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Amino acid residues contributing to function of the heteromeric insect olfactory receptor complex.

Nakagawa T, Pellegrino M, Sato K, Vosshall LB, Touhara K - PLoS ONE (2012)

Bottom Line: Three of the 13 amino acids (D299 and E356 in BmOr-1 and Y464 in BmOrco) altered both current-voltage relationships and K(+) selectivity.We introduced the homologous Orco Y464 residue into Drosophila Orco in vivo, and observed variable effects on spontaneous and evoked action potentials in olfactory neurons that depended on the particular Or-Orco complex examined.Our results provide evidence that a subset of conserved Glu, Asp and Tyr residues in both subunits are essential for channel activity of the heteromeric insect Or-Orco complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Olfactory receptors (Ors) convert chemical signals--the binding of odors and pheromones--to electrical signals through the depolarization of olfactory sensory neurons. Vertebrates Ors are G-protein-coupled receptors, stimulated by odors to produce intracellular second messengers that gate ion channels. Insect Ors are a heteromultimeric complex of unknown stoichiometry of two seven transmembrane domain proteins with no sequence similarity to and the opposite membrane topology of G-protein-coupled receptors. The functional insect Or comprises an odor- or pheromone-specific Or subunit and the Orco co-receptor, which is highly conserved in all insect species. The insect Or-Orco complex has been proposed to function as a novel type of ligand-gated nonselective cation channel possibly modulated by G-proteins. However, the Or-Orco proteins lack homology to any known family of ion channel and lack known functional domains. Therefore, the mechanisms by which odors activate the Or-Orco complex and how ions permeate this complex remain unknown. To begin to address the relationship between Or-Orco structure and function, we performed site-directed mutagenesis of all 83 conserved Glu, Asp, or Tyr residues in the silkmoth BmOr-1-Orco pheromone receptor complex and measured functional properties of mutant channels expressed in Xenopus oocytes. 13 of 83 mutations in BmOr-1 and BmOrco altered the reversal potential and rectification index of the BmOr-1-Orco complex. Three of the 13 amino acids (D299 and E356 in BmOr-1 and Y464 in BmOrco) altered both current-voltage relationships and K(+) selectivity. We introduced the homologous Orco Y464 residue into Drosophila Orco in vivo, and observed variable effects on spontaneous and evoked action potentials in olfactory neurons that depended on the particular Or-Orco complex examined. Our results provide evidence that a subset of conserved Glu, Asp and Tyr residues in both subunits are essential for channel activity of the heteromeric insect Or-Orco complex.

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Mutations in BmOr-1 and BmOrco that affected ion selectivity.(A) Representative current-voltage (I–V) curves of oocytes expressing WT or mutant Or or BmOrco. Red and blue traces represent I–V curves obtained with Na+ and K+ solutions, respectively. (B) Summary of ion permeability ratios of the two BmOr-1 mutants (left) and the BmOrco mutants (right) that had a significant effect on ion selectivity (unpaired Student's t-test, mutant vs. WT **p<0.01; *p<0.05). Data on remaining mutants can be found in Figure S2C. Each bar represents mean ± S.E.M., n = 5–8. Bombykol was applied at the concentration of 1 µM to each oocyte. (C) Schematic showing the two mutations in BmOr-1 and one mutation in BmOrco that affected ion selectivity (see also Figure S2C,S3B).
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pone-0032372-g002: Mutations in BmOr-1 and BmOrco that affected ion selectivity.(A) Representative current-voltage (I–V) curves of oocytes expressing WT or mutant Or or BmOrco. Red and blue traces represent I–V curves obtained with Na+ and K+ solutions, respectively. (B) Summary of ion permeability ratios of the two BmOr-1 mutants (left) and the BmOrco mutants (right) that had a significant effect on ion selectivity (unpaired Student's t-test, mutant vs. WT **p<0.01; *p<0.05). Data on remaining mutants can be found in Figure S2C. Each bar represents mean ± S.E.M., n = 5–8. Bombykol was applied at the concentration of 1 µM to each oocyte. (C) Schematic showing the two mutations in BmOr-1 and one mutation in BmOrco that affected ion selectivity (see also Figure S2C,S3B).

Mentions: The PK/PNa of two BmOr-1 mutants (D299N, E356Q) was slightly decreased compared with that of wild type (Figure 2A,B). The same decrease in PK/PNa compared to wild type was found for the homologous BmOr-3 E365Q mutation (Figure S3B). In contrast, the PK/PNa of BmOrco Y464A was slightly increased compared to wild type (Figure 2A,B). The other 10 candidate mutants did not show altered PK/PNa (BmOr-1: Y170A, D226N, E325Q, D367N, E375N, D378N; BmOrco: E171Q, E329Q, D343N, E422Q) (Figure S2C). Therefore only a small number of the mutations examined here—BmOr-1 D299, BmOr-1 E356, and BmOrco Y464—altered the ion selectivity of the BmOr-1-BmOrco complex (Figure 2C), although the remaining 10 residues may be involved in selectivity of ions other than K+.


