Nakagawa T et al. (2012), Amino acid residues contributing to function of...

XB-ART-44964

PLoS One 2012 Jan 01;73:e32372. doi: 10.1371/journal.pone.0032372.

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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 .

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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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Species referenced: Xenopus
Genes referenced: rps3a

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	Figure 1. Mutations in BmOr-1 and BmOrco that affected reversal potential and rectification index.(A) Schematic of the location of amino acids in BmOr-1 and BmOrco that were mutated in this study. Transmembrane domains were predicted using the PHDhtm algorithm [48]. (B,C) Reversal potential (top) and rectification index (bottom) of oocytes expressing mutant BmOr-1 with wild type (WT) BmOrco (B) or WT BmOr-1 with mutant BmOrco (C). Black and red bars and symbols represent WT and mutants, respectively. Only mutants with a significant effect on both reversal potential and rectification index are depicted (unpaired Student's t-test, mutant vs. WT p<0.05). Data on remaining mutants can be found in Figure S2B. Data are shown as mean Â± S.E.M., nâ=â8â10. Bombykol was applied at the concentration of 1 ÂµM to each oocyte. (D) Schematic showing the eight mutations in BmOr-1 and five mutations in BmOrco that affected both rectification index and reversal potential (see also Figure S2B,S3A).
	Figure 2. Mutations in BmOr-1 and BmOrco that affected ion selectivity.(A) Representative current-voltage (IâV) curves of oocytes expressing WT or mutant Or or BmOrco. Red and blue traces represent IâV 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 Â± S.E.M., nâ=â5â8. Bombykol was applied at the concentration of 1 ÂµM 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).
	Figure 3. Effect of MTSET on ionic permeability of WT and Cys mutant BmOr-1-BmOrco complexes.(A) Representative current traces of oocytes expressing WT or mutant BmOr-1 or BmOrco. Bombykol (1 ÂµM) and MTSET (2.5 mM) were applied at the time indicated by arrowheads and blue squares, respectively. (B) Summary of effects of MTSET on the bombykol response of WT (black bar) and mutant BmOr-1 (gray bars). The mutant with a significant effect on ion permeability is colored in red (unpaired Student's t-test, mutant vs. WT **p<0.01). Data are shown as mean Â± S.E.M., nâ=â5 (see also Figure S4).
	Figure 4. Effect of the Drosophila Orco Y478A mutation on Or-Orco function in heterologous cells and in Drosophila olfactory neurons.(A) Reversal potential (top), rectification index (middle), and permeability ratio (bottom) of oocytes expressing WT Drosophila Orco (black bars and symbols) or Drosophila Orco Y478A (red bars and symbols) along with WT ligand-selective Ors. Unpaired Student's t-test, mutant vs. WT, p<0.05, p<0.01. n.r.â=âno response. Data are shown as mean Â± S.E.M., nâ=â8â10. (B) Anti-Orco antibody staining of WT Drosophila Orco (left) and Drosophila Orco Y478A expressed in Orcoâ/â animals. (C) (Top) Schematic of antennal sensilla showing the sensilla type and associated ligand-selective Ors. (Middle) Representative single-sensillum traces of ab2A, ab2B, ab3A, ab3B, ab5B neurons expressing WT Drosophila Orco or Drosophila Orco Y478A to methyl acetate, ethyl-3-hydroxybutyrate, ethyl butyrate, 2-heptanone, and pentyl acetate, respectively. Blue traces represent ab2A and ab3A neuron; green traces represent ab2B, ab3B, and ab5A and B neurons. (Bottom) Peristimulus time histograms of recordings from WT (black) and mutant (red). Data are presented as mean Â±S.E.M., nâ=â3â7, and raw spikes/sec are plotted. (D) Summary of effects of WT Drosophila Orco (black bars) and Drosophila Orco Y478A (red bars) on spontaneous activity (top; raw spikes/sec) and odor-evoked activity. (bottom; corrected to subtract spontaneous activity). Unpaired Student's t-test, mutant vs. WT, p<0.05, p<0.01, p<0.001. Data are shown as mean Â±S.E.M., nâ=â3â7. (E) (Top) Schematic of ab5 sensilla expressing WT BmOrco or BmOrco Y464A. (Middle) Representative single-sensillum traces of ab5 neurons expressing WT BmOrco or BmOrco Y464A to pentyl acetate. Green traces represent ab5A and ab5B neurons. (Bottom) Peristimulus time histograms of recordings from WT (black) and mutant (red). Data are presented as mean Â±S.E.M., nâ=â4â5, and raw spikes/sec are plotted. (F) Summary of effects of WT BmOrco (black bars) and BmOrco Y464A (red bars) on spontaneous activity (top; raw spikes/sec) and odor-evoked activity. (bottom; corrected to subtract spontaneous activity). Data are shown as mean Â± S.E.M., nâ=â4â5. Unpaired Student's t-test, mutant vs. WT, p<0.05, p<0.01, *p<0.001.

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Bateman, Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange. 2006, Pubmed