Cholesterol differentially modulates the activity of opioid and muscarinic receptors via common non-canonical binding site

G protein-coupled receptors (GPCRs) are membrane proteins representing the largest and most therapeutically targeted receptor class for 30% of currently marketed drugs. Two binding motifs for membrane cholesterol, the ‘cholesterol recognition amino acid consensus (CRAC) domain and ‘cholesterol consensus motif’ (CCM), were postulated. Using a simulation of molecular dynamics we demonstrate the binding of membrane cholesterol to non-canonical sites, distinct from CRAC and CCM, at muscarinic and opioid receptors. We identified a binding site common for muscarinic and opioid receptors at TM6, arginine 6.35 being the principal residue. Effects of depletion of membrane cholesterol on the functional responses of these receptors correlated with effects of mutations arginine 6.35 on functional responses. Exploiting cholesterol-binding sites and variations in receptor-membrane interactions brings novel pharmacotherapeutic opportunities including tissue-specific targeting for sterol-based modulators.