Báo cáo khoa học: Ligand-specific dose–response of heterologously expressed olfactory receptors potx

the olfactory receptor itself.In order to check this hypothesis, we deve-loped an heterologous expression system in mammalian cells to characterize the functional response of receptors t

Ligand-specific dose–response of heterologously expressed

olfactory receptors

Gre´goire Levasseur1, Marie-Annick Persuy1, Denise Grebert1, Jean-Jacques Remy2, Roland Salesse1 and Edith Pajot-Augy1

1

INRA-Biotechnologies, Neurobiologie de l’Olfaction et de la Prise Alimentaire, Re´cepteurs et Communication Chimique,

Jouy-en-Josas, France;2Unite´ Neurogene`se et Morphogene`se au cours du De´veloppement et chez l’Adulte, UMR CNRS 6545, Institut de Biologie du De´veloppement de Marseille, Parc scientifique de Luminy, Marseille, France

Primary olfactory neuronal cultures exposed to odorant

stimulation have previously exhibited concentration-related

effects in terms of intracellular cAMP levels and adenylate

cyclase activity [Ronnett, G.V., Parfitt, D.J., Hester, L.D &

Snyder, S.H (1991) PNAS 88, 2366–2369].Maximal

sti-mulation occurred for intermediate concentrations, whereas

AC activity declined for both low and high odorant

con-centrations.We suspected that this behavior might be

ascribed to the intrinsic response of the first molecular

species concerned by odorant detection, i.e the olfactory

receptor itself.In order to check this hypothesis, we

deve-loped an heterologous expression system in mammalian cells

to characterize the functional response of receptors to

odorants.Two mammalian olfactory receptors were used to

initiate the study, the rat I7 olfactory receptor and the

human OR17-40 olfactory receptor.The cellular response of

transfected cells to an odorant stimulation was tested by a spectrofluorimetric intracellular calcium assay, and proved

in all cases to be dose-dependent for the known ligands of these receptors, with an optimal response for intermediate concentrations.Further experiments were carried out with the rat I7 olfactory receptor, for which the sensitivity to an odorant, indicated by the concentration yielding the optimal calcium response, depended on the carbon chain length of the aldehydic odorant.The response is thus both ligand-specific and dose-dependent.We thus demonstrate that a differential dose–response originates from the olfactory receptor itself, which is thus capable of efficient discrimin-ation between closely related agonists

Keywords: olfactory receptors; olfactory coding; olfactory discrimination; odorants; intracellular calcium

Olfactory receptors (ORs) belong to the large family of

G-protein coupled receptors (GPCRs) characterized by

their seven transmembrane spanning domains.Investigation

of olfactory receptors/odorant interactions is crucial to

understand the molecular basis of olfactory coding.For this

purpose, olfactory receptor genes have been heterologously

expressed in various surrogate cells [1–4], in cell lines with a

neuronal phenotype [5], or derived from the olfactory

epithelium [5–7], or even directly in olfactory epithelium

[8,9].Individual olfactory sensory neurons have also been

tested for their responsiveness to odorant stimulation [10–

15].So far, due to the large number of potential ligands and

the lack of a suitable screening system, only a few OR–

odorant couples have been identified.The rat I7 receptor

[16] was the first mammalian olfactory receptor for which a

preferential ligand (octanal) was identified [8].As such, it has been the subject of subsequent investigations [3,9], involving an impressive range of odorants and reporting a number of stimulating odorants.This raises the possibility that the receptor itself is capable of some olfactory discrimination, as suggested by the response of individual olfactory neurons to a few odorants at given concentrations [12,13], and that this is not only performed in higher olfactory centers (i.e.olfactory bulb).OR17-40 was the first characterized human olfactory receptor, for which helional represented the most effective odorant ligand [6].An heterologous expression system in a mammalian host cell line was thus developed, using the full-length cDNA sequence instead of chimeric constructions.We report a functional expression of the rat I7 olfactory receptor in stably transfected COS cells, and of the human OR17-40 olfactory receptor in stably transfected ODORA cells tested

by a spectrofluorimetric intracellular calcium assay.Both COS-I7 cells and ODORA OR17-40 cells exhibit a dose-dependent response to their ligands with optimal concen-trations in a subpico- to subnano-molar range.Moreover, COS-I7 cells responded differentially to odorants of the same family of aldehydes but with varying carbon chain length, in terms of concentration providing the optimal response.Thus, olfactory receptors themselves can not only efficiently discriminate broad families of odorants, but they are also able to differentiate close odorants of a given family

