Odorant receptors directly activate ... - Wiley Online Library

Journal of Neurochemistry, 2006, 96, 1591–1605

doi:10.1111/j.1471-4159.2006.03667.x

Odorant receptors directly activate phospholipase C/inositol1,4,5-trisphosphate coupled to calcium influx in Odora cells Guang Liu,* Robert M. Badeau,* Akihiko Tanimura and Barbara R. Talamo*,à *Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts, USA Department of Dental Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, Japan àDepartment of Physiology, Tufts University School of Medicine, Boston, Massachusetts, USA

Abstract Mechanisms by which odorants activate signaling pathways in addition to cAMP are hard to evaluate in heterogeneous mixtures of primary olfactory neurons. We used single cell calcium imaging to analyze the response to odorant through odorant receptor (OR) U131 in the olfactory epithelial cell line Odora (Murrell and Hunter 1999), a model system with endogenous olfactory signaling pathways. Because adenylyl cyclase levels are low, agents activating cAMP formation do not elevate calcium, thus unmasking independent signaling mediated by OR via phospholipase C (PLC), inositol-1,4,5trisphosphate (IP3), and its receptor. Unexpectedly, we found that extracellular calcium is required for odor-induced calcium elevation without the release of intracellular calcium, even though the latter pathway is intact and can be stimulated by

ATP. Relevant signaling components of the PLC pathway and G protein isoforms are identified by western blot in Odora cells as well as in olfactory sensory neurons (OSNs), where they are localized to the ciliary zone or cell bodies and axons of OSNs by immunohistochemistry. Biotinylation studies establish that IP3 receptors type 2 and 3 are at the cell surface in Odora cells. Thus, individual ORs are capable of elevating calcium through pathways not directly mediated by cAMP and this may provide another avenue for odorant signaling in the olfactory system. Keywords: calcium imaging, immunohistochemistry, inositol1,4,5-trisphosphate receptor, odorant receptor, phospholipase C, biotinylation. J. Neurochem. (2006) 96, 1591–1605.

Odorant receptors (ORs) in olfactory sensory neurons (OSNs) mediate the response to odorants by depolarization of the OSN membrane, elevation of intracellular calcium, and generation of action potentials in the olfactory axon. Although the adenylyl cyclase (AC)/cAMP pathway is essential for olfaction in vertebrate animals (Brunet et al. 1996; Belluscio et al. 1998; Wong et al. 2000), odorants are known to activate more than one transduction cascade. ORs may use other signals to mediate additional functions: e.g. the OR is essential for convergence of axons (expressing the same OR) onto particular glomeruli in the olfactory bulb, yet the cAMP pathway is not required (Lin et al. 2000; Wang et al. 1998). Other studies of alternative pathways for odorant signaling implicate phosphatidylinositol-3-OH kinase (Spehr et al. 2002), extracellular signal-regulated kinase signaling (Watt and Storm 2001) and phospholipase C (PLC), the focus of the current studies. Biochemical measurements have shown that some odorants increase inositol-1,4,5-trisphos-

phate (IP3) in ciliary preparations (Boekhoff et al. 1990; Breer and Boekhoff 1991) and in OSNs (Breer et al. 1990; Boekhoff and Breer 1992; Ronnett et al. 1993; Bruch 1996). In rat OSNs, intracellular Ca2+ (Cai) elevation by ‘IP3 odors’ was dependent on PLC, and a PLCb2-like subtype was Received August 1, 2005; revised manuscript received November 14, 2005; accepted November 14, 2005. Address correspondence and reprint requests to Barbara Talamo, Department of Neuroscience, Tufts University School of Medicine, Boston, MA 02111, USA. E-mail: [email protected] Abbreviations used: AC, adenylyl cyclase; Cai, intracellular Ca2+ CNGC, cyclic nucleotide-gated channel; ER, endoplasmic reticulum; GPCR, G-protein coupled receptor; HRP, horseradish peroxidase; IBMX, isobutylmethylxanthine; IP3, inositol-1,4,5-trisphosphate; IP3R, IP3 receptor; NCAM, neural cell adhesion molecule; NR, normal Ringer’s solution; OE, olfactory epithelium; OMP, olfactory marker protein; OR, odorant receptor; OSNs, olfactory sensory neurons; PLC, phospholipase C; TRP, transient receptor potential TRPC, TRP channel; XeC, Xestospongin C.

2006 The Authors Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 96, 1591–1605

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identified in ‘IP3 odor’-responsive cells (Noe and Breer 1998). A recent study provides evidence for odorant-activated PLC signaling in a novel class of sensory cells in the olfactory epithelium (OE), microvillar cells that lack adenylyl cyclase III (ACIII) and olfactory marker protein (OMP) but contain PLCb2 and IP3 receptor type 3 (IP3R-3), as well as a plasma membrane transient receptor potential (TRP) channel TRPC-6 (Elsaesser et al. 2005). Electrophysiological studies demonstrate an IP3-gated ion channel (IP3R) permeable to Ca2+ in the plasma membrane of OSNs (Okada et al. 1994; Lischka et al. 1999; Kaur et al. 2001). But thus far no evidence has been provided that ORs directly activate the PLC pathway to stimulate Ca2+ elevation. Therefore, although it is generally accepted that IP3 formation is promoted by odorants, whether it is a primary or secondary response to receptor activation remains unclear. One difficulty with examining signal transduction in primary OSNs is their heterogeneity with respect to ORs expressed, the differentiation state of the cells, and potentially, the heterogeneity of the underlying signaling mechanisms. In this study we utilize an olfactory cell line, Odora, as a model to directly explore the transduction events coupled to a single activated OR. The Odora cell line was generated by transformation of rat OE cells (Murrell and Hunter 1999). Like OSNs, Odora cells transfected with ORs elevate Cai in response to odorants (Murrell and Hunter 1999; Levasseur et al. 2003). However, unlike many other cell lines Odora cells do not require concurrent transfection of additional ‘promiscuous’ G proteins in order to couple the transfected receptor to endogenous signaling pathways (Wetzel et al. 1999; Krautwurst et al. 1998; Kajiya et al. 2001; Ivic et al. 2002). Thus, these OE-derived cells can be used to define transduction events that are directly stimulated by ORs and are therefore valuable in revealing the repertoire of endogenous signaling mechanisms that may be activated by ORs in OSNs, especially signals that might be dwarfed by cAMP and the primary transduction pathway. In the present study, we show that Odora cells transfected with OR U131 mount a Ca2+ response to odorants, but not to forskolin and isobutylmethylxanthine (IBMX), indicating that in Odora cells cAMP elevation is not sufficient by itself to increase cytosolic Ca2+ levels. Moreover, pharmacological studies of cells transfected with the U131 receptor demonstrate that appropriate odorants act via the PLC pathway to elevate Ca2+ without triggering intracellular Ca2+ release, even though control studies with ATP show that the pathway for PLC/IP3-mediated intracellular Ca2+ release is nonetheless intact. The odorant response requires extracellular calcium. One possible mechanism is that Ca2+ crosses the plasma membrane through the IP3R, and we show that all three isotypes of IP3R are present in Odora cells, with IP3R-2 and IP3R-3 present on the plasma membrane as well as intracellular membranes. Western blot analysis indicates that Odora cells share many relevant signaling components with

