Decoding human olfaction by high heterologous expression of odorant receptors detecting signature odorants

Humans have 391 odorant receptor (OR) genes. Due to their poor functional expression in vitro, reproducible receptor-ligand pairs are known for only a minority of them, and the pharmacology of human olfaction is still poorly understood. Using a well-expressed OR as a template, the C-terminal domains of two weakly expressed class I and class II ORs were engineered by several rounds of mutations, leading to dramatically increased sensitivity paralleled by increased cell-surface expression. These optimized C-terminal domains facilitated high surface expression and strongly increased the sensitivity of the assay for many ORs. A human OR library with these C termini allowed us to identify novel ORs for key natural signature odorants such as (-)ambrox, the key odorant from the sperm whale secretion Ambergris; the pepper note rotundone; (+)-nootkatone from grapefruit; and various floral, fruity, and food-related impact odorants. ORs for off-odors, such as the OR detecting 2,4,6-trichloroanisole-the cork note in wine-could equally be identified. Many of these newly de-orphanized ORs have sensitivities in the nanomolar range and unique specificities based on screening of stereoisomers and structural analogs. Thus, a comparison of OR activation by 13 differently smelling stereoisomers of ambrox reveals an unprecedented view of the stereoselectivity of a human OR. These data refine our view of the sensitivity and specificity of human ORs and challenge the combinatorial model of odor coding at the OR level.