Optimization of Insect Odorant Receptor Trafficking and Functional Expression Via Transient Transfection in HEK293 Cells.

Fabio Miazzi,Bill S Hansson,Dieter Wicher,Markus Knaden,Silke Sachse,Sofia Lavista-Llanos,Carolin Hoyer

doi:10.1093/chemse/bjz062

Fabio Miazzi, Bill S Hansson + Show 5 more

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https://doi.org/10.1093/chemse/bjz062

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Journal: Chemical senses	Publication Date: Aug 31, 2019
Citations: 10	License type: CC BY 4.0

Affiliation: Max Planck Institute for Chemical Ecology

Abstract

Insect odorant receptors (ORs) show a limited functional expression in various heterologous expression systems including insect and mammalian cells. This may be in part due to the absence of key components driving the release of these proteins from the endoplasmic reticulum and directing them to the plasma membrane. In order to mitigate this problem, we took advantage of small export signals within the human HCN1 and Rhodopsin that have been shown to promote protein release from the endoplasmic reticulum and the trafficking of post-Golgi vesicles, respectively. Moreover, we designed a new vector based on a bidirectional expression cassette to drive the functional expression of the insect odorant receptor coreceptor (Orco) and an odor-binding OR, simultaneously. We show that this new method can be used to reliably express insect ORs in HEK293 cells via transient transfection and that is highly suitable for downstream applications using automated and high-throughput imaging platforms.

Highlights

Insect odorant receptors (ORs) are 7 transmembrane-domain proteins, with an inverted topology compared to G protein-coupled receptors (GPCRs) (Benton et al 2006; Butterwick et al 2018), responsible for the detection of a vast number of chemically diverse odorants including pheromones (Hallem and Carlson 2006; Touhara and Vosshall 2009)
2.9 ± 1.05% of cells transfected with hOr47a/odorant receptor coreceptor (Orco) showed a ∆[Ca2+]i ≥ 6.75 nM after a 100 μM pentyl acetate stimulation, while 21.61 ± 4.16% of cells transfected with E.hOr47a/Orco and 30.51 ± 6.56% of cells transfected with R.E.hOr47a/Orco reached such threshold
When comparing cell responses after a 100 μM VUAA1 stimulation, only 30.24 ± 7.21% of cells transfected with hOr47a/Orco showed an ∆[Ca2+]i ≥ 6.25 nM, while 61.49 ± 5.95% and 71.76 ± 3.29% of cells transfected with E.hOr47a/Orco and R.E.hOr47a/Orco, respectively could be defined as “responding” (Figure 1e and Supplementary Figure 1)

Summary

Introduction

Insect odorant receptors (ORs) are 7 transmembrane-domain proteins, with an inverted topology compared to G protein-coupled receptors (GPCRs) (Benton et al 2006; Butterwick et al 2018), responsible for the detection of a vast number of chemically diverse odorants including pheromones (Hallem and Carlson 2006; Touhara and Vosshall 2009). Chemical Senses, 2019, Vol 44, No 9 et al 2007; Tsitoura et al 2010; German et al 2013), and mammalian cells (e.g., HEK293 and CHO) using vectors for transient expression (Sato et al 2008; Wicher et al 2008) or as stable lines (Grosse-Wilde et al 2006; Jones et al 2011; Corcoran et al 2014) Each of these methods offers several advantages, and bears disadvantages: the “empty neuron system” allows the expression of OR proteins in an environment that is very similar to their native olfactory sensillum. The selection of a monoclonal cell population represents—to date—the most effective approach to deal with the limited functional expression of ORs in the plasma membrane in heterologous systems due to an impaired intracellular trafficking of ORs both in insect (German et al 2013) and mammalian cells (HaltydeLeon et al 2016)

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