OE, and immunohistochemical staining localizes IP3Rs and IP3-cascade signaling components to OSNs. Hence, we have identified a unique IP3-mediated signaling pathway directly activated by ORs; this transduction mechanism is likely to provide another route for OR-mediated signaling in vivo, one that does not substitute for the cAMP signal but may modulate it or contribute additional information.

Materials and methods Materials The sources of chemicals used in this study are as follows: fura-2 AM and pluronic F-127 were from Molecular Probes, Eugene, OR, USA; Xestospongin C (XeC) was from Calbiochem, La Jolla, CA, USA; U73122, U73343 and IBMX were from RBI/Sigma, Natick, MA, USA; forskolin, as well as protease inhibitors including phenylmethylsulfonyl fluoride, pepstatin, leupeptin, chymostatin, soybean trypsin inhibitor and dithiothreitol were purchased from Sigma, St Louis, MO, USA; ATP was from Boehringer Mannheim, Penzberg, Germany; antipain was obtained from Worthington, Freehold, NJ, USA. Cell culture and transfection Odora cells (line AP-7) (kindly provided by Dr Dale Hunter, Boston, MA, USA) were cultured as previously described (Murrell and Hunter 1999). All cultures are grown for the same period of time under conditions that produce cells that are either ‘differentiated’ or ‘differentiated and transfected with receptor U131’. For expression of U131 and ACIII, cells were differentiated at 39C for 5 days and then transfected with an expression vector (pJG 3.6) encoding hemagglutinin-tagged (HA) OR U131 (HA-U131) (McClintock et al. 1997) (kindly provided by Dr Tim McClintock, Lexington, KY, USA) or ACIII in pCIS vector (Bakalyar and Reed 1990) (kindly provided by Dr R. Reed, Baltimore, MD, USA) using Superfect transfection reagent (Qiagen Inc., Valencia, CA, USA). In other reported studies, modification of the N-terminus of the OR did not affect ligand binding or downstream signaling (Flag-tagged OR I7) (Ivic et al. 2002). The transfection efficiency for Odora cells was estimated at between 10 and 30% by cotransfection with enhanced green fluorescence protein (pEGFP-C1, BD Biosciences, San Diego, CA, USA) in cells grown for IP3 assay as described below. Fortyeight hours after transfection, cells were either harvested for biochemical study or loaded with fura-2 AM for calcium imaging experiments. Untransfected cells grown under the same conditions were used as controls for imaging or biochemical studies. Odora cell membrane preparation for western blot Odora cells in the culture dish were washed twice with phosphatebuffered saline and then incubated with 2 mL EDTA for 5 min at room temperature. Cells were harvested by scraping and collected by centrifugation. The cell pellet was suspended in 500 lL buffer A (20 mM Tris-HCl, pH 7.4, 2 mM MgCl2, 1 mM EDTA, 0.5 mM dithiothreitol, 5 lg/mL leupeptin, 1 lg/mL each of aprotinin and pepstatin, 6 lg/mL each of chymostatin and antipain, and 100 lg/ mL phenylmethylsulfonyl fluoride), sonicated, and spun at 100 000 g for 30 min at 4C. The pellet was resuspended in buffer A without protease inhibitor but including 5% glycerol.


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Tissue membrane preparation for western blot Tissues from OE, whole brain minus cerebellum, cerebellum, liver, or lung were removed from adult Sprague-Dawley rats and then homogenized in 5 mL HEPES/saline buffer, pH 7.4 (145 mM NaCl, 5 mM KCl, 1.6 mM K2HPO4, 2 mM MgSO4, 20 mM HEPES, 7.5 mM D-glucose) containing 6 lg/mL chymostatin, 1 lg/mL each of leupeptin, pepstatin, aprotinin, antipain, and 100 lg/mL phenylmethylsulfonyl fluoride. After a 10 min spin at 1500 g, 4C, to remove blood and debris, the sample was spun at 40 000 g for 30 min at 4C. The membrane fraction was prepared by resuspending the pellet in the above HEPES/saline buffer without protease inhibitors, but including 1 mM EDTA and 5% glycerol. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot Protein samples were separated by denaturing polyacrylamide gel (range from 6 to 10%) electrophoresis in sodium dodecyl sulfate and then transferred to PolyScreen PVDF membrane (NEN, Boston, MA, USA). ACIII was run on a 6% gel for Fig. 5(a)(i) (see Results section). To better resolve the ACIII bands, a lower amount of OE protein was loaded on 8% gels and run at lower current in Fig. 5(a)(ii). For all, the membranes were first blocked in TBST buffer (50 mM Tris, pH 7.4 containing 200 mM NaCl and 0.1% Tween-20) containing 5% non-fat milk for 1 h at 37C, and then incubated with primary antibody for 1 h at room temperature followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for another 1 h at room temperature. In all cases, bound antibody was detected by chemiluminescence (NEN kit). Antibodies were obtained as follows and used at dilutions indicated: polyclonal rabbit antibodies to ACIII, Gaolf, Gai-2, Gao, PLCc-1 and PLCc-2, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA (1 : 200); IP3R-1, Alexis Biochemical, San Diego, CA, USA (1 : 1000); PLCb-1, b-2 and b-3, Santa Cruz Biotechnology Inc. (1 : 300); Gai-1 and Gai-3, Calbiochem (1 : 1000). Mouse monoclonal antibody to Na+/K+ ATPase a1 was from Santa Cruz (SC-21712, 1 : 200) and mouse monoclonal antibody to calnexin was from BD Transduction Laboratories, Lexington, KY, USA (1 : 1000). Recombinant Gai1–3 proteins were obtained from Calbiochem. Whole cell lysates from RAW 164.7, A-10 and Hela cells are from Santa Cruz. Secondary antibodies were HRP-goat anti-mouse IgG from Santa Cruz and HRP-goat anti-rabbit IgG from New England Biolabs, Beverly, MA, USA or Cell Signaling Technology, Beverly, MA, USA. Western blots with antibodies to IP3R-2 and IP3R-3 were performed by a different procedure and were carried out as described (Tanimura et al. 2000). Briefly, after protein transfer, the membranes were blocked for 2 h in ‘Block Ace’ solution (93.5 mM trisodium citrate, 6.2 mM citrate and 3% non-fat milk) containing 10% goat serum prior to incubation for another 2 h with affinity-purified rabbit anti-IP3R-2 antibody (120 ng/mL), or rabbit anti-IP3R-3 antibody (100 ng/mL). Membranes were washed and then were incubated with HRPconjugated goat anti-rabbit IgG for 1 h at room temperature. Biotinylated proteins on blots were incubated with avidin-HRP (2 lg/mL) (Pierce, Rockford, IL, USA) in TBST buffer for 1 h at room temperature and visualized by chemiluminescence. Dye loading and calcium imaging The isolation of primary mouse OSNs and loading with the calcium dye, fura-2 AM, were performed as described (Bozza and

