.

2. Plating Cells for Transfection

1. Aspirate media from a 100% confluent 10....

Results

A primary screen tested 328 ORs against 26 odors at a concentration of 100 µM. This odor concentration has been demonstrated to effectively activate a large proportion of ORs with known ligands¹⁰. First, normalized luciferase activity was calculated by dividing the firefly luciferase reading by the Renilla luciferase reading. Next, baselined values were calculated by subtracting the normalized luciferase readings for the no odor control from the normalized luciferase readings for each well (Fi.......

Discussion

Odorant identity is encoded by olfactory receptor activation patterns, but receptor activation patterns, including which receptors are activated and to what degree, are known for fewer than 6% of human olfactory receptors^1-11. Efforts to characterize olfactory receptors have been limited by their labor-intensive methods or applicability to only a subset of the olfactory receptor family^{17,23,24,33,34}. The Hana3A heterologous expression system supports the robust expression of the majority of tested o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hana3A cells	Avaiable from the Matsunami Laboratory upon request
RTP1S-pCI	Avaiable from the Matsunami Laboratory upon request
M3-R-pCI	Avaiable from the Matsunami Laboratory upon request
pCRE-luc	Agilent	219076	LUC
pSV40-RL	Promega	E2231	RL
Minimum Essential Media, Eagle	Sigma Aldrich	M4655	MEM
FBS	Life Technologies	16000-044	FBS
PBS (without Ca2+ and Mg2+)	Cellgro	21-040-CV	PBS
Trypsin (0.05% Trypsin EDTA)	Life Technologies	25300	Trypsin
CD293	Life Technologies	11913-019	CD293
96-well PDL white/clear plate	BD BioCoat	356693	plates
Lipid transfection reagent: Lipofectamine 2000	Life Technologies	11668-019	Lipofectamine
Firefly luciferase substrate, firefly luciferase quencher/Renilla luciferase substrate: Dual-Glo Assay	Promega	E2980	dual glo
Synergy S2	BioTek	SLAD	BioTek S2
Microplate reader software: Gen5 Data Analysis Software	BioTek	Gen5	Gen5
BIOSTACK	BioTek	BIOSTACK2WR	BioStack
Multiflo	BioTek	MFP	MultiFlo
300 μl GripTips	Integra	4433	GripTips
12.5 μl GripTips	Integra	4414	GripTips
300 μl GripTips ViaFlo96	Integra	6433	XYZ tips
12.5 μl GripTips 384 XYZ	Integra	6403	XYZ tips
384ViaFlo	Integra	6030	384ViaFlo
TE buffer	Macherey Nagel	740797.1
DMSO	Sigma Aldrich	D2650-100ML	DMSO
Forskolin	Enzo Life Sciences	BML-CN100-0010	FOR