G-Protein in Retina

   The equilibrium concentration of MII, the G protein-activating form of photoactivated rhodopsin, increased as a function of ethanol concentration in lipid bilayers, and polyunsaturated phospholipid acyl chains increased ethanol's effect by 40%. Enhanced formation of MII correlated with the effects of ethanol on acyl chain packing properties, supporting a phospholipid-mediated mechanism for the action of ethanol in modulating integral membrane receptor conformation. Mitchell DC, Litman BJ. Effect of ethanol on metarhodopsin II formation is potentiated by phospholipid polyunsaturation. Biochemistry 1994 Nov 1;33(43):12752-6.

Photo-activation of rhodopsin triggers transient binding of the peptide, resulting in measurable dipolar contributions to 1J(NH) and 1J(CH) splittings. The N-terminal eight residues of the bound undecapeptide adopt a near-ideal alpha-helical conformation terminated by an alpha(L) type C-cap, with Gly9 at the C' position in the center of the reverse turn. The angle between the helix axis and the membrane normal is 40 degrees (4) degrees. Peptide protons that make close contact with the receptor are identified by analysis of the NOESY cross-relaxation pattern and include the hydrophobic C terminus of the peptide. Koenig BW, Kontaxis G, Mitchell DC, Louis JM, Litman BJ, Bax A. Structure and orientation of a G protein fragment in the receptor bound state from residual dipolar couplings. J Mol Biol 2002 Sep 13;322(2):441-461.

Cholesterol mediates the function of the G protein-coupled receptor, rhodopsin, by reducing acyl chain packing free volume, rather than interacting specifically with rhodopsin. Niu SL, Mitchell DC, Litman BJ. Manipulation of cholesterol levels in rod disk membranes by methyl-beta-cyclodextrin: effects on receptor activation. J Biol Chem 2002 Jun 7;277(23):20139-45. The association constant (K(a)) for metarhodopsin II (MII) and transducin (G(t)) binding was reduced in both 18:0,22:6PC and 18:0,18:1PC by inclusion of 30 mol% cholesterol in the membrane. These findings demonstrate that membrane compositions can alter the signaling cascade by changing protein-protein interactions occurring predominantly in the hydrophilic region of the proteins, external to the lipid bilayer. A similar loss in efficiency of receptor-G protein binding may contribute to the loss of cognitive skills, odor and spatial discrimination, and visual function associated with n-3 fatty acid deficiency. Niu SL, Mitchell DC, Litman BJ. Optimization of receptor-G protein coupling by bilayer lipid composition II: formation of metarhodopsin II-transducin complex. J Biol Chem 2001 Nov 16;276(46):42807-11.

Incorporation of 30 mol% cholesterol slowed the rate of MII-G(t) complex formation by about 400% in 18:0,18:1PC, but by less than 25% in 18:0,22:6PC bilayers. In 18:0,22:6PC, with or without cholesterol, MII-G(t) formed rapidly after MII formed. In contrast, cholesterol in 18:0,18:1PC induced a considerable lag time in MII-G(t) formation after MII formed. These results demonstrate that membrane composition is a critical factor in determining the temporal response of a G protein-coupled signaling system. Mitchell DC, Niu SL, Litman BJ. Optimization of receptor-G protein coupling by bilayer lipid composition I: kinetics of rhodopsin-transducin binding. J Biol Chem 2001 Nov 16;276(46):42801-6.

Signaling in DHA bilayers was far less affected by the addition of cholesterol than in bilayers containing less unsaturated phospholipids. These studies point up the importance of DHA acyl chains in promoting optimal function in G protein-coupled signaling pathways. The results reported here suggest that visual and cognitive deficits observed in n-3 deficiency may result from decreased efficiency in related neurotransmitter and visual signaling pathways in the absence of DHA. J Mol Neurosci 2001 Apr-Jun;16(2-3):237-42; discussion 279-84. The role of docosahexaenoic acid containing phospholipids in modulating G protein-coupled signaling pathways: visual transduction. Litman BJ, Niu SL, Polozova A, Mitchell DC.