Kauer 1998). For Odora cells, cultures grown on 22 · 40 mm coverslips were washed twice with normal Ringer’s solution (NR) (140 mM NaCl, 5 mM KCl, 10 mM glucose, 1 mM Na pyruvate, 1 mM CaCl2, 1 mM MgCl2 and 10 mM HEPES, pH 7.2), and then loaded with fura-2AM (4 lM) and 0.01% pluronic F-127 in NR for 1 h at 33C for fluorescence ratio imaging. Fluorescence images were collected every 4 s as described (Bozza and Kauer 1998) or using software from Axon Imaging Workbench 2.2 (Axon Instruments, Inc., Foster City, CA, USA). Occasionally certain high light conditions triggered a brief shutoff of the CCD camera, which then automatically resumed operation. These artifacts are represented by a gap in the data during the seconds when 340 nm and 380 nm values went to zero, as noted in appropriate figure legends. Mouse OSNs or Odora cells mounted in a chamber (RC22, Warner Instruments, Hamden, CT, USA) were constantly perfused with NR or stimuli delivered by a solenoid valveregulated perfusion array (Warner Instruments). Odorant mixtures and other reagents in Ringer’s solution were applied for 16–20 s unless otherwise indicated. The stock solutions of U73122, U73343, XeC and IBMX were made in dimethylsulfoxide. Forskolin stock was made in 95% ethanol. Fresh dilutions into Ringer’s solution were made each day. The final concentrations of dimethylsulfoxide and ethanol in these solutions are < 0.1% (v/v) and do not elicit any calcium signals. The percentage inhibition by pharmacological drugs in Fig. 3 (see Results section) is calculated by comparing the response in the presence of drugs to the control response immediately preceding it. Inositol-1,4,5-trisphosphate assay IP3 formation by Odora cells was measured with a [3H]IP3 assay kit (Amersham Pharmacia, Piscataway, NJ, USA) following the manufacturer’s protocol. In brief, Odora cells from five 100 mm plates (1.5 · 105 cells plated/dish, grown and differentiated as described above) were resuspended in NR and preincubated with 10 mM LiCl for 10 min at room temperature. Immediately after the first sample was removed (0 time point), the cells were stimulated with odorant Mix B (50 lM of each odor component in Table 1), or with ATP (100 lM), or NR for times indicated. Incubations were terminated by addition of an equal volume of 15% trichloroacetic acid. The formation of IP3 in the sample was assayed by displacement of [3H]IP3 bound to IP3-binding protein that was provided in the assay kit. Standard displacement curves were run with every assay. Biotinylation of intact Odora cells and avidin isolation of biotinylated proteins Intact Odora cells growing on 100 mm Petri dishes were biotinylated using the membrane-impermeant amino reagent, sulfo-NHS-biotin (Pierce), and membrane proteins (300 lg/ 150 lL) were isolated on avidin beads as described (Tanimura et al. 2000). Membrane samples were used for avidin purification immediately or stored at )80C until use. For avidin isolation, an aliquot of solubilized biotinylated membranes was saved as the ‘total extract’; the remaining sample was incubated with 100 lL prewashed avidin-beads (Sigma) and the resulting supernatant comprised the ‘avidin-unbound’ fraction; the ‘avidin-bound’ fraction was eluted from washed beads with 50 lL of electrophoresis sample buffer.


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Table 1 Chemical components of eight odorant mixtures (all odorant components were made at 50 lM in normal Ringer’s solution) Mix A

Mix B

Mix C

Mix D

Mix E

Mix F

Mix G

Mix H

Benzaldehyde N-Butyl acetate N-Pentyl-alcohol Propionic acid

benzyl alcohol N-butanol N-heptanoic acid N-heptanol N-hexanol N-nonanoic acid N-pentanoic acid

(+)-carvone cinnamaldehyde citral 2-ethyl fenchol (–)-limonene

cineole ethyl-n-butyrate hexyl acetate isobutyraldehyde (1R)-(+)-a-pinene

acetophenone citralva (+/–)citronellal eugenol geraniol hedione (+/–)menthone phenyl ethyl alcohol

ethyl vanillin isopentanoic acid lilial lyral phenyl ethyl amine triethylamine

benzyl alcohol N-butanol 2-ethyl fenchol N-hexanol N-heptanol isopropanol 1-pentanol 3-pentanol

benzyl acetate N-butyl acetate ethyl acetate N-hexyl acetate methyl acetate N-pentyl acetate propyl acetate

