title: A conserved aspartic acid is important for agonist (VUAA1) and odorant/tuning receptor-dependent activation of the insect odorant co-receptor (Orco).
abstract: Insect odorant receptors function as heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor, and a conserved co-receptor (Orco). An Orco agonist, VUAA1, is able to activate both heteromeric and homomeric Orco-containing channels. Very little is known about specific residues in Orco that contribute to cation permeability and gating. We investigated the importance of two conserved Asp residues, one in each of transmembrane domains 5 and 7, for channel function by mutagenesis. Drosophila melanogaster Orco and its substitution mutants were expressed in HEK cells and VUAA1-stimulated channel activity was determined by Ca(2+) influx and whole-cell patch clamp electrophysiology. Substitution of D466 in transmembrane 7 with amino acids other than glutamic acid resulted in a substantial reduction in channel activity. The D466E Orco substitution mutant was ~2 times more sensitive to VUAA1. The permeability of the D466E Orco mutant to cations was unchanged relative to wild-type Orco. When D466E Orco is co-expressed with a conventional tuning odorant receptor, the heteromeric complex also shows increased sensitivity to an odorant. Thus, the effect of the D466E mutation is not specific to VUAA1 agonism or dependent on homomeric Orco assembly. We suggest the gain-of-activation characteristic of the D466E mutant identifies an amino acid that is likely to be important for activation of both heteromeric and homomeric insect odorant receptor channels.
authors.affiliation: School of Biological Sciences, University of Auckland, Auckland, New Zealand.
authors.forename: Brijesh N
authors.initials: BN
authors.lastname: Kumar
authors.forename: Robert W
authors.initials: RW
authors.lastname: Taylor
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Pask
authors.forename: Laurence J
authors.initials: LJ
authors.lastname: Zwiebel
authors.forename: Richard D
authors.initials: RD
authors.lastname: Newcomb
authors.forename: David L
authors.initials: DL
authors.lastname: Christie
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000852:Anopheles / Q000378:metabolism*; D029721:Drosophila Proteins / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D018035:Receptors, Odorant / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013864:Thioglycolates / Q000494:pharmacology*; D014230:Triazoles / Q000494:pharmacology*
chemical_list: D029721:Drosophila Proteins; D019476:Insect Proteins; C572612:N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)thio)acetamide; C496847:OR22a protein, Drosophila; D018035:Receptors, Odorant; D013864:Thioglycolates; D014230:Triazoles
references.citation: FEBS Lett. 2007 Dec 11;581(29):5601-4
references.pmid: 18005664
references.citation: PLoS One. 2011 Apr 07;6(4):e18605
references.pmid: 21490930
references.citation: Science. 2005 Mar 11;307(5715):1638-42
references.pmid: 15692016
references.citation: Proc Natl Acad Sci U S A. 2011 May 24;108(21):8821-5
references.pmid: 21555561
references.citation: PLoS One. 2012;7(3):e32372
references.pmid: 22403649
references.citation: J Biol Chem. 2001 Dec 14;276(50):46983-8
references.pmid: 11598117
references.citation: PLoS Biol. 2006 Feb;4(2):e20
references.pmid: 16402857
references.citation: J Mol Biol. 2001 Jan 19;305(3):567-80
references.pmid: 11152613
references.citation: Nature. 2008 Apr 24;452(7190):1002-6
references.pmid: 18408712
references.citation: PLoS One. 2012;7(1):e30304
references.pmid: 22272331
references.citation: J Gen Physiol. 2011 Mar;137(3):289-97
references.pmid: 21357733
references.citation: Front Cell Neurosci. 2013 Jan 24;7:1
references.pmid: 23355802
references.citation: Chem Senses. 2011 Nov;36(9):781-90
references.pmid: 21677030
references.citation: Annu Rev Physiol. 2003;65:133-59
references.pmid: 12471162
references.citation: Curr Biol. 2005 Feb 22;15(4):R119-21
references.pmid: 15723778
references.citation: Nature. 2010 Mar 4;464(7285):66-71
references.pmid: 20130575
references.citation: Proc Int Conf Intell Syst Mol Biol. 1998;6:175-82
references.pmid: 9783223
references.citation: PLoS Genet. 2012;8(8):e1002930
references.pmid: 22952454
references.citation: Chem Senses. 2011 Jan;36(2):149-60
references.pmid: 20956733
references.citation: Cell. 2004 Jun 25;117(7):965-79
references.pmid: 15210116
references.citation: Insect Biochem Mol Biol. 2008 Aug;38(8):770-80
references.pmid: 18625400
references.citation: Nature. 2008 Apr 24;452(7190):1007-11
references.pmid: 18408711
references.citation: PLoS One. 2012;7(5):e36784
references.pmid: 22590607
references.citation: Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4418-23
references.pmid: 20160092
references.citation: Nat Rev Neurosci. 2010 Mar;11(3):188-200
references.pmid: 20145624
references.citation: Neuron. 2004 Sep 2;43(5):703-14
references.pmid: 15339651
references.citation: ACS Chem Biol. 2012 Oct 19;7(10):1647-52
references.pmid: 22924767
references.citation: Front Cell Neurosci. 2011 Jun 16;5:5
references.pmid: 21720521
references.citation: PLoS One. 2011;6(12):e28774
references.pmid: 22174894
references.citation: Chem Senses. 2011 Jul;36(6):497-8
references.pmid: 21441366
references.citation: Nature. 2006 Sep 14;443(7108):230-3
references.pmid: 16921383