Introduction

Olfactory receptors are responsible for transduction of extracellular signals, enabling a cell or organism to respond to an environment. Role of G proteins in olfactory receptor transduction remains unclear. In order to understand the role of G proteins in olfactory signaling of insect ORs we aimed to develop an in vitro olfactory receptor assay for Drosophila ORs (dORs) – the odor induced calcium imaging of HEK293T cells expressing dORs.

Assays for vertebrate ORs have been developed in yeast, Xenopus oocytes, HEK293 cells (human embryonic kidney cells), COS-7 cells (derived from the kidney of the African Green Monkey, Cercopithecus aethiops: resembles fibroblast cells in humans)

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and Sf9 cells (a clonal isolate of insect Spodoptera frugioerda Sf21 cells) (Knight et al., 2003;

Krautwurst et al., 1998; Levasseur et al., 2003; Matarazzo et al., 2005; Mezler et al., 2001;

Minic et al., 2005; Wetzel et al., 1999). Similar methods were also used to characterize insect ORs. Xenopus oocytes, HeLa cells (derived from human cervical cancer cells), HEK293 cells , S2 cells (Schneider 2 cells, derived from a primary culture of late stage (20–24 h old) Drosophila melanogaster embryos) and Sf9 cells were used for heterologous expression and the receptors were characterized by electrophysiological (voltage clamp) (Nakagawa et al., 2012; Nichols et al., 2011; Wetzel, 2001) or by calcium imaging (Grosse-Wilde, 2006; Grosse-Wilde et al., 2007; Kiely et al., 2007; Neuhaus et al., 2005;

Smart et al., 2008) or by both techniques (Sato et al., 2008; Wicher et al., 2008).

These assays were used to determine ligands for orphan receptors, used to study the molecules involved in olfactory transduction, used to study the channel properties of ORs including the characterization of the ion conducting pore, used to characterize Orco, used for structural studies and the heterologously expressed ORs were also used as an electronic nose sensor. The receptor for honey bee queen pheromone component 9-oxo-2-decenoic acid was identified through heterologous expression (Xenopus oocytes) (Wanner et al., 2007) and was used to characterize the ORs from B. mori (Or1 and Or3) in the presence or absence of Orco (BmOr2) (Nakagawa et al., 2005). Also the identity of a putative pheromone receptor of H. virescences was confirmed by heterologous expression (HEK293 cells, calcium imaging) (Grosse-Wilde et al., 2007). In recent years heterologous expression of insect ORs was used to study the mechanisms of insect olfactory signaling. ORs belonging to various groups of insects (D. melanogaster, A.

gambiae, B. mori) were expressed in heterologous cell system (Xenopus oocytes, HEK293, HeLa and Sf9 cells) and by using electrophysiological or calcium imaging techniques, the role of second messengers activated by different groups of G proteins in OR signal transduction cascades was studied (Deng et al., 2011; Sargsyan et al., 2011; Sato et al., 2008; Smart et al., 2008; Wicher et al., 2008). Also the ion conducting pore of Drosophila ORs (dORs) and Anopheles gambiae ORs (AgORs) including Orco was characterized through heterologous expression (Xenopus oocytes, electrophysiology for dORs (Nakagawa et al., 2012; Nichols et al., 2011) and HEK cells, calcium imaging and electrophysiology for AgORs (Pask et al., 2011)). By using this assay Orco agonist and antagonist was first described and characterized (HEK293 T-Rex cells, electrophysiology and calcium mobilization assays (Jones et al., 2011; Jones et al., 2012)), the channel properties of Orco were characterized (Nakagawa et al., 2012; Nichols et al., 2011; Pask et al., 2011; Sato et al., 2008; Wicher et al., 2008) and also the metabotropic regulation of Orco was described (Sargsyan et al., 2011). The membrane topology of insect ORs were predicted by this assay (Smart et al., 2008; Tsitoura et al., 2010) and also the dimerization of Drosophila ORs was elucidated by this assay (Neuhaus et al., 2005). Moreover

