A partially purified preparation of isolated chemosensory cilia from the olfactory epithelium of the bullfrog, Rana catesbeiana

Anholt, Robert R. H.; Aebi, U.; Snyder, S. H.

doi:10.1523/jneurosci.06-07-01962.1986

Cited by 107 publications

(69 citation statements)

References 18 publications

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“…Olfactory cilia from 30 mice were isolated by the calcium shock method (35). The cilia were treated with hypotonic solution (36) followed by ultracentrifugation over a sucrose gradient to isolate membranes.…”

Section: Methodsmentioning

confidence: 99%

ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification

Stephan

Shum²,

Hirsh³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

305

377

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For vertebrate olfactory signal transduction, a calcium-activated chloride conductance serves as a major amplification step. However, the molecular identity of the olfactory calcium-activated chloride channel (CaCC) is unknown. Here we report a proteomic screen for cilial membrane proteins of mouse olfactory sensory neurons (OSNs) that identified all the known olfactory transduction components as well as Anoctamin 2 (ANO2). Ano2 transcripts were expressed specifically in OSNs in the olfactory epithelium, and ANO2::EGFP fusion protein localized to the OSN cilia when expressed in vivo using an adenoviral vector. Patch-clamp analysis revealed that ANO2, when expressed in HEK-293 cells, forms a CaCC and exhibits channel properties closely resembling the native olfactory CaCC. Considering these findings together, we propose that ANO2 constitutes the olfactory calciumactivated chloride channel.Anoctamin ͉ cilia ͉ olfaction ͉ signal transduction ͉ TMEM16B

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Section: Methodsmentioning

confidence: 99%

ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification

Stephan

Shum²,

Hirsh³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

305

377

View full text Add to dashboard Cite

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“…Isolation of Olfactory Cilia-Partially purified preparations of chemosensory cilia from mouse olfactory epithelium were isolated with the calcium shock method (10).…”

Section: Methodsmentioning

confidence: 99%

G Protein-coupled Receptor Kinase 3 (GRK3) Gene Disruption Leads to Loss of Odorant Receptor Desensitization

Peppel¹,

Boekhoff²,

McDonald³

et al. 1997

Journal of Biological Chemistry

149

115

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G protein-coupled receptor kinases (GRKs) 2 and 3 (␤-adrenergic receptor kinases 1 and 2 (␤ARK1 and -2)) mediate the agonist-dependent phosphorylation and uncoupling of many G protein-coupled receptors. These two members of the GRK family share a high degree of sequence homology and show overlapping patterns of substrate specificity in vitro. To define their physiological roles in vivo we have generated mice that carry targeted disruption of these genes. In contrast to GRK2-deficient mice, which die in utero (Jaber, M., Koch, W. J., Rockman, H., Smith, B., Bond, R. A., Sulik, K. K., Ross, J. JR., Lefkowitz, R. J. Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974 -12979), GRK3 deletion allows for normal embryonic and postnatal development. GRK3 is expressed to a high degree in the olfactory epithelium, where GRK2 is absent. Here we report that cilia preparations derived from GRK3-deficient mice lack the fast agonist-induced desensitization normally seen after odorant stimulation. Moreover, total second messenger (cAMP) generation in these cilia preparations following odorant stimulation is markedly reduced when compared with preparations from wildtype littermates. This reduction in the ability to generate cAMP is evident even in the presence of nonodorant receptor stimuli (GTP␥S and forskolin), suggesting a compensatory dampening of the G protein-adenylyl cyclase system in the GRK3 (؊/؊) mice in the olfactory epithelium. These findings demonstrate the requirement of GRK3 for odorant-induced desensitization of cAMP responses.Many G protein-coupled receptors (GPCRs) 1 show diminished ability to signal and couple to G proteins after prolonged or repeated agonist stimulation. This phenomenon, referred to as agonist-mediated desensitization, occurs very rapidly and is initiated via receptor phosphorylation by G protein-coupled receptor kinases (GRKs) that serve to uncouple the receptor from its G protein (1).Whereas the function of these proteins has been mostly studied in vitro and in tissue culture the physiological relevance of the mechanisms initiated by them have just begun to be explored. While there is some evidence of substrate specificity among the different members of the GRK family, most show activity toward a wide variety of agonist-occupied receptors in vitro. This, in addition to their ubiquitous tissue expression, has made it difficult to precisely determine the role of the GRKs in vivo. To clarify the physiological role of the individual members of this family, we have generated mice that carry targeted disruptions of the GRK2 or GRK3 (␤ARK2) genes. GRK2 deletion is embryonically lethal as homozygous mice die in utero before gestational day 15.5 of severe cardiac malformations (2). Whereas GRKs 2 and 3 show 81% amino acid identity (3) and an overlapping pattern of tissue expression (4), GRK3 apparently is not able to compensate for the loss of GRK2 in embryogenesis. In most tissues examined GRK2 is the predominant form. However, in the olfactory epithelium GRK2 is virtually a...