Immunoprecipitation of inositol-1,4,5-trisphosphate receptor type 3 Direct immunoprecipitation of IP3R-3 from biotinylated cell membranes was carried out according to the published protocol (Tanimura et al. 2000). Immunohistochemistry Postnatal-day-18 Sprague-Dawley rats were perfused with 4% paraformaldehyde. Nasal tissue was dissected out, rinsed, and cryoprotected in 30% sucrose overnight at 4C and then frozen in OCT mounting medium (Sakura Finetek, Inc. Torrance, CA, USA). Then 12-lm sections of olfactory tissues were cut on a cryostat. For staining, the slides were washed with phosphate-buffered saline, incubated with blocking solution (phosphate-buffered saline containing 5% normal donkey serum, 0.2% Triton X-100) and then with primary antibodies diluted in blocking solution for 1 h at room temperature plus overnight at 4C. Slides were then washed with phosphate-buffered saline and incubated for 1 h at room temperature with secondary antibodies Alexa 488-conjugated donkey anti-rabbit IgG (Molecular Probes) or Cy3-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). After washing with phosphate-buffered saline, slides were dehydrated through ethanol and xylene, and then coverslipped in Permount (Sigma). Primary antibodies used in immunohistochemistry are the same as the ones used for western blots except as follows: rabbit antibody reactive with all three types of IP3R, Calbiochem no. 407143 (1 : 200); goat anti-IP3R-1 and goat anti-IP3R-2, Santa Cruz (1 : 50); rabbit anti-IP3R-3, Affinity BioReagents, Golden, CO, USA (1 : 100); rabbit anti-Gai-2, Calbiochem (1 : 50); rabbit antibody to neural cell adhesion molecule (NCAM), Chemicon International, Inc., Temecula, CA, USA (1 : 200). Goat polyclonal antibody to OMP was kindly provided by Dr Frank Margolis (Baltimore, MD, USA). To identify OSNs, all rabbit antibodies to proteins of interest were co-incubated with goat-anti-OMP antibody. When goat antibodies were used, OSNs were marked with rabbit anti-NCAM antibody. Negative controls were run for all experiments by omitting primary antibody. For all antibodies, specificity of binding was established on western blots by preincubation of the antibody with the cognate peptide. As an additional control we also have determined that there is no cross-reaction between the non-paired primary and secondary antibodies in double label experiments. Fluorescence images were obtained by fluorescence microscopy with a Zeiss imaging system using Mac Openlab 3.5.2

software. The confocal fluorescence image of PLCb-2 was visualized with a Leica TCS SP2 AOBS system. Optical images were prepared for publication with Adobe Photoshop 7.0.

Results

Odora cells transfected with odorant receptor U131 become responsive to odorants Differentiated Odora cells transfected with ORs target functional receptor to the plasma membrane and respond to odorants (Murrell and Hunter 1999). To identify the signal transduction pathways activated by OR in these cells, here we first tested Odora cells that had been transfected with receptor U131 for responsiveness to eight odorant mixtures (Table 1) by utilizing calcium fluorescence ratio-imaging of individual cells. Only odorant Mix B is able to elevate Cai, indicating selectivity as previously shown for 7- and 9-carbon fatty acids (Murrell and Hunter 1999) (Fig. 1a, left panel). Most cells do not respond to high K+ solution. Untransfected cells do not respond to Mix B (data not shown). Unexpectedly, chemicals that activate the cAMPmediated signal transduction pathway fail to elevate Cai in Odora cells; forskolin (10 lM), an activator of AC and IBMX (500 lM), an inhibitor of phosphodiesterase are ineffective (Fig. 1a, right panel). This differs from primary mouse OSNs, where forskolin (Fig. 1b, left panel) and IBMX (Fig. 1b, right panel) cause a robust Cai increase. Further, in Odora cells it appears that the endogenous level of ACIII is low, limiting activation of the AC/cAMP pathway. Overexpression of ACIII restores the ability of forskolin to elevate Cai, indicating that the cAMP/cyclic nucleotide-gated channel (CNGC) system is intact in Odora cells (Fig. 1c). Odor-induced calcium elevation requires extracellular Ca2+ and acts through phospholipase C/inositol-1,4,5trisphosphate pathway Further examination of the Cai response to odorants in receptor U131-expressing Odora cells showed that the odorant response requires extracellular Ca2+ (Fig. 2a), and is abolished when extracellular Ca2+ is removed (10 cells,



Fig. 1 Odorant responses of Odora cells transfected with HA-U131 receptor and primary olfactory sensory neurons (OSNs). (a) U131transfected Odora cells respond to odorant Mix B but not to drugs that activate cAMP formation. Calcium responses were monitored in Odora cells transfected with U131 receptor and stimulated with eight different odorant mixtures (see Table 1) and KCl (K, 100 mM), in the order indicated (left panel). Right panel, forskolin (FORS, 10 lM) and isobutylmethylxanthine (IBMX: 500 lM) do not induce an intracellular Ca2+ (Cai) increase in U131-transfected Odora cells that respond to odorant Mix B. (b) Mouse primary OSNs elevate Cai when the AC/ cAMP pathway is activated. Mouse OSNs show Cai responses to high

concentrations of KCl (100 mM), forskolin (10 lM) (left panel) and varied concentrations of IBMX (right panel). IBMX concentrations: 1 lM (IBMX1); 5 lM (IBMX5); 50 lM (IBMX50); 250 lM (IBMX250) and 500 lM (IBMX500). (c) Overexpression of adenylyl cyclase III (ACIII) rescues the response to forskolin in Odora cells. Odora cells transfected with ACIII show a robust Cai increase to repeated application of forskolin (10 lM). They also respond to ATP (10 lM) and occasionally to high K+ solution, as seen here. The gaps show removal of a camera artifact. All traces are representative of at least three independent experiments.