Correspondence to E.Pajot-Augy, INRA-Biotechnologies,

Neuro-biologie de l’Olfaction et de la Prise Alimentaire, Re´cepteurs et

Communication Chimique, 78352 Jouy-en-Josas Cedex, France.

Fax: + 33 1 34 65 22 41, Tel.: + 33 1 34 65 25 63,

E-mail: pajot@jouy.inra.fr

Abbreviations: OR, olfactory receptor; GPCR, G-protein coupled

receptor; COS, Cercopithecus aethiops SV40 transformed;

PLC, phospholipase C; NaCl/P i , phosphate buffer saline;

HEK, human embryonic kidney; FBS, fetal bovine serum.

(Received 17 February 2003, revised 18 April 2003,

accepted 15 May 2003)

Materials and methods

Constructs

The I7 full-length sequence was amplified by PCR from

genomic rat DNA with cloned Pfu DNA polymerase

(Stratagene) and inserted in a pGEM-T vector (Promega)

for subcloning and control sequencing (Genome Express)

I7 was inserted in the pCMV-Tag3 expression vector

(Stratagene), in frame with the translational starting sequence

(10-base Kozak consensus sequence) and the 10 amino acids

long tag from the human c-myc gene of this vector, using sites

PstI and KpnI of the MCS.Similarly, OR17-40 full-length

sequence was cloned into a pGEM-T vector, then inserted in

the pCMV-Tag3 expression vector using sites BamHI and

XhoI of the MCS.Therefore, the c-myc epitope is located at

the 5¢-terminus of olfactory receptor sequences

Cell lines and transfection

COS-7 cells (Cercopithecus aethiops kidney cells transformed

by an origin-defective mutant of SV-40) were grown in

Dul-becco’s Modified Eagle’s Medium containing 10%

decom-plemented foetal bovine serum in a 5% (v/v) CO2atmosphere

at 37C, and transfected at a 50% confluence using

ExGen 500 from Euromedex in six-well dishes.Geneticin

(G418) was used at a final concentration of 0.5 mgÆmL)1to

select stable clones.Culture media, G418 and trypsin-EDTA

were from Gibco BRL, fetal bovine serum (FBS)from Perbio

ODORA cells [17] consist of a conditionally immortalized

cell line derived from the olfactory sensory neuron lineage,

obtained from rat olfactory epithelium.They were grown at

33C in the same medium as COS cells.Transfection, and

selection of stable clones, were performed similarly

Human embryonic kidney (HEK) 293 cells were grown

and transfected in the same conditions as COS cells

For further experiments, transfected cells were used 24 h

after transfection

RT-PCR on extracted RNA

RNAs from established stable clones were prepared from 107

cells following the modified procedure of Chomczynski [18]

proposed by Puissant and Houdebine [19].RT-PCR was

performed on DNase-treated RNA samples (RQ1 DNase,

RNase-free from Promega).First-strand synthesis was

achieved with Gibco BRL SuperScript kit using oligo(dT)12)18

as the primer.Specific primers were designed with I7 or

OR17-40 sequences and used to specifically amplify the target cDNA

by PCR on the first strand: 5-ATggAgCAgAAACTC

ATCTCTgAA-3¢ and 5¢-TTCTgCAgCTAACCAATTTTg

CTgCCTTTgTT-3¢ for I7, 5-CgggATCCATgCAgCCA

TgACCgCCTCCC-3¢ for OR17-40

Each PCR consisted of 40 cycles: 94C/60 C/72 C with

1 min steps, with a final elongation of 10 min at 72C.PCR

products were sequenced (Genome Express)

