Name: live-cell measurement of odorant receptor activation using a real-time camp assay
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Description: Watch this Scientific Journal Video about Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay at JoVE.com

The work was supported by the Chinese National Science Foundation (31070972), Science and Technology Commission of Shanghai Municipality (16ZR1418300), the Program for Innovative Research Team of Shanghai Municipal Education Commission, the Shanghai Eastern Scholar Program (J50201), and the National Basic Research Program of China (2012CB910401).

1. Culturing and Maintenance of Hana3A Cells
<ol>
	<li>Maintain cells in 10 mL of minimum essential medium (MEM) with 10% fetal bovine serum (FBS), 100 &#181;g/mL penicillin-streptomycin, and 1.25 &#181;g/mL amphotericin B in a 100-mm cell culture dish in a 37 &#176;C cell culture incubator with 5% CO2. With every other passage, add 1 &#181;g/mL puromycin to maintain stable transfection of plasmids (See Introduction). 
	NOTE: Perform all steps involving cell culture in a class II biolog...

<ol>
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Accurately measuring an OR&#39;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 ...

The sense of smell plays an important role in animals&#39; survival as they rely on their olfactory abilities to obtain food, avoid predators and danger, distinguish species, and select mate1,2. 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, respectively3,4,5. ORs activated by odorants lead to increased intracellular cAMP levels via the sequential activation of olfactory G-protein (Golf) and type III adenylyl cyclase (ACIII). The resulting increased level of intracellular cAMP could function as a second messenger, which opens the nucleotide-gated channel on the cell surface, triggering influx of cations including Ca2+ and action potentials, and ultimately initiating neuro-potential transmission and olfactory perception. The process of detecting and discriminating a large number of odorants by ORs is regarded as the first step of olfactory perception6,7.
Since Buck and Axel8 first successfully cloned odorant receptors and elucidated the mechanism of olfactory perception initiated by ORs, deorphanization of the OR family became one of the hotspots in this field. Various in vivo, ex vivo and in vitro methods to measure OR activation have been reported9,10,11,12. A traditional method that used Ca2+ imaging followed by single-cell RT-PCR on OSNs enabled the identification of different ORs to aliphatic odorants13,14,15. More recently, the advent of large-scale transcriptome analyses promoted the development of more high-throughput in vivo methods. The Kentucky assay identified eugenol- and muscone-responsive mouse ORs with the use of the S100a5-tauGFP reporter mouse strain and microarray analysis9. Based on the decrease in OR mRNA levels after odorant exposure, the DREAM technology employed a transcriptomic approach to determine OR activation profiles in both vertebrate and non-vertebrate species16. Similarly, given the phosphorylation of S6 in neuronal activations,the Matsunami group sequenced mRNAs from phosphorylated ribosome immunoprecipitations to identify responsive ORs12. Finally, the Feinstein group reported super sniffer mice that could serve as a platform to study odor coding in vivo, known as the MouSensor technology17.
In the in vitro realm, the challenge of culturing OSNs makes a heterologous expression system that mimics OR functional expression in vivo an ideal solution to conduct large-scale screening of odorous chemicals for ORs. Nevertheless, since cultured cell lines of non-olfactory origins differ from native OSNs, OR proteins are retained in the endoplasmic reticulum and unable to traffic to the plasma membrane, resulting in OR degradation and loss of receptor function18,19. To solve this problem, extensive works have been made to replicate OR functional expression on the cell membrane in heterologous cell lines. Krautwurst et al. first attached the first 20 amino acids of rhodopsin (Rho-tag) to the N-terminal of OR protein and this promoted the cell-surface expression of some ORs in human embryonic kidney (HEK) cells20. By conducting a serial analysis of gene expression (SAGE) library analysis from single OSNs, Saito et al. first cloned the receptor-transporting protein (RTP) family members, RTP1 and RTP2, and the receptor expression enhancer protein 1 (REEP1) that facilitated OR trafficking to the cell membrane and enhanced odorant-mediated responses of ORs in HEK293T cells21. Based on these findings, the Matsunami group successfully established the Hana3A cell line, stably transfected with RTP1, RTP2, REEP1, and G&#945;olf in HEK293T, and transiently transfected with Rho-tagged ORs, for efficient OR functional expression. Subsequent studies revealed 1) a shorter form of RTP1, RTP1S, that could more robustly promote OR function than the original RTP1 protein and 2) the type 3 muscarinic acetylcholine receptor (M3R) that could enhance OR activity via inhibition of &#946;-arrestin-2 recruitment, both of which were introduced to the heterologous expression system to optimize experimental output22,23.
Several detection methods have been used to quantify receptor activation in heterologous systems. The secreted placental alkaline phosphatase (SEAP) assay works with a reporter enzyme transcriptionally regulated by cAMP response elements (CREs), making it an attractive option for assessing OR activation. The fluorescence is readily detected in a sample of the culture medium after the incubation with SEAP detection reagent24. Using this method, the functions of ORs as well as a secondary class of chemosensory receptors expressed in the OE-the trace amine-associated receptors (TAARs) have been characterized25,26,27. Another common method, the luciferase assay, uses a firefly luciferase reporter gene under the control of the cAMP response element (CRE). Measuring luminescence generated by luciferase production provides an efficient and robust means of quantifying OR activation10,11,28.
Real-time cAMP assays have also been widely used in dynamically monitoring the function of heterologous or endogenous GPCRs. One example of such advanced assays takes advantage of a genetically encoded biosensor variant, which possesses a cAMP-binding domain fused to a mutant form of luciferase. When cAMP binds, the conformational change leads to the activation of luciferase, luminescence from which can then be measured with a chemiluminescence reader29,30. The real-time cAMP technology has been reported suitable for the de-orphaning of human odorant receptors in HEK293 and NxG 108CC15 cells31,32,33, as well as in the HEK293T-derived Hana3A cells34,35. The Krautwurst group also described in detail the real-time cAMP technology to be suitable for bi-directional large-scale OR screening approaches32,33.
Here we describe a protocol for measuring OR activation using a real-time cAMP assay in Hana3A cells. In this protocol, the luminescence of pre-equilibrated live cells is kinetically measured for 30 min following treatment with specific volatile compounds, representing a more efficient and accurate analysis of OR activation that are less susceptible to artifacts that occur in the cellular environment with prolonged time and odor-induced cell toxicities. This real-time measurement allows for a large-scale screening of both ORs and ligands, as well as characterization of specific OR-ligand pairs of interest. Using this method, we successfully identified OR5AN1 as the receptor for the musk compound muscone by performing a screening against 379 human ORs and subsequently confirming the positive screening result.

