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Phosphorylation alters the pH-dependent active state equilibrium of rhodopsin by modulating the membrane surface
S K Gibson1, J H Parkes, P A Liebman
1Department of Biochemistry and Biophysics, University of Pennsylvania Medical Center, Philadelphia 19104-6059, USA.
Abstract:
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.
Insights
Phosphorylation paradoxically enhances G protein-coupled receptor signaling by shifting the metarhodopsin I-II equilibrium toward the active MII state. This shift is explained by altered membrane surface potential due to phosphorylation, affecting surface pH and MII formation kinetics.
Area of Science:
- Biochemistry
- Biophysics
- Molecular Biology
Background:
- G protein-coupled receptors (GPCRs) are crucial signaling proteins.
- Phosphorylation typically reduces GPCR activity and lifetime.
- Light-activated rhodopsin's metarhodopsin I-II (MI-MII) equilibrium shifts towards MII (active state) upon phosphorylation, a paradoxical observation.
Purpose of the Study:
- To investigate the mechanism behind phosphorylation-induced shift in the MI-MII equilibrium.
- To elucidate the role of membrane surface potential and pH in this process.
- To determine the protonation stoichiometry and pK of the site involved in MII formation.
Main Methods:
- Spectroscopic analysis of light-activated rhodopsin.
- Varying phosphorylation stoichiometry and ionic strength.
- Application of Gouy-Chapman theory to model membrane surface potential and pH.
- Kinetic analysis of MI-MII relaxation.
Main Results:
- Phosphorylation increases the apparent pK for MII formation, proportional to phosphorylation stoichiometry.
- Decreasing ionic strength amplifies the effect of phosphorylation on pK.
- Gouy-Chapman theory quantitatively explains the pK changes via altered membrane surface potential and surface pH.
- Observed fractional H+ dependence of MII formation is an artifact of changing surface charge during titration.
- MII formation is consistent with titration of a single H+ binding site with 1:1 stoichiometry and an intrinsic pK of 6.3 at 0.5°C.
- Protonation of this site before light activation enhances MII formation rate.
Conclusions:
- Phosphorylation-induced changes in membrane surface potential and pH drive the MI-MII equilibrium shift.
- The apparent fractional H+ dependence is an artifact, with actual 1:1 H+ stoichiometry for MII formation.
- A single protonation site with distinct pKs before and after activation governs MII formation kinetics.