Immunofluorescence microscopy

Cells were cultured on glass slides, coated with either FBS

or 0.01% polyL-Lysine.They were washed with NaCl/P

(Na2HPO4 8 mM, KH2PO4 1.5 mM, NaCl 150 mM, KCl

3 mM) for 4· 5 min.Fixation was performed with 2.5% paraformaldehyde in NaCl/Pifor 20 min at room tempera-ture.Cells were washed again with NaCl/Pifor 4· 5 min No permeabilization was performed.Preincubation was carried out for 1 h at room temperature in NaCl/Pi+ 2% BSA (Sigma).A mouse monoclonal anti-(c-myc Ig) (Roche) was used in combination with a FITC-coupled secondary anti-body (Jackson Immunoresearch Laboratories).The primary antibody was diluted at 1/800 from the 1 mgÆmL)1stock solution and incubated for 18 h at 4C in NaCl/Pi incuba-tion buffer.Cells were washed four times in NaCl/Pi+ 0 2% BSA, then incubated for 1 h at room temperature in the dark with a 1/800 dilution of FITC-coupled goat anti-(mouse IgG).Cells were washed four times in NaCl/Pi+ 0 2% BSA After a final NaCl/Pi rinsing, slides were mounted with Vectashield (Vector), and kept at 4C in the dark.They were examined under a fluorescent microscope (Leica DMRB) equipped with the appropriate filter for fluorescein, or on a Carl Zeiss LSM 310 confocal laser scanning microscope at

488 nm excitation using helium-neon ion laser, and optimal depth resolution.It was checked with another membrane receptor with the same c-myc tag at its N-terminus, expressed

in the same type of cells, that this procedure indeed induces only a membrane-located fluorescence (data not shown, prolactin receptor expression vector by courtesy of I.Gourdou-Jacovella, NOPA, INRA Jouy-en-Josas) Odorants

Octanal, heptanal, nonanal, octanol and octanoic acid were from Sigma-Aldrich.Helional, lyral and lilial were free gifts from Roche.Stock solutions (10)1M) were prepared each day in dimethylsulfoxide, and 10)4M dilutions in water were made extemporaneously, directly from the 10)1M stock solution.EtOH was used instead of dimethylsulfoxide for lyral and lilial, with further extemporaneous dilutions starting from 10)3Mdilutions in water.Further dilutions were prepared extemporaneously by successive 1 : 10 dilu-tions in water.Diacetyl (Sigma-Aldrich) soludilu-tions were prepared directly in water

Spectrofluorimetric intracellular calcium assay Stable cells were seeded at about 200 cellsÆmm)2on glass coverslips of adequate size coated previously with either FBS or 0.01% poly(L-lysine), grown until a uniform layer of subconfluent cells was obtained, and placed in a 1% FBS medium 24 h before experiments.Cells to be transfected were seeded at about 100 cellsÆmm)2 on glass coverslips previously coated with either FBS or 0.01% poly(L-lysine), grown for 24 h, transfected as described above, and used

24 h later.Prior to the assay, cells were washed in a Hank’s Hepes buffer, pH 7.4 (137 mM NaCl, 5.4 mM KCl, 0.441 mMKH2PO4, 0 16 mMNaH2PO4, 0 885 mMMgCl2, 5.55 mMglucose, 1.25 mMCaCl2, 25 mMHepes; buffer A) They were then loaded with 1 lMfluorescent marker fura-2-acetoxy-methyl [20] (Molecular Probes) for 30 min in the dark at room temperature, and washed three times in buffer A.Fura-2-acetoxy-methyl is an EGTA-derived cal-cium chelator that enters the cells and is transformed in Fura-2 by nonspecific esterases.Coverslips were introduced

in an adapted cuvette, with excitation and emission beams

at 45 relative to the surface

Experiments were performed on a Hitachi F-2500

spec-trofluorimeter using a double wavelength excitation

(k1¼ 340 nm, k2 ¼ 380 nm, excitation slits at 10 nm)

