Rhodopsin Activation in Membranes: Thermodynamic Model of the Two Protonation Switches

Abstract

Activation of the G protein-coupled receptor (GPCR) rhodopsin is initiated by light-induced isomerization of the retinal ligand, which triggers two protonation switches in the conformational transition to the active receptor state. Disruption of an interhelical salt bridge occurs by deprotonation of the retinal protonated Schiff base (PSB) together with uptake of a proton from the solvent by Glu134 of the conserved cytoplasmic E(D)RY motif. Using a combination of UV-visible and Fourier-transform infrared (FTIR) spectroscopy of rhodopsin, we access deprotonation of the PSB and activating conformational changes of the helix bundle separately, and study the pH-dependent activation mechanism of rhodopsin in different membrane environments. The data are analyzed using a thermodynamic framework based on the Hofmann-Hubbell scheme derived for rhodopsin in detergent, distinguishing between PSB deprotonation, activating conformational changes, and cytoplasmic proton uptake in the transitions from Meta I to Meta IIa, Meta IIb, and Meta IIbH+, respectively. While a classical Henderson-Hasselbalch-like equilibrium between Meta I and Meta IIbH+ is observed at 10 °C, more complex titration curves with non-zero alkaline endpoints are found above 20 °C. These reflect partial population of an entropy-stabilized Meta IIb state, in which the PSB salt bridge is broken and activating helix movements have taken place, but where Glu134 remains unprotonated. This partial activation is converted to full activation only by coupling to the pH-dependent protonation of Glu134 from the solvent, which enthalpically stabilizes the active receptor conformation. In a membrane environment, protonation of Glu134 is therefore a thermodynamic rather than a structural requirement for activating helix movements. In light of conservation of the E(D)RY motif in rhodopsin-like GPCRs, protonation of this carboxylate may serve a similar function in signal transduction of other members of this important receptor family.