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Fig. 2 Odorant responses require extracellular Ca2+ and are modulated by phospholipase C (PLC). Odora cells transfected with U131 receptor were assayed by calcium imaging. (a) Removal of extracellular Ca2+ blocks the odorant response. Odora cells respond to repeated presentations of odorant Mix B (B). Stimulation with Mix B in extracellular medium containing 1 mM EGTA and no added Ca2+ (0Ca) fail to elicit a Cai response. The response to B returns when extracellular Ca2+ is restored. (b) Blockade of PLC or inositol-1,4,5trisphosphate receptor (IP3R) inhibits the odor response. PLC inhibitor, U73122 (10 lM) (B/U122) reversibly blocks the Mix B response, as does application of Xestospongin C (XeC: 1 lM) (B/XeC). ATP here at 50 lM gives a very robust Ca2+ response. These traces are representative of three or more independent experiments.

n ¼ 3). Removal of extracellular Na+ does not block the calcium response, as would have been the case if Ca2+ entry occurred via Na+/Ca2+ exchange secondary to Na+ influx (10 cells; n ¼ 3; not shown). Moreover, Fig. 2(b) shows that PLC inhibitor U73122 fully blocks the odorant response (13 cells, n ¼ 3). The response recovers but the percentage of pharmacological inhibition cannot be quantitated readily because the odorant response itself sometimes desensitizes (Figs 2a and 3a). U73343, an inactive analogue of U73122 that is frequently used as a control compound, essentially gives no inhibition (6 cells, n ¼ 3; not shown). Further, XeC, an antagonist of IP3R (Gafni et al. 1997), also reversibly blocks the odor-stimulated Cai signals (6 cells, n ¼ 3)

Fig. 3 ATP releases intracellular Ca2+ through phospholipase C (PLC) in Odora cells. (a) ATP releases intracellular calcium. Odora cells transfected with U131 receptor elevate intracellular Ca2+ (Cai) in response to both odorant Mix B and ATP (10 lM), but not to high K+ solution. Removal of extracellular Ca2+ (0Ca) does not eliminate the response to ATP. Note: The second and third applications of ATP were 4 s longer than the first application of ATP (12 s). (b) ATP response is inhibited by blockade of inositol-1,4,5-trisphosphate receptor (IP3R) and PLC. Untransfected Odora cells were imaged for Cai responses to ATP (10 lM in all cases) and drugs were evaluated in Ringer’s solution containing ATP plus either 1 lM XeC (ATP/XeC), 10 lM U73122 (ATP/U122), or 10 lM U73343 (ATP/U343). XeC reversibly inhibits the ATP response. In addition, U73122 effectively blocks the ATP response, whereas U73343 has no effect. In our experiments, the effects of U73122 appear to be only partially reversible. A gap is present in (a) where a camera artifact was removed. These traces are representative of three or more independent experiments.

(Fig. 2b). These results clearly implicate both PLC and IP3R in the calcium signaling through OR U131. To establish that the machinery for release of intracellular Ca2+ through PLC and IP3 is intact, we stimulated Odora cells with 10 lM ATP (Fig. 3), which elicits a robust Cai response, seen in more than 90% of Odora cells. The ATP response (purinergic receptor P2y2 type, K. Washburn and B. Talamo, personal communication) occurs in the absence of extracellular Ca2+, evidence that Ca2+ is released from



internal calcium stores (13 cells, n ¼ 6) (Fig. 3a). Moreover, the ATP response also appears to be PLC/IP3-mediated (Fig. 3b). Application of U73122 reduces the amplitude of the ATP response by 97% (17 cells, n ¼ 5), whereas U73343 does not alter the Ca2+ response to ATP (10 cells, n ¼ 5). XeC also dramatically and reversibly inhibits the ATP response by 90% (8 cells, n ¼ 3). Thus, although PLC and IP3R participate in both ATP and odorant responses in Odora cells, the pathways activated by ATP and odorants mobilize Ca2+ through different routes. Odorant stimulates inositol-1,4,5-trisphosphate formation in Odora cells To further confirm the participation of the PLC/IP3R pathway, we measured IP3 formation in Odora cells stimulated with ATP or odorants. Unlike ATP, which stimulates nearly all of the cells, 24% of the Odora cells (36 out of 150 cells tested) respond to odorants, as expected based on the transfection efficiency. Figure 4 shows that values for IP3 formation elicited by ATP or Mix B are elevated at all time points of stimulation and significantly different from control (ANOVA, p < 0.005). IP3 rises within 15 s and remains elevated. In control experiments with untransfected cells (Fig. 4), no change in IP3 concentration is observed in response to normal Ringer (NR) or in response to odorant Mix B (MixB-CTL). This excludes possible non-specific effects of odorants or Ringer’s solution on the stimulation of IP3 formation.

Fig. 4 ATP and odorant mix B each stimulate inositol-1,4,5-trisphosphate (IP3) accumulation in odora cells. IP3 accumulation was measured in untransfected Odora cells stimulated with 100 lM ATP (ATP) or normal Ringer’s solution (NR). ‘MixB-CTL’ shows stimulation of untransfected cells with Mix B. The ‘MixB’ trace shows stimulation of U131-transfected Odora cells with Mix B. Stimulation of untransfected Odora cells by ATP or stimulation of U131-transfected cells by Mix B enhances accumulation of IP3, whereas neither NR nor Mix B elevates IP3 above baseline in untransfected cells. For untransfected cells, the basal levels of IP3 are 4.8 ± 0.9 pmol/plate (n ¼ 10); for transfected cells, the basal levels of IP3 are 6.5 ± 0.2 pmol/plate (n ¼ 3). 6 pmol of IP3 corresponds to about 2033 dpm displaced from the binding protein (100% bound ¼ 7000 dpm). (Values are the means ± SE for three or more independent experiments. Data were normalized to values at 0 time and analyzed by single factor ANOVA, p < 0.005).