Emission intensities F(k1) (calcium-chelating Fura-2) and

F(k2) (nonchelating Fura-2) were monitored at 510 nm for

10 min (emission slit at 10 nm).Each measurement was

calibrated by final injection of 25 lMdigitonin (Sigma) to

obtain the maximum of calcium-chelating Fura-2 [providing

Fmax(k1) and Fmin(k2)], followed by an injection of EGTA

4 mMTris 30 mM, pH 8, to reach the minimum

non-chelating Fura-2 [providing Fmin (k1) and Fmax (k2)]

The intracellular calcium concentration is provided by the

spectrofluorimeter using: [Ca2+]i(nM)¼ K · (R) Rmin)/

(Rmax) R)where, Rmin¼ [Fmin(k1)) Z1]/[Fmin(k2)) Z2]

and Rmax¼ [Fmax (k1)) Z1)/[Fmax (k2) – Z2] and

R¼ [F(k1)) Z1)/F(k2) – Z2] and K ¼ Kd· F0/Fs, where

Kdis Fura-2 dissociation constant (224 nM), F0is the 510 nm

emission signal (380 nm excitation) in the absence of

calcium, and Fs is the 510 nm emission signal (380 nm

excitation) with a saturating concentration of calcium Z1

and Z2 are the intrinsic fluorescence intensities of the sample

excited at k1 and k2

Odorant stimulation was performed by injection of a

30-lL volume of a given dilution of the odorant in the

spectrofluorimeter cuvette containing 3 mL buffer A,

indu-cing a further odorant dilution of 1/100.A magnetic stirrer

ensures efficient homogenization of odorant in the medium

in less than 2 s.As there is no buffer aspiration and thus no

rinsing of the odorant, a new coverslip must be used for each

odorant stimulation and for each

concentration.Measure-ments were performed several times at each concentration

and for each odorant.Experiments with solvents at the

same dilution used in odorant samples were also performed

under the same conditions with new coverslips.Data plots

mention first quartile, median (second quartile) and third

quartile of all significant data.In the case of a single

odorant, all data points are also plotted as a scatter chart

Results

Clonal transfected cell lines

The presence of I7 mRNA in COS-I7 cells and of OR17-40

mRNA in ODORA OR17-40 cells was tested by RT-PCR

in the stable clones, after a DNase treatment to eliminate a

potential genomic DNA contamination.The expected bands

(980 bp for I7 and 950 bp for OR17-40) were detected on

agarose–ethidium bromide gels for COS-I7 cells and

ODORA OR17-40 cells, respectively (Fig.1, lanes 2 and

4).Negative controls were obtained by RT-PCR performed

on mRNAs from untransfected cells (Fig.1, lanes 1 and 3),

and by PCR on nonretro-transcripted mRNAs (not shown)

Sequencing the PCR product confirmed that the RT-PCR

products indeed had the expected OR sequences

Fluorescence microscopy

In order to visualize the recombinant expression of I7

and OR17-40 olfactory receptors, we performed

immuno-fluorescence microscopy on nonpermeabilized COS-I7 and

ODORA OR17-40 stable clonal cell lines, using an anti-(c-myc Ig) and a fluorescein isothiocyanate (FITC)-coupled secondary antibody.Stable COS-I7 cells never exhibited any detectable labeling, nor did stable ODORA OR17-40 cells Detection was thus attempted on various types of cells transiently transfected with I7 or OR17-40 expression vectors.Observations were performed with a confocal microscope.COS cells transiently transfected with I7 showed

no specific labeling either.In the case of OR17-40, positive labeling was observed in a number of transiently transfected cell lines (Fig.2).About 1% of OR17-40 transiently trans-fected COS cells showed a positive labeling (Fig.2A).A cortical localization is particularly visible in HEK293

