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 Ca2+ 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.
Highlights
Odorant receptors (Ors) are one of the main insect chemosensory receptor families required to sense olfactory cues in the environment [1]
As conserved acidic amino acids are known to have a role in ion permeation and gating of cation channels [23,24,25], we have investigated the importance of conserved Asp residues in predicted TMs 5 and 7 of DmelOrco
It is likely that an Asp, or an acidic amino acid, at position 466 is critical for DmelOrco function
Summary
Odorant receptors (Ors) are one of the main insect chemosensory receptor families required to sense olfactory cues in the environment [1]. The conventional Ors are highly divergent and provide selectivity to a broad range of odorant compounds [11] Their expression is restricted to specific olfactory receptor neurons (ORNs) [11,12,13]. Orco is capable of forming functional channels in the absence of a conventional receptor [6,16]. These homomeric channels can be activated directly by the agonist VUAA1 [16] and its analogues [17]. Studies with several heteromeric Or complexes indicate that the presence of an odorant-specific Or can alter the properties of the channel pore [20,21,22]
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