The effective range of the rhodopsin-lipid interactions facilitating cluster formation exceeds two adjacent lipid layers. In similar mixed bilayers containing no cholesterol, cluster formation was absent at temperatures above lipid phase transition, indicating a crucial role of cholesterol in microdomain formation. Polozova A, Litman BJ. Cholesterol dependent recruitment of di22:6-PC by a G protein-coupled receptor into lateral domains. Biophys J 2000 Nov;79(5):2632-43. Mitchell DC, Gawrisch K, Litman BJ, Salem N Jr. Why is docosahexaenoic acid essential for nervous system function? Biochem Soc Trans 1998 Aug;26(3):365-370.

When rhodopsin was photolyzed in the presence of several n-alcohols, increased MII formation was observed in the order ethanol > butanol > hexanol, whereas longer chain n-alcohols inhibited MII formation with decanol > octanol. The magnitude of the stimulatory effects was greater in a more highly unsaturated phospholipid. Alcohols, which enhanced MII formation also increased phospholipid acyl chain packing free volume, while those that decreased this bilayer property inhibited MII formation. An apparent discontinuity in the effect of these alcohols results when their potency is calculated in terms of the total aqueous alcohol concentration. In sharp contrast, a continuous variation in their behavior is observed, when their potency is calculated in terms of the amount of alcohol partitioned in the membrane. Our findings strongly support a lipid-mediated mechanism of action for alcohols on rhodopsin and, by analogy, for other G protein-coupled receptors. Mitchell DC, Lawrence JT, Litman BJ. Primary alcohols modulate the activation of the G protein-coupled receptor rhodopsin by a lipid-mediated mechanism. J Biol Chem 1996 Aug 9;271(32):19033-6.

The extent of formation of metarhodopsin II (MII), the G protein--activating photointermediate of rhodopsin, increased progressively with the level of acyl chain unsaturation at the sn-2 position. Added cholesterol reduced the amount of MII. The acyl chain packing free volume of the rhodopsin containing lipid vesicles was characterized by derived from measurements of the time-resolved fluorescence anisotropy decay of the hydrophobic membrane probe 1,6-diphenyl-1,3,5-hexatriene. The relationship among sn-2 acyl chain unsaturation, cholesterol content, and MII formation is explained on the basis of variation in a fractional volume parameter with bilayer lipid composition and a novel model for the packing of phospholipids containing polyenoic acyl chains, such as 22:6n-3.Litman BJ, Mitchell DC. A role for phospholipid polyunsaturation in modulating membrane protein function. Lipids 1996 Mar;31 Suppl:S193-7.

Phospholipid composition had a marked effect on ethanol potency, with the presence of polyunsaturated phospholipid acyl chains increasing ethanol potency by 40%. The effects of ethanol on lipid acyl chain packing in POPC and PDPC were investigated using frequency domain anisotropy decay measurements of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene. Enhanced formation of MII due to the presence of ethanol was correlated with the effects of ethanol on acyl chain packing properties. These findings support a phospholipid-mediated mechanism for ethanol action in modulating integral membrane receptor conformation.Mitchell DC, Litman BJ. Effect of ethanol on metarhodopsin II formation is potentiated by phospholipid polyunsaturation. Biochemistry 1994 Nov 1;33(43):12752-6.

For mixtures of nonlamellar-forming dioleoylphosphatidylethanolamine (DOPE) with dioleoylphosphatidylcholine (DOPC), calculations of the membrane surface pH confirmed that an increase in DOPE favored the meta II state. Rhodopsin may function to balance forces within the bilayer with a negative spontaneous curvature. A new biophysical principle is introduced: matching of the spontaneous curvature of the lipid bilayer to the mean curvature of the lipid/water interface adjacent to the protein, which balances the lipid/protein solvation energy. In this manner, the thermodynamic driving force for the meta I-meta II conformational change of rhodopsin is tightly controlled by mixtures of nonlamellar-forming lipids having distinctive material properties.Botelho AV, Gibson NJ, