Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3

Abstract

Amino acid substitutions within the conserved polypeptide sequence of the insect olfactory receptor co-receptor (Orco) have been demonstrated to influence its pharmacological properties. By sequence analysis and phylogenetic investigation, in the Lepidopteran subgroup Ditrysia we identified a fixed substitution in the intracellular loop-3 (ICL-3) of a conserved histidine to glutamine. By means of HEK293 cells as a heterologous system, we functionally expressed Orco from the Ditrysian model Cydia pomonella (CpomOrco) and compared its functional properties with a site-directed mutagenized version where this ICL-3-glutamine was reverted to histidine (CpomOrcoQ417H). The mutagenized CpomOrcoQ417H displayed decreased responsiveness to VUAA1 and reduced response efficacy to an odorant agonist was observed, when co-transfected with the respective OR subunit. Evidence of reduced responsiveness and sensitivity to ligands for the mutagenized Orco suggest the fixed glutamine substitution to be optimized for functionality of the cation channel within Ditrysia. In addition, contrary to the wild type, the mutagenized CpomOrcoQ417H preserved characteristics of VUAA-binding when physiologic conditions turned to acidic. Taken together, our findings provide further evidence of the importance of ICL-3 in forming basic functional properties of insect Orco- and Orco/OR-channels, and suggest involvement of ICL-3 in the potential functional adaptation of Ditrysian Orcos to acidified extra-/intracellular environment.

Chemosensory Receptors in the Larval Maxilla of Papilio hospiton

“…To test the expression of Papilio OR1 subunits before testing candidate ligands, we used the main Orco agonist VUAA1, which has been reported for activating both homomeric and heteromeric Orco-based receptor channels (Jones et al, 2011). Dose-response characteristics to VUAA1 (Figure 5), which are consistent with the pharmacologic parameters reported in our previous investigations using CpomOrco + ORs (Cattaneo et al, 2017a;Bobkov et al, 2021) suggested the heterologous coexpression of Papilio OR1-subunits with CpomOrco. Despite expression pieces of evidence of both PhospOR1 and PmachOR1 in HEK-cells, we did not identify any ligand able to activate these subunits within the panel we used (Figure 6).…”

“…We are aware of the possibility to perform functional trials in HEK cells using synthetic codonoptimized sequences (Roberts et al, 2021). However, the choice of CpomOrco was based on the conservation of this subunit with orthologs within and outside the order of Lepidoptera that allows for the generation of functional chimeric Orco/OR channels (Bobkov et al, 2021). For example, many Lepidoptera ORs have been deorphanized through the heterologous expression of insect ORs in empty neurons of D. melanogaster, where dipteran Orco/lepidoteran OR channels are generated (Gonzalez et al, 2016).…”

“…The full-length CDSs of PhospOR1, PhospPain, and their orthologs PmachOR1 and PmachPain were cloned following protocols we described with more details in our previous investigations (Cattaneo et al, 2017a;Bobkov et al, 2021). We amplified retro-transcribed cDNA samples with primers shown in Supplementary Table 1, which were designed on the P. machaon sequences (GenBank XM_014512458.1 and XM_014500087.1).…”

“…Activation of HEK293A cells transfected with CpomOrco + PhospOR1, CpomOrco + PmachOR1, CpomOrco + CpomOR3, the sole CpomOrco, PhospPain, PmachPain, and dTRPA1(B) was tested using the same procedures we previously described (Cattaneo et al, 2017a,b, Bobkov et al, 2021. Petri dishes were incubated for 1 h at room temperature in 1.0 mL HEK Ca ++ Ringer (mM: 140 NaCl, 5 KCl, 2 CaCl 2 , 10 HEPES, pH 7.4) containing the fluorescent calcium indicator Fluo-4AM (Invitrogen) at 5-15 µM prepared with 0.06-0.2% Pluronic F-127 (Invitrogen).…”

“…The non-specific Orco-agonist VUAA1: acetamide, N- (4ethylphenyl)-2-[[4-ethyl-5-(3-pyridinyl)-4H-1,2,4-triazol-3yl]thio]-, CAS 525582-84-7 (Glixx Laboratories, Southborough, MA, United States), selected among the ligands we previously reported active on CpomOrco/OR channels (Cattaneo et al, 2017a;Bobkov et al, 2021), was dissolved in dimethyl sulfoxide (DMSO, Sigma Aldrich, St. Louis, MO, United States) and stored as a stock solution (200 mM) at -20 • C. The final working concentrations (10-1,000 µM) of VUAA1 were always prepared right before the experiments. Amplitudes of the calcium responses were used to generate dose-response characteristics and values were normalized to the response amplitude recorded at 1,000 µM VUAA1.…”