OR17-40 cells, in which about 10% of the cells exhibited this pattern (Fig.2B).As for OR17-40 transiently transfected ODORA cells, only 1& of the cells yielded a discrete punctuate labeling

at the level of the plasmic membrane (Fig.2C)

Intracellular calcium assay Characteristics of the calcium response to odorant stimulation Figure 3 shows representative curves obtained during spectrofluorimetric intracellular calcium assays on COS-I7 cells from a stable clone, each with a single odorant stimulation performed, respectively, at heptanal 10)13M, octanal 10)10Mand nonanal 10)12M(final concentrations), and on a ODORA OR17-40 stable clone stimulated with helional 10)12M(final concentration).The response of the cells consists of a transient peak of intracellular calcium concentration, with a maximum reached about 10 s after injection, and prompt return to the baseline.The late broad increase results from the calibration procedure

Specificity and dose-dependence of the calcium response: I7 response from COS-I7 cells Specificity of olfactory receptors responses to odorant stimulation was investigated

Fig 1 Detection of rat I7 mRNA in RT-PCR products from cell strains Lane 1, native COS 7 cells; lane 2, COS-I7 clone obtained through stable transfection (see conditions in the text).A 980 bp band corresponding to the expected sequence size of the PCR product is detected.Lane 3, native ODORA cells; lane 4, ODORA OR17-40 clone obtained through stable transfection.A 950 bp band corres-ponding to the expected sequence size of the PCR product is detected Size marker is DRIgest III from Amersham.

on both rat I7 and human OR17-40 receptors.I7 was

expressed in COS cells that represent a widely used,

multipurpose cell factory

A number of odorants were tested on COS-I7 cells from

the same clone.A series of aliphatic aldehydes (heptanal,

octanal, nonanal), aromatic aldehydes (lyral, lilial) and

odorants with same carbon chain length but a different

chemical function from octanal (octanol and octanoic acid),

and diacetyl were used.We obtained a very specific response

of I7 receptor with the three aliphatic aldehydes, but not with other odorants (Fig.4).Solvents at the same dilution used in odorant samples did not induce any stimulation of the cells.Negative controls were obtained with native COS cells, for which no calcium response was ever obtained with any odorant stimulation (aldehydes, lyral, lilial or diacetyl) ATP disodium salt (10)4 ) was used as positive control

Fig 2 Confocal fluorescence microscopy on OR17-40 transiently transfected cells Immunolabeling was performed with a mouse monoclonal anti-(c-myc Ig), and a FITC-coupled secondary antibody.A, COS cells; B, HEK293 cells; C, ODORA cells.

The different COS-I7 clones gave comparable responses

to odorant stimulations.In a given clone, plotting [Ca2+]i

increase in response to various concentrations of heptanal

yields a narrow bell-shaped curve, with a maximum for a

10)13Mconcentration of heptanal (Fig.4), and no response

for higher concentrations

Specificity and dose-dependence of the calcium response: OR17-40 response from ODORA OR17-40 cells In an effort to reproduce more closely natural conditions,

OR17-40 was expressed in ODORA cells that are more representative of the native tissue expressing olfactory receptors.Stable ODORA OR17-40 cells were submitted

to helional or other odorant stimulation.Only helional induced a response from the cells.No response was obtained with solvents used at the same dilution as in odorant samples Native ODORA cells never exhibited any response to any odorant stimulation tested.Again, the dose–response profile

to helional stimulation is a narrow bell-shaped curve, with a maximum for 10)11M helional, almost no response for

10)10Mand above, few responses for 10)12Mhelional, and

no response for lower concentrations (Fig.5)

In addition, stimulation experiments were attempted on ODORA, COS and HEK cells transiently transfected with OR17-40 expression vector, as immunodetection was able

to reveal various levels of receptor expression at the membrane in those cells.Although the same global response pattern was observed as in ODORA OR17-40 stable cells (not shown), both response level and reproducibility were too low to allow accurate data processing in any of those transiently transfected cells

