‘Canonical’, cAMP dependent olfactory signal transduction cascade in rodents
Odorant receptors can be found expressed on cilia or microvilli of OSNs (Ottoson, 1956).
Independent of the receptor family, the basic molecular transduction machinery in the majority of ORNs consists of four principal (macro-)molecular complexes: a receptor protein, a heterotrimeric G-protein, a membrane-associated enzyme and a set of ion channels (Schematic 1). Already before the OR gene family was discovered, specific mechanisms of odor detection and parts of the signal transduction machinery had already been known (Schild and Restrepo, 1998). The G-protein Gαolf was the first identified G protein of olfactory transduction. Its absence leads to an anosmic phenotype in mice (Belluscio et al., 1998;
12 Jones and Reed, 1989). In further knockout models, the essential roles of adenylate cyclase III (ACIII) and a cyclic-nucleotide gated channel in ORNs were confirmed (Baker et al., 1999;
Brunet et al., 1996; Wong et al., 2000). The latter results and pioneering physiological experiments (Firestein et al., 1991; Nakamura and Gold, 1987), solidified the cAMP-dependent olfactory transduction pathway associated with the OR family (Firestein et al., 1991; Nakamura and Gold, 1987). Upon odorant binding to ORs, the GTP bound dissociated Gαolf subunit binds to adenylate-cyclase III initiating the conversion from ATP to second messenger cAMP (Bakalyar and Reed, 1990; Pace et al., 1985). The second messenger cAMP subsequently triggers the opening of cyclic-nucleotide gated cation channels, causing an influx of sodium and calcium into the ORN (Nakamura and Gold, 1987; Firestein et al., 1991; Lowe and Gold, 1993). The increase in calcium conductance was shown to be essential to trigger further amplification of the generator potential by a Ca2+-activated chloride channels(Kleene and Gesteland, 1991; Kurahashi and Yau, 1993; Lowe and Gold, 1993).
The resulting Ca2+-activated chloride ion net efflux depolarizes the cell towards its reversal potential at around 0 mV (Kurahashi and Yau, 1993). The resulting net depolarization is sufficiently more positive than the resting membrane potential to trigger action potentials, the unitary electrical events encoding odor information.
‘Non-canonical’, cAMP independent olfactory transduction cascade in rodents
Olfactory signal transduction pathways independent of cAMP were already postulated and partially described early in vertebrates like fish and amphibians (Schild and Restrepo, 1998).
With the emergence of the VR family and the investigation of VRN signal transduction, the cAMP independent transduction pathway was back on the radar in rodents. The mutually exclusive expression of two heterotrimeric G proteins (Gαo and Gαi2) in two distinct subsets of VRNs were first hints towards the essential cAMP-independent transduction cascades in VRs (Halpern et al., 1995). While Gao has been confirmed to act in V2R mediated signal transduction in basal VRNs (Chamero et al., 2011), the role of Gαi2 in V1R transduction has yet to be validated. In addition to the soluble Gα subunit, the membrane tethered ßy-subunits contribute to the heterogeneity of signal transduction cascades (Rünnenburger et al., 2002;
Ryba and Tirindelli, 1995; Sathyanesan et al., 2013; Wu et al., 1996). Instead of the adenylate-cyclase, phopholipase C (PLC) serves as a major effector enzyme that produces second messenger molecules. The membrane-associated PLC is activated by binding to the freed ßy subunit upon GDP-GTP exchange in the Gα subunit (Rünnenburger et al., 2002).
PLC catalyzes the hydrolysis of phosphatidylinositol-4,5-bis-phosphate (PIP2) in the inner plasma membrane leaflet, setting free membrane-bound diacylglycerol (DAG) and water-soluble inositol-1,4,5-trisphosphate (IP3; Holy, 2000; Lucas et al., 2003; Rünnenburger et al., 2002; Spehr et al., 2002). The local elevation of second messengers leads to calcium influx via two main mechanisms: IP3 dissipates from the membrane and triggers calcium influx into the cytosol from intracellular compartments (Kim et al., 2011; Yang and Delay, 2010). DAG diffuses laterally through the plasma-membrane of the VRN microvilli and activates TRPC2,
13 a member of the transient receptor potential channel family upon binding (Liman et al., 1999;
Lucas et al., 2003; Spehr and Munger, 2009). The increased cation conductance of activated TRPC2 leads to further calcium level elevation in the microvilli of VRNs. Subsequently, calcium ions trigger Ca2+-activated chloride net efflux through ANO1or ANO2 chloride channels (Amjad et al., 2015; Dibattista et al., 2012; Kim et al., 2011; Lucas et al., 2003;
Münch et al., 2018; Yang and Delay, 2010). Depolarization through ANO1/2 is possible due to the high chloride concentration in VRN microvilli (Kim et al., 2015; Untiet et al., 2016).
TAAR receptor transduction is not studied in detail yet. The receptor topology and involvement of Gαolf in its signal transduction point to a canonical OR like signal transduction cascade (Liberles and Buck, 2006). FPRs (Liberles et al., 2009; Rivière et al., 2009) and MS4A receptors (Greer et al., 2016) will not be discussed further on the signal transduction level in this work.
Signal transduction in aquatic species
The existence of cAMP dependent and independent signal transduction pathways in fish was already confirmed early on in catfish ORN cilia (Restrepo et al., 1993). While several studies in fish have contributed to the understanding of signal transduction, few of them were able to characterize the two pathways as detailed and convincing as Sato and co-workers (Sato et al., 2005). Analogous to their mammalian counterparts, ciliated ORNs in zebrafish express Gαolf and a cyclic nucleotide gated channel subunit (here CGNA2), main components of the canonical cAMP transduction pathway (Hansen et al., 2003; Sato et al., 2005). The microvillous ORN type was distinguishable from the ciliated type by its expression of TRPC2 (Munger et al., 2009; Sato et al., 2005).
Both cAMP-dependent and independent signal transduction pathways are present in amphibians (Schild and Restrepo, 1998). Signal transduction independent of cAMP has been confirmed in Xenopus laevis ORNs (Manzini and Schild, 2003a). Key players of the cAMP-independent signal transduction cascade including PLC, DAG and TRPC2 were found to be associated with the microvillous ORN type of the larval olfactory mucosa (Gliem et al., 2013;
Manzini and Schild, 2003a; Sansone et al., 2014a, 2014b).
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Schematic 2 Odorant receptor dependent signal transduction pathways
Vertebrate odorant receptor classes (V1Rs, V2Rs, TAARs and ORs) use heterotrimeric G-proteins (soluble Gα- and membrane tethered ßy-subunit) to transduce information from the exterior to the inside of the cell. Upon odorant binding the odorant receptor undergoes conformational changes that lead to exchange of bound GDP with GTP in the G-protein and its subsequent dissociation into the respective Gα- (V1R: Gαi2, V2R: Gαo, TAAR & OR: Gαolf) and ßy-subunits. In the V1R and V2R transduction cascade the ßy-subunit, activates PLC which creates the second messengers IP3 and DAG from PIP2. DAG triggers cation-conducting TRPC2 channels, depolarizing the neuron. In the OR and TAAR signal transduction cascade, the GTP-bound Gαolf subunit activates ACIII which catalyzes cAMP production from ATP. Cation influx through CNG channels is triggered upon cAMP binding and the resulting Ca2+ influx induces ANO-mediated Cl- efflux, boosting depolarization of the neuronal plasma membrane.
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