Amino acid residues contributing to function of the heteromeric insect olfactory receptor complex.

Nakagawa T, Pellegrino M, Sato K, Vosshall LB, Touhara K - PLoS ONE (2012)

Mutations in BmOr-1 and BmOrco that affected ion selectivity.(A) Representative current-voltage (I–V) curves of oocytes expressing WT or mutant Or or BmOrco. Red and blue traces represent I–V curves obtained with Na+ and K+ solutions, respectively. (B) Summary of ion permeability ratios of the two BmOr-1 mutants (left) and the BmOrco mutants (right) that had a significant effect on ion selectivity (unpaired Student's t-test, mutant vs. WT **p<0.01; *p<0.05). Data on remaining mutants can be found in Figure S2C. Each bar represents mean ± S.E.M., n = 5–8. Bombykol was applied at the concentration of 1 µM to each oocyte. (C) Schematic showing the two mutations in BmOr-1 and one mutation in BmOrco that affected ion selectivity (see also Figure S2C,S3B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3293798&req=5

pone-0032372-g002: Mutations in BmOr-1 and BmOrco that affected ion selectivity.(A) Representative current-voltage (I–V) curves of oocytes expressing WT or mutant Or or BmOrco. Red and blue traces represent I–V curves obtained with Na+ and K+ solutions, respectively. (B) Summary of ion permeability ratios of the two BmOr-1 mutants (left) and the BmOrco mutants (right) that had a significant effect on ion selectivity (unpaired Student's t-test, mutant vs. WT **p<0.01; *p<0.05). Data on remaining mutants can be found in Figure S2C. Each bar represents mean ± S.E.M., n = 5–8. Bombykol was applied at the concentration of 1 µM to each oocyte. (C) Schematic showing the two mutations in BmOr-1 and one mutation in BmOrco that affected ion selectivity (see also Figure S2C,S3B).
Mentions: The PK/PNa of two BmOr-1 mutants (D299N, E356Q) was slightly decreased compared with that of wild type (Figure 2A,B). The same decrease in PK/PNa compared to wild type was found for the homologous BmOr-3 E365Q mutation (Figure S3B). In contrast, the PK/PNa of BmOrco Y464A was slightly increased compared to wild type (Figure 2A,B). The other 10 candidate mutants did not show altered PK/PNa (BmOr-1: Y170A, D226N, E325Q, D367N, E375N, D378N; BmOrco: E171Q, E329Q, D343N, E422Q) (Figure S2C). Therefore only a small number of the mutations examined here—BmOr-1 D299, BmOr-1 E356, and BmOrco Y464—altered the ion selectivity of the BmOr-1-BmOrco complex (Figure 2C), although the remaining 10 residues may be involved in selectivity of ions other than K+.

Bottom Line: Three of the 13 amino acids (D299 and E356 in BmOr-1 and Y464 in BmOrco) altered both current-voltage relationships and K(+) selectivity.We introduced the homologous Orco Y464 residue into Drosophila Orco in vivo, and observed variable effects on spontaneous and evoked action potentials in olfactory neurons that depended on the particular Or-Orco complex examined.Our results provide evidence that a subset of conserved Glu, Asp and Tyr residues in both subunits are essential for channel activity of the heteromeric insect Or-Orco complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Olfactory receptors (Ors) convert chemical signals--the binding of odors and pheromones--to electrical signals through the depolarization of olfactory sensory neurons. Vertebrates Ors are G-protein-coupled receptors, stimulated by odors to produce intracellular second messengers that gate ion channels. Insect Ors are a heteromultimeric complex of unknown stoichiometry of two seven transmembrane domain proteins with no sequence similarity to and the opposite membrane topology of G-protein-coupled receptors. The functional insect Or comprises an odor- or pheromone-specific Or subunit and the Orco co-receptor, which is highly conserved in all insect species. The insect Or-Orco complex has been proposed to function as a novel type of ligand-gated nonselective cation channel possibly modulated by G-proteins. However, the Or-Orco proteins lack homology to any known family of ion channel and lack known functional domains. Therefore, the mechanisms by which odors activate the Or-Orco complex and how ions permeate this complex remain unknown. To begin to address the relationship between Or-Orco structure and function, we performed site-directed mutagenesis of all 83 conserved Glu, Asp, or Tyr residues in the silkmoth BmOr-1-Orco pheromone receptor complex and measured functional properties of mutant channels expressed in Xenopus oocytes. 13 of 83 mutations in BmOr-1 and BmOrco altered the reversal potential and rectification index of the BmOr-1-Orco complex. Three of the 13 amino acids (D299 and E356 in BmOr-1 and Y464 in BmOrco) altered both current-voltage relationships and K(+) selectivity. We introduced the homologous Orco Y464 residue into Drosophila Orco in vivo, and observed variable effects on spontaneous and evoked action potentials in olfactory neurons that depended on the particular Or-Orco complex examined. Our results provide evidence that a subset of conserved Glu, Asp and Tyr residues in both subunits are essential for channel activity of the heteromeric insect Or-Orco complex.

Show MeSH
Related in: MedlinePlus