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-coupled receptors, yet paradoxically shifts the metarhodopsin I-II (MI-MII) equilibrium (K(eq)) of light-activated rhodopsin toward MII, the conformation that activates G protein. In this report, we show that phosphorylation increases the apparent pK for MII formation in proportion to phosphorylation stoichiometry. Decreasing ionic strength enhances this effect. Gouy-Chapman theory shows that the change in pK is quantitatively explained by the membrane surface potential, which becomes more negative with increasing phosphorylation stoichiometry and decreasing ionic strength. This lowers the membrane 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 fractional H(+) dependence is an artifact of altering the membrane surface charge during a titration, resulting in a fractional change in membrane surface pH compared to bulk pH. Gouy-Chapman calculations of membrane pH at various phosphorylation levels and ionic strengths suggest MII formation behavior consistent 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 suggests this same site has an intrinsic pK of 5.0 prior to light activation and its protonation before activation greatly enhances the rate of MII formation.
Gibson
S K
SK
Department of Biochemistry and Biophysics, University of Pennsylvania Medical Center, Philadelphia 19104-6059, USA.
Parkes
J H
JH
Liebman
P A
PA
D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D009994:Osmolar Concentration; D010766:Phosphorylation; D010782:Photolysis; D011522:Protons; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D012374:Rod Cell Outer Segment / Q000502:physiology; D013053:Spectrophotometry; D013499:Surface Properties
D008565:Membrane Proteins; D011522:Protons; D012243:Rhodopsin