FRET Sensors Reveal the Retinal Entry Pathway in the G Protein-Coupled Receptor Rhodopsin

Abstract

The visual photoreceptor rhodopsin (Rho) represents a curious case among G protein-coupled receptors (GPCRs) because its primary ligand 11-cis-retinal (11CR) is an inverse agonist, which partitions into the bilayer and enters through an intermembranous pathway. Light activates Rho by converting 11CR to all-trans-retinal (ATR), which serves as an agonist ligand. We used genetic code expansion and bioorthogonal labeling to engineer Rho mutants that serve as FRET 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.