Cholesterol differentially modulates the activity of opioid and muscarinic receptors via a common binding site

G protein-coupled receptors (GPCRs) are membrane proteins that represent the largest and most therapeutically targeted receptor class, accounting for 30% of currently marketed drugs. Two binding motifs for membrane cholesterol, the cholesterol recognition amino acid consensus (CRAC) domain and the cholesterol consensus motif (CCM), have been postulated. Using a simulation of the molecular dynamics of cholesterol association with the receptor, we predicted 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 to muscarinic and opioid receptors at TM6, with arginine 6.35 as the major residue. Membrane cholesterol depletion mimics the effects of R 6.35 mutations, confirming its role in receptor modulation. Targeting cholesterol-binding sites offers novel pharmacotherapeutic strategies, including tissue-specific sterol-based modulation.