Differential dose–response to a family of linear aldehydes for I7 In the case of I7, the response of the receptor to a family of linear aldehydes was investigated.For each aldehyde, a bell-shaped dose–response curve was obtained.The maximal signal amplitude is of the same order of magnitude for each aldehyde studied.However, the concentration–response curves are shifted along the concentration axis as a function of the odorant carbon chain length.COS-I7 clones cells exhibit a response to heptanal in

a low concentration range (10)14to 10)12M), to nonanal in

an intermediate concentration range (10)13to 10)10M), and

to octanal for higher odorant concentrations over a broader range (10)12to 10)7M)

Fig 3 Spectrofluorimetric intracellular calcium assays The curves

show representative responses of cells to single odorant stimulation.

COS-I7 cells were stimulated with aldehydic odorants, and ODORA

OR17-40 cells with helional.The final concentration indicated for the

respective odorants is reached by injection of a dilution of the odorant

at t 0 indicated by the arrow.A typical curve resulting from pure

dimethylsulfoxide (or ethanol) injection is also shown.

Fig 4 Differentialdose-response of rat I7 receptor expressed in a

COS-I7 clone Dose-response curves were plotted by measuring the

intracellular calcium concentration increase in response to stimulation

by aldehydic odorants over a large concentration range (final

con-centrations).Heptanal, j; octanal, •; nonanal, m.First quartile,

median (second quartile), and third quartile of all significant data are

plotted.The symbols for the medians are enlarged and curves are

drawn from these points for each odorant.

Fig 5 Dose-response of human OR17-40 receptor expressed in an ODORA-OR17-40 clone stimulated by helional over a large concen-tration range (finalconcenconcen-trations) All significant data points are plotted.First quartile, median (second quartile), and third quartile are shown, and the curve based on the median is traced.

Expression of olfactory receptors in surrogate cells is

necessary to study molecular interaction with odorants

and signalling pathways used for olfactory coding.A

number of attempts have already been reported in

hetero-logous systems, as well as in cells presenting neuronal

phenotypes (primary neurons cultures or immortalized

olfactory cell lines), to express olfactory receptors properly

inserted into the plasma membrane

[1,4,5,7,17,21].How-ever, it is still a matter of debate whether nonengineered,

native receptors, can indeed be functionally expressed.As

we expect to use the designed system to study not only

ligand/receptor interaction, but also the functional

charac-terization and desensitization of stimulated signalling

path-ways, we assumed that any modification of the expressed

protein could interfere with its functional interactions

Therefore, unlike in previous studies, we expressed the

olfactory receptor without any molecular manipulation of

the coding sequence of the receptor, such as addition of an

import sequence to enhance protein translocation to the

membrane [2,6,22–24], or engineering chimeric constructs

with only part of the coding sequences of olfactory receptors

[3,24].Only the c-myc tag was added at the 5¢-terminus of I7

sequence

Olfactory receptor specific and dose-dependent response

The cells expressing recombinant I7 exclusively exhibit a

response to odorants of the aldehyde family (namely

heptanal, octanal and nonanal) consistent with the results

of previous in vivo [8] or in vitro studies [3,9].However, in

those studies, octanal was reported as being the main ligand

for rat I7 receptor.Further analysis shows that these results

and ours are in fact complementary.Zhao and Araneda’s

experiments on adenovirus-infected olfactory epithelium

were conducted with varying carbon chain length aldehydes

using a single odorant concentration of 10)3M.At this

concentration, octanal was reported to show the largest

response – with a response amplitude (electro-olfactogram)

