Protonation Switches in GPCR Activation: Physiologically Significant Rhodopsin Photointermediates

Abstract

Rhodopsin is a paradigm for GPCRs, yet unlike other class A members, its bound chromophore's UV/vis absorbance provides excellent time-resolved information about GPCR activation steps. At least three species equilibrate on the millisecond time scale after rhodopsin photoexcitation in a membrane lipid environment. The first equilibrium is pH-independent and involves Meta I480, the visible absorbing, protonated Schiff base (PSB) species, and Meta IIa, the UV absorbing, deprotonated SB species. The second equilibrium, involving spectrally silent proton uptake by Meta IIa to produce Meta IIb, accounts for the fact that low pH causes anomalous disappearance of the protonated SB species, Meta I480. The equilibria affect production of the G protein-activating species R∗ and are of great interest. However they must be studied promptly because inactivation steps follow, and long illumination increases secondary photolysis of photoproducts. We used time-resolved absorbance measurements of bovine rhodopsin on the microsecond-to-hundred millisecond time scale to study kinetics of lumirhodopsin decay and the effect of membrane environment on the first equilibrium constant, K1, and on the pKa of the second equilibrium. Reconstituted membranes of rhodopsin with POPC, DOPC, or a mixture of DOPC and DOPE were studied at 30°C. We also extended previous 20°C studies of the pH dependence of the equilibria in the native disk membranes, to determine how increased temperature affects lumirhodopsin decay through the purely transient 380 nm absorbing species, Meta I380, into the final equilibrium mixture. Meta I380 has recently attracted substantial interest, since time-resolved circular dichroism measurements on the microsecond timescale suggest the chromophore has a different conformation than in later 380-nm photointermediates. Our results suggest SB deprotonation precedes other activating changes in the protein. Significant details are now emerging that give new insights into rhodopsin activation and complement FTIR and spin-label approaches.