Phosphorylation alters the pH-dependent active state equilibrium of rhodopsin by modulating the membrane surface potential

Phosphorylation reduces the lifetime and activity of activated G protein-co upled receptors, let paradoxically shifts the metarhodopsin I-II (MI-MII) e quilibrium (K-eq) of light-activated rhodopsin toward MII, the conformation that activates G protein. In this report, we show that phosphorylation inc reases the apparent pK for MII formation in proportion to phosphorylation s toichiometry. Decreasing ionic strength enhances this effect. Gouy-Chapman theory shows that the change in pK is quantitatively explained by the membr ane surface potential, which becomes more negative with increasing phosphor ylation stoichiometry and decreasing ionic strength. This lowers the membra ne surface pH compared to the bulk pH, increasing K-eq and the rate of MII formation (k(1)) while decreasing the back rate constant (k(-1)) of the MI- MII relaxation. MII formation has been observed to depend on bulk pH with a fractional stoichiometry of 0.6-0.7 H+/MII. We find that the apparent frac tional Hf dependence is an artifact of altering the membrane surface charge during a titration, resulting in a fractional change in membrane surface p H compared to bulk pH. Gouy-Chapman calculations of membrane pH at various phosphorylation levels and ionic strengths suggest MII formation behavior c onsistent with titration of a single H+ binding site with 1:1 stoichiometry and an intrinsic pK of 6.3 at 0.5 degrees C. We show evidence that suggest s this same site has an intrinsic pK of 5.0 prior to light activation and i ts protonation before activation greatly enhances the rate of MII formation .