of 1.7 relative to the control, whereas aldehydes with shorter

or longer chains exhibited lower responses (1.5 for nonanal,

1.45 for decanal, 1.35 for heptanal) These results compare

to our own results at the highest odorant concentrations

used (10)10or 10)9M), which induced the largest calcium

response with octanal, a less intense response with nonanal,

and no response at all with heptanal.Nevertheless, shifting

to lower and more physiological concentrations highlighted

a different ranking of the odorants, heptanal singled out as

the preferential odorant at a 10)13Mconcentration, more

efficient than nonanal, and octanal no longer inducing any

response at this concentration.These observations allow us

to conclude that heptanal can in fact be defined as the

preferential odorant ligand for rat I7, inducing a response at

the lowest concentration.Moreover, the concentration

range of odorants giving rise to signal detection is in the

submicro- to subpico-molar concentration range that seems

to be close to reported physiological detection limits for

some odorants in humans (10)7to 10)11M[6,25]) or in dogs

(10)14to 10)17M [25,26]).All previous studies have been

performed using much higher odorant concentrations –

thus, far above the physiological range – and a much

narrower concentration range than in the present work:

1 lMto 100 mMrange [8], 1–30 lMrange [3], 640 lM[23]

We have also performed heterologous expression of I7 in a yeast system, where a specific dose-dependent response was obtained exclusively in response to heptanal stimulation, in the 10)8 to 10)5M range [27], which corroborates the present results in terms of preferential ligand, even though the odorant concentration range needed for stimulating the receptor response in the yeast system is much higher than in COS cells.This modulation could arise from modifications

in the lipidic environment differing among cellular types [28]

In the case of OR17-40 human olfactory receptor, only helional, among all other odorants tested, elicited a response from the cells expressing the receptor.This is true for stable ODORA cells, but also for transiently transfected ODORA cells or COS cells.This specificity had already been reported, but only for an odorant concentration of 50 lM [22], whereas in our experimental set-up, an optimum was obtained for 10)11M helional, a dose far below those usually tested in other systems

Bell-shaped dose-dependent response The bell-shaped odor dose–response curves obtained here for both human OR17-40 and rat I7 olfactory receptors expressed in various cell types clearly differs from classical pharmacological GPCR dose–response curves exhibiting a plateau at high ligand concentration.However, some previous studies already seemed to yield a similar dose– response curve, though shifted to higher concentrations: in Krautwurst’s study [3], involving expression in HEK293 cells of a chimeric receptor including the N-terminus of rhodopsin and full-length I7 sequence and Ga15,16, octanal induced a response at 10 lMbut also a smaller response at

1 lM and 30 lM.Other measurements reported in the literature fall short of answering the question of the shape of the dose–response curves [12] as the concentration ranges for the odorants that were explored lie within the submil-limolar to milsubmil-limolar range, thus far from physiological concentrations and far from the concentrations used in this study.For the highest concentrations, experiments are prevented by the toxic effect of both the solvent and the odorant chemical itself, and by solubility problems.Kajiya

et al.[29] also reported cAMP elevation and [Ca2+]i increase when using recombinant olfactory receptors expressed in HEK293; dose–response curves seemed to downturn at the highest ligand concentration (1 mM for mOR-EG, 3 mM for mOR-EV).Similarly, increasing odorant concentrations elicit increasingly larger responses from isolated olfactory neurons [11,14], while even higher concentrations seem to yield relatively smaller responses [14].Moreover, the results obtained by Ronnett et al , on populations of primary olfactory neuronal cultures exposed

to odorant stimulation, had previously exhibited concen-tration-related patterns in terms of intracellular cAMP levels and adenylate cyclase activity, where maximal stimu-lation occurred for intermediate concentrations, whereas adenylate cyclase activity declined for both low and high odorant concentrations [30].In the present study, we followed the response of populations of cells expressing a single olfactory receptor, either I7 or OR17-40.The results