Odora cells resemble olfactory epithelium in expression of olfactory relevant signaling proteins We compared the expression of olfactory signaling proteins in Odora cells and OE to establish that Odora cells are a relevant model for investigating signaling mechanisms underlying the activation of an identified OR (Fig. 5). Western blot analysis shows that Odora cells do express ACIII and Gaolf (Fig. 5a), the two key components in the cAMP pathway. Overexpression of U131 in Odora cells does not change the expression level of either ACIII or Gaolf. ACIII is detected both as the native, unglycosylated form (band above 105 kDa marker) and higher MW glycosylated forms as previously reported (Bakalyar and Reed 1990; Wei et al. 1996) in rat OE and Odora cells (Fig. 5a). These data are consistent with previous immunohistochemical demonstrations of ACIII and Gaolf (Murrell and Hunter 1999). Gai types 1–3 as well as Gao are expressed in both Odora and OE (Fig. 5b). The low level of Gai-2 in OE is consistent with immunohistochemistry showing Gai-2 in only a subset of rat OSNs (Wekesa and Anholt 1999). In contrast, Gaq is not detected in either OE or Odora cells (data not shown). Both Gai and Gao heterotrimers activate various PLC isozymes (Dorn et al. 1997). PLCb-1, b-2 and b-3 are all detected in both Odora cells and OE (Fig. 5c). PLCb-4 is not detectable in either (data not shown). Only the 100 kDa form of PLCb-1 is observed in Odora cells, but two alternative forms of PLCb-1 at apparent MWs of 100 kDa and 150 kDa are detected in rat OE; these two variants are commonly observed in freshly isolated tissues (Tang et al. 2000). Of the two PLCc isoforms only PLCc-1 is detected in both OE and Odora cells (Fig. 5c); PLCc-2 is not observed (data not shown). In addition, overexpression of U131 does not change the expression levels of any of these G-proteins and PLC isoforms (data not shown). Inositol-1,4,5-trisphosphate receptors are present in the plasma membrane of Odora cells The calcium signaling studies implicate IP3R in odormediated signal transduction. We first looked for expression of the candidate IP3R isotypes by western blotting. Figure 6(a) shows that Odora cells express all three types of known IP3R, with apparent MWs of 240–260 kDa. Compared to rat OE, Odora cells appear to express a higher level of IP3R-3, and somewhat lower levels of IP3R-1 and IP3R-2. For IP3R to directly mediate influx of extracellular Ca2+ through its cation channel, it must be present on the plasma membrane. To detect surface IP3Rs, we selectively biotinylated externally located amino group-containing macromolecules on intact Odora cells using the membrane-impermeant amino reagent, sulfo-NHS-biotin. Trypan blue exclusion confirmed that more than 95% of the sulfo-NHS-biotin treated Odora cells were viable (data not shown). Western blots of the biotinylated proteins with IP3R subtype-specific


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Fig. 5 Odora cells express olfactory-relevant signaling proteins. Equal amounts of membrane proteins (20 lg) isolated from rat olfactory epithelium (OE), brain or cerebellum, Odora cells or U131-transfected Odora cells (U131) were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting. (a) Odora cells express both adenylyl cyclase III (ACIII) and Gaolf. Levels are much lower than those in OE. Transfection with U131 receptor apparently does not change the expression level of ACIII and Gaolf (i and iii). To better show the expression of ACIII in OE, another blot with a lower

amount of OE membrane protein (10 lg) is shown along with a control A-10 whole cell lysate (ii). Both glycosylated and unglycosylated forms of ACIII are seen. (b) Odora cells and rat OE also express Gao and isoforms of Gai. Recombinant Gai-1, 2 and 3 (r-Gai) were run as controls. (c) Odora cells express various forms of phospholipase C (PLC), which are also found in OE. Membranes from rat cerebellum, rat brain, RAW 164.7 whole cell lysates (RAW164) were used as positive controls for blots with antibodies to PLC isoforms.

antibodies establish that a portion of IP3R-2 and IP3R-3 is present at the cell surface as indicated by pull-down in the avidin-bound fractions (Fig. 6b). For IP3R-1, the results were inconclusive, as a very faint band was observed in the avidinbound fraction from biotinylated or non-biotinylated cells, possibly representing non-specific background binding to avidin (data not shown). To confirm the specificity of cell surface labeling, we verified that the biotinylation protocol only labels plasma membrane proteins and does not label intracellular sites in intact Odora cells (Fig. 6c). Na+/K+ ATPase, a plasma membrane marker, is detected only in avidin-bound fractions (left panel) whereas calnexin, an abundant 90 kDa integral membrane protein located in the

endoplasmic reticulum (ER), is not detected in the avidinbound fractions (right panel). Next, to confirm that the biotinylated surface protein pulled down by the avidin beads is indeed IP3R, we immunoprecipitated IP3R-3 and probed for biotin. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the same band is labeled by the IP3R-3 antibody and avidin-HRP. Localization of phospholipase C/inositol-1,4,5-trisphosphate cascade signaling proteins in olfactory sensory neurons OE contains more than four types of cells. If the signaling components of the PLC/IP3 pathway in the OE are relevant to



Fig. 6 Inositol-1,4,5-trisphosphate receptors type 2 (IP3R-2) and type 3 (IP3R-3) are present in the plasma membrane of Odora cells. (a) All three types of IP3Rs are expressed in olfactory epithelium (OE) and Odora cells. Membrane proteins (30 lg each) isolated from Odora cells, OE and rat tissues enriched in an IP3R subtype (as controls) were analyzed by immunoblotting with IP3R subtype-specific antibodies. (b) IP3R-2 and IP3R-3 are detected on the external surface of Odora cells. Membrane proteins isolated by avidin pull-down from externally biotinylated Odora cells were analyzed by immunoblotting with IP3R-2 or IP3R-3 specific antibody. From left to right, lane 1: total extract; lane 2: avidin-bound fraction; lane 3: avidin-unbound fraction. Based on the original amount of total extract, the relative amounts of samples loaded in lanes 1, 2, and 3 were 1 : 4 : 1. Both IP3R-2 and IP3R-3 are detected in avidin-bound membrane fractions. (c) Biotinylation occurs only at the cell surface. Left panel: A plasma membrane

marker, Na+/K+ ATPase, is present only in the biotinylated avidinbound fraction. Hela whole cell lysate (Hela-WCL) from Santa Cruz was used as a positive control. Right panel: Calnexin, an intracellular protein, is not biotinylated and is present only in the avidin-unbound fraction. A blot of membranes from externally biotinylated cells probed with anti-IP3R-2 antibody as in (b) (not shown) was stripped and reblotted with antibody to calnexin. (d) Imunoprecipitation of biotinylated IP3R-3. Membrane proteins isolated by avidin pull-down from externally biotinylated Odora cells were treated with rabbit anti-IP3R-3 antibody and the resulting immunoprecipitates (IPP) and control sample were analyzed by blotting with the same anti-IP3R-3 antibody (left panel) or avidin-HRP (right panel). From left to right, lane 1: rat lung membrane protein (control); lane 2: total extract; lane 3: immunoprecipitates (IPP). Data shown in all figures are representative of at least three independent experiments.