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“…Isolation of Olfactory Cilia-Olfactory cilia preparations were obtained using the calcium-shock method (21,22). Briefly, after a short wash of the olfactory epithelium in ice-cold saline solution (120 mM NaCl, 5 mM KCl, 1.6 mM K 2 HPO, 25 mM NaHCO 3 , 7.5 mM glucose, pH 7.4), the tissue was subjected to Ringer's solution containing 10 mM calcium and gently stirred for 5 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Odorants Selectively Activate Distinct G Protein Subtypes in Olfactory Cilia

Schandar

Laugwitz

Boekhoff

et al. 1998

Journal of Biological Chemistry

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Chemoelectrical signal transduction in olfactory neurons appears to involve intracellular reaction cascades mediated by heterotrimeric GTP-binding proteins. In this study attempts were made to identify the G protein subtype(s) in olfactory cilia that are activated by the primary (odorant) signal. Antibodies directed against the ␣ subunits of distinct G protein subtypes interfered specifically with second messenger reponses elicited by defined subsets of odorants; odor-induced cAMP-formation was attenuated by G␣ s antibodies, whereas G␣ o antibodies blocked odor-induced inositol 1,4,5-trisphosphate (IP 3 ) formation. Activation-dependent photolabeling of G␣ subunits with [␣- Chemoelectrical signal transduction is considered to be mediated via intracellular reaction cascades triggered by G protein-coupled receptors (1). Biochemical studies over the last decade have revealed that odorants elicit the formation of either cAMP or IP 3 1 in olfactory preparations (2-6). Whereas the functional implications of the dual transduction pathways in the crustacean olfactory system are well established (7,8), in vertebrates the relative importance of the two pathways in olfactory signaling remains controversial (9, 10). Heterotrimeric GTP-binding proteins play a key role in signal transduction processes, coupling activated receptors to the appropriate effector system. A variety of different G␣ subtypes have been identified in vertebrate olfactory epithelium including G s short , G il , G i2 , G i3 , G o , and G q (11-17). Even an olfactory-specific isoform of G s (G olf ) has been discovered (18). However, it is currently unclear how many and which type of G proteins are involved in olfactory signal transduction. To approach the question of which G protein subtype(s) may mediate the transduction processes in olfactory sensory cells, it is necessary to identify the G protein that is activated upon stimulation with distinct odor ligands. This can be accomplished by an activationdependent labeling procedure (19), in which receptor-activated G protein ␣ subunits are photolabeled using the hydrolysisresistant GTP-analogue [␣-32 P]GTP azidoanilide. Subsequent immunoprecipitation of G␣ subunits with subtype-specific antibodies permits identification of G protein subtypes that are labeled upon stimulation of olfactory cilia preparations with distinct odorants. The data indicate that cAMP-and IP 3 -inducing odorants result in labeling of different G protein subtypes. EXPERIMENTAL PROCEDURES MaterialsSprague-Dawley rats were purchased from Charles River, Sulzfeld. The odorants citralva (3,7-dimethyl-2,6-octadiennitrile), hedione (3-oxo-2-pentyl cyclopentaneacetic acid methyl ester), eugenol (2-methoxy-4-(2-propenyl)phenol), lilial (para-butyl-␣-methyl hydrocinnamic aldehyde), lyral (4-(4-hydroxy-4-methyl pentyl)-3-cyclohexene-10-carboxyldehyde), and ethylvanillin (3-ethoxy-4-hydroxybenzaldehyde) were provided by DROM, Baierbrunn. Isovaleric acid (3-methylbutanoic acid) and pyrrolidine (tetrahydropyrrole) were purchased from Sigma. The ra...

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A partially purified preparation of isolated chemosensory cilia from the olfactory epithelium of the bullfrog, Rana catesbeiana

Cited by 107 publications

References 18 publications

ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification

ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification

G Protein-coupled Receptor Kinase 3 (GRK3) Gene Disruption Leads to Loss of Odorant Receptor Desensitization

Odorants Selectively Activate Distinct G Protein Subtypes in Olfactory Cilia

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