obtained tend to support the interpretation that the

bell-shaped dose–response curve indeed arises from an intrinsic

response of the olfactory receptor itself

Desensitization mechanisms may be evoked to account

for the shape of the dose–response curves

Desensitization of the receptors depends on their

phos-phorylation, as well as downstream mechanisms with

contribution of GRKs and beta-arrestins [31], and on their

internalization [32].We infer that some inhibition of the

receptor or saturation of its transduction pathway might

occur at high concentration, as it had been evoked for

isolated olfactory neurons [14].In the present experimental

set-up with no rinsing after odorant application, this could

involve a blocked, saturated conformation of the receptor,

with bound ligand but no activation of the transduction

pathway.In other experimental set-ups with extensive

washing following the stimulation, highly concentrated

odorants may nonetheless elicit some response from the

receptors.At the other end of the concentration range, the

present experiments clearly established the threshold

odor-ant concentration, above which the receptor is able to

trigger a cellular response

Olfactory receptor discrimination ability

The I7 olfactory receptor exhibits a differential

dose-dependent response to odorants of a same chemical family

It was already known that an olfactory receptor could

recognize a number of odorants, that a given odorant could

be detected by a number of different receptors, and that

different odorants could be recognized by different

combi-nations of olfactory receptors (combinatorial receptor

coding [12,33]).This observation can also be related to

the behavior of individual ORNs, which have a different

reaction profile according to the odorant tested, and a

different concentration threshold for each odorant [11,14]

Here again, as in the case of the shape of the dose–response

curves, we demonstrate that a single receptor exhibits an

elaborate discrimination ability, responding differentially to

closely related odorants, with a different coding of the

olfactory information in terms of odorant concentration

This may arise from a modulation of the odorant–receptor

interactions depending on odorant chain length, within the

putative ligand-binding site determined by transmembrane

domains IV–VII, which will be further investigated by

bio-informatic docking studies

Visual estimation of the olfactory receptors expression

level

Comparison between immunofluorescence results and

spec-trofluorimetric calcium measurements performed in the

various cell lines indicate that no direct correlation exists

between immunodetection and functional response levels

Indeed, transiently transfected HEK cells yield no better

intracellular calcium results than stable ODORA OR17-40

cells.Expression of olfactory receptors in only limited

amounts could lead to adequate membrane trafficking and

functional expression, whereas overexpression could be

detrimental to functionality, leading to intracellular receptor

aggregation or to unphysiological receptor coupling as already observed in the case of other GPCRs [34]

A few experiments performed on stable COS-I7 cells with heptanal 10–13M using calcium imaging in B.Dufy’s laboratory in Bordeaux (CNRS UMR 5543, France) showed that less than 10% of the cells are in fact responsive

to the stimulation by this odorant.Similar observations were made from other cell types (e.g CHO) stably transfected with the same expression vector (results not shown).This confirms that the absence of immunodetection

of the receptor at the cell surface is not synonymous to an absence of functional response.The observations also suggest that not all cells of a stable cell line under continuous Geneticin selection pressure may at a given time yield a functional response.Several phenomena could be respon-sible for this behavior: the physiological state of the cell may influence receptor expression efficiency, the adequacy of the effector pathway may limit the responsiveness, and consti-tutive activity of olfactory receptors could induce receptor internalization and recycling without odorant stimulation [35].Thus, future experiments involving our nonengineered receptors could largely benefit from implementing calcium imaging experiments as an alternative technique to calcium spectrofluorimetry

Taken together, our results indicate that olfactory recep-tors themselves exhibit a complex pharmacology.Consid-ering the high number of olfactory receptor genes and the large spectrum of odorants detected by a given receptor, this adds another level of complexity to the olfactory receptor world.The combination of these properties could account for the exquisite adaptation of the olfactory perception to the amazing complexity of the odor space

Acknowledgements

We are grateful to Miche`le Lieberherr for supporting G.L.concerning the spectrofluorimetric intracellular calcium measurements, and for fruitful discussion.The authors wish to warmly thank Bernard Dufy, Pierre Vacher and Thomas Ducret (CNRS UMR 5543, Bordeaux 2 University) for their generous offer to perform calcium imaging experiments using their equipment, their skills, and their time.We acknowledge the generous gift of helional, lyral and lilial samples by Roche (Dubendorf, Switzerland) through the courtesy of Boris Schilling.This research was supported in part by Institut National de

la Recherche Agronomique.G.L.is a doctorant with a grant from the French Ministe`re de l’Education Nationale, de la Recherche et de la Technologie.

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