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Fig. 7 Immunohistochemical analysis of signaling proteins of phospholipase C/inositol-1,4,5-trisphosphate (PLC/IP3) pathway in olfactory epithelium (OE). Immunostaining was performed on rat OE sections. (a) Localization of Gai/o in olfactory sensory neuron (OSN) zone and axon bundles. Antibody to olfactory marker protein (OMP) (lower panel) was co-stained with antibody to (i) Gai-1, (ii) Gai-2, (iii) Gai-3, and (iv) Gao. (b) PLCb-2 is localized in ciliary and OSN cell layers and axon

bundles by confocal microscopy. (i) Green fluorescence: PLCb-2. (ii) Red fluorescence: OMP. (iii) Overlay: yellow image shows co-localization of PLCb-2 and OMP. (c) PLCb-3 (i) and OMP (ii) staining identify PLCb-3 in OSN cell body and in axon bundles. Negative controls with primary antibody omitted were run for every condition and showed no specific staining signals. Scale bar represents 100 lm. Arrows indicate ciliary zone; axon bundles, AB; olfactory sensory neuron layer, OSN.



antibody staining shows that IP3R-2 co-localizes with neural cell adhesion molecule (NCAM) in the neuronal zone and in nerve bundles (Fig. 8b). The ciliary zone also stains for IP3R-2, although NCAM is not distributed into cilia. Of the other two types of IP3R, IP3R-1 appears to weakly stain only axon bundles, whereas IP3R-3 is mostly seen in axon bundles and cell bodies in the deeper layer of OE (data not shown). Discussion

Fig. 8 Immunohistochemical analysis of inositol-1,4,5-trisphosphate receptors (IP3Rs). (a) Staining with antibody reactive with all three types of IP3R (i) labels IP3R1–3 in ciliary layer, olfactory sensory neuron (OSN) layer and axon bundles (AB). (ii) Shows co-staining of olfactory marker protein (OMP). (b) Specific antibody for IP3R-2 (i) labels ciliary layer, OSN layer and AB. Neural cell adhesion molecule (NCAM) staining (ii) labels only sensory neurons and axon bundles, but not cilia or cell surface. No specific staining was observed in the absence of primary antibody. Scale bar represents 100 lm.

olfactory receptor pathways, they must be localized to OSNs. Immunohistochemical analysis showed that Gai-1 and Gai-2 co-localize with OMP in the ciliary and dendritic zones of the rat OE (Fig. 7a). Cell bodies of OSNs also stain strongly for Gai-1 whereas Gai-3 is only present at low levels. All three forms of Gai as well as Gao are strongly represented in axon bundles. PLC isoform PLCb-2 (confocal images, Fig. 7b) co-stains with OMP and is localized to ciliary and sensory neuron zones as well as axon bundles, whereas PLCb-3 (Fig. 7c) stains cell bodies of OSN and axon bundles. Some components are not present in OSNs: PLCb-1 is not detected in OE, but is present in cells of respiratory epithelium, whereas PLCc-1 can only be detected in nerve bundles, where it appears to be localized in glial cells (data not shown). The positive band on western Blot for PLCb-1 in OE (Fig. 5c) may be due to the presence of respiratory epithelium in dissected ‘OE’ tissue. Immunohistochemical staining of IP3Rs with a pan-IP3R polyclonal antibody that recognizes all three types of IP3Rs, in conjunction with antibody to OMP (Fig. 8a), shows IP3Rs at the surface of the OE in the ciliary layer, the sensory neuron zone and in axon bundles. IP3R subtype-specific

The present study provides strong evidence that ORs can directly mediate odor stimulation of the PLC/IP3 pathway in U131-expressing Odora cells. Other studies have reported that mice deficient in Gaolf or a subunit of the CNGC do not respond to odorants reported to activate the IP3 pathway (Brunet et al. 1996; Belluscio et al. 1998), raising the possibility that the IP3 pathways may be activated secondarily to cAMP or that they do not directly transduce odor detection and recognition, but rather participate in other biological events. A possible modulatory role for alternative signaling pathways is highlighted by recent studies indicating that OR signaling is dynamically regulated (Duchamp-Viret 1999, 2000; Hallem et al. 2004). Further, a mechanism for OR-mediated targeting of axons to glomeruli has not yet been demonstrated and potentially could involve alternative pathways such as PLC/IP3. The presence in olfactory axons of all three isoforms of Gai and Gao, as well as PLCb-2, -3 and IP3R1-3 along with OR is consistent with this possibility, although activation of signaling in the axon and identification of the local ligand for OR has not been reported. Nonetheless, there is direct evidence for PLC signaling in a subpopulation of sensory cells in the OE that responds to odorant, expresses components of the PLC pathway and fails to respond to forskolin, confirming the presence of cAMPindependent odorant signaling pathways (Elsaesser et al. 2005). In Odora cells the level of ACIII is inadequate for Cai elevation in response to odorants, revealing OR coupling to PLC and allowing us to explore its properties. Expression of endogenous signaling proteins such as Gao, Gai, and PLC in these cells provides a possible molecular basis for coupling of OR to the PLC/IP3 pathway. Activation of PLC by odorants is further established by biochemical measurements of IP3 formation in U131-transfected cells and by inhibition of the Cai response by blockers of PLC and IP3R, U73122 and XeC, respectively. Odorant activates the PLC/IP3 pathway and calcium elevation, dependent on extracellular calcium in Odora cells. The absence of a Cai response to odorant when extracellular Ca2+ is removed is not due to defective coupling of IP3 to intracellular Ca2+ release, since ATP does activate a P2y receptor through PLC to release intracellular Ca2+. The machinery for activating these pathways is present in sensory neurons as well as in the