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heterologously expressed Drosophila ORs was used as an electronic nose sensor and was shown to have broad range of sensitivities than other sensors like metal oxide E-nose sensors (Berna et al., 2009). In these studies insect ORs were expressed alone (Berna et al., 2009; Kiely et al., 2007; Smart et al., 2008) or with Orco (Deng et al., 2011; Nakagawa et al., 2012; Neuhaus et al., 2005; Nichols et al., 2011; Pask et al., 2011; Sato et al., 2008;

Tsitoura et al., 2010; Wanner et al., 2007; Wicher et al., 2008) and with (Grosse-Wilde et al., 2007; Jones et al., 2011; Jones et al., 2012; Wetzel, 2001) or without exogenous G proteins.

Expression of insect ORs in Xenopus oocytes requires longer time; further, only transient expression is possible. In contrast, mammalian or insect cell lines permit both transient and stable expression of insect ORs. Transient expression of ORs can be achieved in 1-2 days, which is much shorter when compared to expression in oocytes (4-7 days). Electrophysiological experiments alone can be used to characterize insect ORs in Xenopus oocytes, whereas both electrophysiological and calcium imaging techniques can be used to characterize insect ORs in cell lines. For the electrophysiological experiments, Xenopus oocytes are best suited, because of their large size (~1 mm); handling and manipulation of the oocytes is also easy. Xenopus oocytes and mammalian or insect cell lines have their own advantages and disadvantages as an expression system for insect ORs. Mammalian or insect cell lines are well suited for the expression of insect ORs when time is considered as a major factor for expression and non electrophysiological experiments are preferred for functional studies (e.g. calcium imaging).

Only very few studies deal with insect ORs expressed in insect cell lines (Sf9 or S2 cells) when compared to mammalian cell lines. Insect cell lines would appear overall more suited for expression of insect ORs. However, although the transfection efficiency of insect and mammalian cell lines are comparable, the percentages of cells responding to odorants were much lower in insect cell lines (~4% of cells responded to an odor (Kiely et al., 2007)). Thus HEK293 and HeLa cells are largely used to express insect ORs (Deng et al., 2011; Sato et al., 2008; Smart et al., 2008; Wicher et al., 2008). Some studies used modified HEK293 cells (Flp-In T-Rex293/Gα₁₅) for functional studies (Grosse-Wilde, 2006; Grosse-Wilde et al., 2007). For functional studies in mammalian cell lines insect ORs were expressed alone or together with the co-receptor (Orco) and with or without the exogenous G proteins (Deng et al., 2011; Grosse-Wilde, 2006; Grosse-Wilde et al., 2007; Neuhaus et al., 2005; Sato et al., 2008; Smart et al., 2008; Wicher et al., 2008).

Expression of Orco together with OR was shown to enhance the sensitivity of the response up to 1000 fold (Neuhaus et al., 2005; Wicher et al., 2008).

We used HEK293T cells as a heterologous system for the functional studies of dORs. HEK293T cells are similar to the parental HEK293 cells but they stably express the SV40 large T antigen. We transiently transfected the cells with dOR and Orco but

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not any exogenous G proteins. For the validation of the assay we expressed dOr22a and Orco or dOr92a and Orco and measured odor-induced calcium changes. About 50% of the cells were transfected with dORs and most of the transfected cells were functional (about 30% of cells responded to odor which is about 60% of transfected cells). For efficient OR expression, co-expression of Orco was required. Single cells were analyzed semiautonomously using user-defined workflows in the KNIME software (Konstanz Information Miner - http://www.knime.org/). This approach is highly efficient and less time consuming than the nonautonomous analysis methods described in the literature (Kiely et al., 2007; Smart et al., 2008). Odor responses were significant and reproducible.

This assay can be used in future to study the role of G proteins in olfactory signaling or to de-orphanize receptors or to study the mechanisms of odor adaptation. In this chapter detailed explanation of the technique is provided and in the next chapter by using this method as one of the techniques we studied the role of G proteins (G_o/i subgroup) in olfactory signaling of dORs (dOr22a and Orco).