<table width="1240">
	<tbody>
		<tr height="25">
			<td height="25" style="width:405px;height:25px;">Amphotericin B</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;">A2942</td>
			<td style="width:623px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">FBS</td>
			<td>Gibco</td>
			<td>10099-141</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">GloSensor cAMP reagent</td>
			<td>Promega</td>
			<td>E1290</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">pGloSensor-20F cAMP plasmid</td>
			<td>Promega</td>
			<td>E1171</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Hana3A cells</td>
			<td></td>
			<td></td>
			<td>available from authors upon request</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HBSS, w/o calcium chloride and magnesium chloride</td>
			<td>GIBCO</td>
			<td>14175095</td>
			<td style="width:623px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HEPES</td>
			<td>Hyclone</td>
			<td>SH30237</td>
			<td style="width:623px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Lipofectamine2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">M3R plasmid</td>
			<td></td>
			<td></td>
			<td>cloned into a mammalian expression vector such as pCI</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MEM, w/EBSS and w/L-glutamine</td>
			<td>Hyclone</td>
			<td>SH30024</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Muscone</td>
			<td>Santa Cruz</td>
			<td>sc-200528</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Musk tibetene</td>
			<td>Sigma-Aldrich</td>
			<td>S359165</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">OR plasmids</td>
			<td></td>
			<td></td>
			<td>cloned with a Rho-tag into a mammalian expression vector such as pCI</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PBS, w/o calcium or magnesium</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Penicillin-streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Plasmid miniprep kit</td>
			<td>Tiangen</td>
			<td>DP103-03</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Puromycin</td>
			<td>Sigma</td>
			<td>P8833</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">RTP1S plasmid</td>
			<td></td>
			<td></td>
			<td>cloned into a mammalian expression vector such as pCI</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Trypsin-EDTA</td>
			<td>Hyclone</td>
			<td>SH30236</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">0.2-mL PCR tube</td>
			<td>Axygen</td>
			<td>PCR-02-C</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1.5-mL Eppendorf tube</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">15-mL 17 mm x 120 mm conical tube</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">8-well and/or 12-well multichannel pipetman</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">96-well flat-bottomed white cell culture plate</td>
			<td>Greiner</td>
			<td>655098</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">100 mm x 20 mm cell culture dish</td>
			<td>BD Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Class II biological safety cabinet with laminar flow</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell culture incubator, w/ 5% CO2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Centrifuge, with swinging bucket rotor for 15-ml conical tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Infinite F200 plate reader</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Phase-contrast microscope with x10 and x20 objectives</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Spectrophotometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sterile reagent reservoirs for multichannel distribution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sterile paper towel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

live-cell measurement of odorant receptor activation using a real-time camp assay

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.

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 mice37,38. 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...

Watch this Scientific Journal Video about Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay at JoVE.com

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

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.

