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.