Entry Pathway for the Inverse Agonist Ligand in the G Protein-Coupled Receptor Rhodopsin

While the number of high-resolution structures of ligand-bound G protein-coupled receptors (GPCRs) has been steadily climbing, ligand binding and unbinding pathways remain largely undefined. The visual photoreceptor rhodopsin (Rho) represents a curious case among GPCRs because its primary ligand 11-cis-retinal (11CR) is an inverse agonist, which partitions into the bilayer and is likely to enter its orthosteric binding pocket through an intermembranous pathway. Light activates Rho by converting 11CR to all-trans-retinal (ATR), which serves as an agonist ligand. The light-triggered switch from the inactive to the active conformation creates two openings in the transmembrane region, suggesting pathways for exit of ATR and subsequent entry of 11CR to regenerate Rho. However, stabilization of an active ligand-free opsin conformation has been found to inhibit 11CR binding. Here we address this paradox of opsin regeneration with 11CR. We used genetic code expansion to engineer Rho mutants that serve as fluorescence sensors for measuring 11CR binding kinetics and energetics. We found mutations that alter a channel between transmembrane helices 5 and 6 (TM5/6) dramatically affect 11CR binding kinetics, but not ATR release kinetics. Our data provide direct experimental evidence for 11CR entry between TM5/6 in Rho that involves dynamic allosteric control of the ligand entry channel. Our findings can be extended to other visual pigments and a wide range of GPCRs with hydrophobic ligands that are hypothesized to enter their binding pockets through transmembrane pores.