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

The overall goal of this real-time high-throughput cyclic AMP assay is to screen odorants against odorant receptors and vice versa. Since mice are useful for investigating odorant receptor functioning in that it allows for larger scale screenings of both odorant receptors and the ligands as well as pharmacological cartelizations of a receptor-ligand pairs of interest. The main advantage of this technique is that we can assess odorant-to-receptor activation efficiently and reliably.Heterologous odorant-to-receptor expression system using live cells. Begin by observing Hana3A cells under a phase-contrast microscope to ensure cell viability and to estimate confluence. Then aspirate all medium from the cell culture dish.And wash the cells by adding 10 milliliters of phosphate buffered saline onto the plate, swirling the dish, and aspirating the PBS. Next, add the three milliliters of 0.5%trypsin-EDTA onto the plate. Leave the trypsin on the cells for one minute or until all cells are afloat.Inactivate the trypsin by adding 5 milliliters of MEM with 10%FBS and pipetting up and down to break up chunks of cell mass. After performing a cell count, transfer 2x10 to the 6th cells per 96-well plate to be transfected to a 15 milliliter tube. It is important that you calculate the correct number of cells to be prepared onto 96-well plate to avoid over or undergrowth of cells prior to stimulation.Centrifuge the tubes at 200 times G for five minutes. And then aspirate the supernatant without disturbing the cell pallet. Add six milliliters of medium without antibiotics for each 96-well plate and pipette up and down without creating air bubbles to break up chunks of the cell mass.Transfer the suspended cells into a reservoir. Using a multi-channel pipette, pipette 50 microliters of cells into each well of the 96-well plates. Incubate overnight at 37 degrees Celsius with 5%carbon dioxide.Prior to transfection observe the plated cells under a phase-contrast microscope to ensure a proper confluence of approximately 30 to 50%per well and then return to the incubator. Follow the row tagged odorant-to-receptor construct, the accessory factor constructs, and the cyclic AMP biosensor variant construct. Then prepare a plasma transfection mixture in 500 microliters of MEM for each 96-well plate.Next, prepare a transfection mixture containing 18 microliters of lipid-mediated transfection reagent in 500 microliters of MEM. Mix the transfection mixture with the plasma mixture by pipetting up and down. Then incubate at room temperature for 15 minutes.After the incubation, stop the reaction by adding 5 milliliters of MEM with 10%FBS to the transfection mix. Next, spread out a thick layer of sterile paper towels in the cell culture hood. Take a 96-well plate with cells from the incubator.Gently and repeatedly, tap the plate upside down on the pile of paper towels so that the medium is completely absorbed by the paper towel. Transfer 50 microliters of the combined transfection mixture to each well of the 96-well plate and incubate overnight for 18 to 24 hours at 37 degrees Celsius with 5%carbon dioxide. You can manage the transfection and the stimulation schedule should persist on instrument for more than one 96-well plate is tested in one experiment so that all plates are mirrored after same transfection and distillers exposure interval.Before stimulation, observe the transfected cells under a phase-contrast microscope to ensure a proper confluence of 50 to 80%per well before returning to the incubator. Then prepare stimulation medium by adding 10 millimolar heaps and five millimolar glucose to Hanks'balanced salt solution. Thaw 55 microliters alloquats of real-time cyclic AMP assay substrate reagent alloquats on ice.Prepare 2%equal abration solution by mixing 55 microliters of the substrate reagent and 2, 750 microliters of stimulation medium. After spreading out a thick layer of paper towels on the bench, gently and repeatedly tap the plate upside down on the paper towels so that the transfection medium is absorbed by the paper towels. Wash the cells by pipetting 50 microliters of stimulation medium to each well.Gently and repeatedly tap out the stimulation medium from the 96-well plate. Pipette 25 microliters of 2%equillibration solution into each well and incubate at room temperature in the dark for two hours. Prior to the end of the incubation time, dilute thawed odorant stock solutions to working concentration and stimulation medium.Before odorant addition, use a chemiluminescence plate reader to measure the basal luminescence level of the plate. Quickly remove the plate from the plate reader and add 25 microliters of the odorant dilutions to each well. Then immediately start continuous luminescence measurement of all wells for 20 cycles within 30 minutes.379 unique human odorant receptors were screened against 30 micromolar muscone using the real-time cyclic AMP assay. Colored blocks along the X axis indicate different human odorant-receptor families. The Y axis represents normalized luminescence 30 minutes after odorant addition when the response reached the maximum for the positive control.The response of OR5AN1 is indicated. This figure shows real-time measurements of OR5AN1 activation by different concentrations of muscone and the result from a no-odor negative control. The arrow indicates the time point of oderant addition.Here, a concentration response curve of OR5AN1 against muscone 30 minutes after oderant addition is shown. The technique described here takes a total of three days starting with a stock plate of culture tiers on the first day. Once mastered, the process on the last day can be completed in three hours for each 96-well plate if it is performed properly.After watching this video, you should have a good understanding of how to to manage the activation of oderant receptors using a cyclic AMP assay to harness the results.

Research

JoVE Journal

Neuroscience

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

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

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

SwarmSight: Real-time Tracking of Insect Antenna Movements and Proboscis Extension Reflex Using a Common Preparation and Conventional Hardware

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...