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model Odora cells, as shown by immunocytochemistry and western blot. In the OE, IP3-cascade proteins are stained at the ciliary surface, although we are not able to distinguish between possible localization in cilia versus microvilli; they also are found in cell bodies of OSN and axon bundles. A variety of explanations could account for differences in expression levels in Odora cells and in OE. Possibilities include the heterogeneity of OSNs as well as microvillar cells in the OE tissue, varying expression in different compartments of the cell or different subpopulations of OSNs, different stages of maturation in OSNs that may be captured in the Odora line, or differences in the immortalized cell line. Information about signaling characteristics of specific ORs in olfactory cells is limited. Functional expression of OR I7 and OR 14–40 has been reported in Odora cells (Levasseur et al. 2003), but the signaling pathways are not known. Native mouse OSNs respond to short chain fatty acids (Malnic et al. 1999; Hamana et al. 2003) or to odorant Mix B (Bozza and Kauer 1998), although the signaling cascades have not been examined. In the case of the microvillar sensory cells, the odorant signaling pathway through PLC is known, but the identity of the OR is not (Elsaesser et al. 2005). Although the C-terminus of OR appears to be important for coupling to particular G proteins (Katada et al. 2004), the specificity and selectivity of individual receptors for coupling with downstream PLC or AC pathways and the role of the G protein/downstream cascade in shaping the response (Shirokova et al. 2005) needs further exploration in the context of signaling components actually expressed in particular OSNs. Various explanations may account for differences in the PLC-mediated responses to ATP and odorants in Odora cells. All three types of IP3R are present in Odora cells and external biotinylation shows that IP3R-2 and 3 are represented at the cell surface as well as internally, raising the possibility that various isotypes of IP3Rs in Odora cells might differentially couple with G-protein coupled receptors (GPCRs) such as P2y and ORs to mediate mobilization of Ca2+ through distinctive pathways. Selective coupling has been reported in sympathetic neurons where bradykinin receptor B2 and muscarinic receptor (M1AChR) both activate PLC-b, but only IP3 formed by bradykinin causes intracellular release of calcium from the ER (Delmas et al. 2002). The authors suggest that differential coupling is mediated through separate specialized signaling microdomains in the membrane. Odorant activation of the PLC/IP3 pathway in primary OSNs and ciliary preparations (which may include microvilli) has been established, but the requirement for OR or independence of the cAMP pathway has not been examined until the recent demonstration of cAMP-independent microvillar cell responses to odorant (Elsaesser et al. 2005). Odorants may elicit cross-talk between AC/cAMP and

PLC/IP3 pathways as reported (Muller et al. 1998; Vogl et al. 2000). In Odora cells, forskolin or IBMX stimulation does not give a calcium signal, but a role for cAMP in PLCdependent odorant signaling cannot be ruled out. It is possible that an odorant-stimulated rise in cAMP is a trigger for coupling of the OR to PLC, even though cAMP levels are too low to stimulate Ca2+ entry in Odora cells. This shift of coupling from AC to PLC occurs for some other GPCRs via activation of protein kinase A (Daaka et al. 1997; Lawler et al. 2001). Activation of PLC classically results in Ca2+ release from internal stores via an IP3R on the ER (Putney. 1999) as for ATP. The absence of this response and the requirement for extracellular calcium in Odora cells is most easily explained by postulating that Ca2+ enters through the plasma membrane. Recent reports show that IP3R can mediate calcium entry independent of release of internal calcium stores (Tong et al. 2004; van Rossum et al. 2004). We hypothesize two possible mechanisms for IP3R-dependent, odor-activated Ca2+ entry in Odora. First, Ca2+ may enter through an IP3R channel on the plasma membrane. Previous investigators localized an unidentified subtype of IP3R to the ciliary layer of rat OE and to dendrites (Cunningham et al. 1993) and to the plasma membrane of lobster OSN by light and electron microscopy immunostaining (Munger et al. 2000). IP3R-3 appears to be localized in the subapical regions as well as soma and axon of microvillar cells (Elsaesser et al. 2005). Odorant was shown to release internal calcium stores in microvillar sensory cells, but Ca2+ influx mechanisms were not explored. A direct role for IP3R in Ca2+ influx has been reported in various cells (Ueda et al. 1996; Kiselyov et al. 1997; Ueda and Inoue 2000; Barrera et al. 2004) and IP3Rs are located at the plasma membrane in several different cell types (Tanimura et al. 2000; Barrera et al. 2004). Additional work will be needed to explore the role of IP3Rs in olfactory calcium signaling and examine the possibility that local release of Ca2+ from the ER via IP3R stimulates influx in the absence of a global change in cytosolic Ca2+, as suggested by Berridge (Berridge 2004). A second possibility is that IP3R mediates Ca2+ entry through interaction with a different channel on the plasma membrane, such as a TRP channel as shown in other systems (Kiselyov et al. 1998; Boulay et al. 1999; Ma et al. 2000; Kanki et al. 2001; Brann et al. 2002). Channels of the TRP family are involved in many examples of sensory transduction (Hardie and Minke 1992; Caterina et al. 1997, 1999; Colbert et al. 1997; Liman et al. 1999; McKemy et al. 2002; Perez et al. 2002), including the vomeronasal system, where TRPC2 appears to interact with IP3R-3 (Brann et al. 2002) via the PLC pathway (Berghard and Buck 1996; Wang et al. 1997; Taniguchi et al. 2000; Lucas et al. 2003). PLC and IP3R also complex or scaffold with channels such as TRPC and other components of the Ca2+ signaling pathway (Tang et al. 2000; Li and Montell 2000; Lockwich et al. 2001). It should be



considered that IP3R may trigger Ca2+ entry through the plasma membrane via conformational changes that are independent of Ca2+ release from the ER pool or ion permeability functions of the IP3R (van Rossum et al. 2004). In summary, we show that OR U131 can directly activate PLC and Cai signaling in the absence of cAMP-mediated Ca2+ signals, and for the first time demonstrated that an identified OR can mobilize Ca2+ elevation via endogenous PLC signaling pathways, dependent on the presence of extracellular Ca2+. This likely indicates that Ca2+ enters across the plasma membrane. Further, our study localizes G proteins and PLC-cascade signaling proteins to the zones of the OE responsible for odorant responses – the cilia/ microvillar surface, cell bodies and axons of OSNs – and identifies both IP3R-2 and IP3R-3 at the surface of Odora cells. These studies provide the functional basis for identifying which G proteins couple to OR to activate this pathway and for characterization of the molecular entities and mechanisms for odorant-activated Ca2+ elevation. Extension of these studies to OSNs will allow the evaluation of the roles of these systems in vivo. Acknowledgements We thank Dr Karina Meiri and Dr John Kauer for their helpful comments on the manuscript. This work is supported in part by grants DARPA DAAK 60–97-K-950 and NIH R55DC 05229 to BRT.

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