title: Mutational analysis of cysteine residues of the insect odorant co-receptor (Orco) from Drosophila melanogaster reveals differential effects on agonist- and odorant-tuning receptor-dependent activation.
abstract: Insect odorant receptors are heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor (ORx) and a highly conserved co-receptor known as Orco. Orco is found only in insects, and very little is known about its structure and the mechanism leading to channel activation. In the absence of an ORx, Orco forms homomeric channels that can be activated by a synthetic agonist, VUAA1. Drosophila melanogaster Orco (DmelOrco) contains eight cysteine amino acid residues, six of which are highly conserved. In this study, we replaced individual cysteine residues with serine or alanine and expressed Orco mutants in Flp-In 293 T-Rex cells. Changes in intracellular Ca(2+) levels were used to determine responses to VUAA1. Replacement of two cysteines (Cys-429 and Cys-449) in a predicted intracellular loop (ICL3), individually or together, gave variants that all showed similar increases in the rate of response and sensitivity to VUAA1 compared with wild-type DmelOrco. Kinetic modeling indicated that the response of the Orco mutants to VUAA1 was faster than wild-type Orco. The enhanced sensitivity and faster response of the Cys mutants was confirmed by whole-cell voltage clamp electrophysiology. In contrast to the results from direct agonist activation of Orco, the two cysteine replacement mutants when co-expressed with a tuning receptor (DmelOR22a) showed an ∼10-fold decrease in potency for activation by 2-methyl hexanoate. Our work has shown that intracellular loop 3 is important for Orco channel activation. Importantly, this study also suggests differences in the structural requirements for the activation of homomeric and heteromeric Orco channel complexes.
authors.affiliation: School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Turner
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232, and.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Derryberry
authors.affiliation: School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Brijesh N
authors.initials: BN
authors.lastname: Kumar
authors.affiliation: School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Thomas
authors.initials: T
authors.lastname: Brittain
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232, and.
authors.forename: Laurence J
authors.initials: LJ
authors.lastname: Zwiebel
authors.affiliation: School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand,; Plant & Food Research, Private Bag 92169, Auckland 1142, New Zealand.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Newcomb
authors.affiliation: School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand,. Electronic address: d.christie@auckland.ac.nz.
authors.forename: David L
authors.initials: DL
authors.lastname: Christie
mesh_terms: D000495:Allosteric Site; D000818:Animals; D019921:Biotinylation; D002118:Calcium / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D004252:DNA Mutational Analysis; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics*; D004331:Drosophila melanogaster; D000939:Epitopes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D007473:Ion Channels / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009812:Odorants; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018035:Receptors, Odorant / Q000737:chemistry / Q000235:genetics*; D013864:Thioglycolates / Q000737:chemistry; D014230:Triazoles / Q000737:chemistry
chemical_list: D029721:Drosophila Proteins; D000939:Epitopes; D007473:Ion Channels; C572612:N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)thio)acetamide; C496847:OR22a protein, Drosophila; C492465:Orco protein, Drosophila; D018035:Receptors, Odorant; D013864:Thioglycolates; D014230:Triazoles; D003545:Cysteine; D002118:Calcium
keywords: 7-Helix Ligand-gated Channel; Channel Activation; Cysteine Residues; Insect; Ion Channel; Mutagenesis; Neurobiology; Olfaction; Receptor Structure-Function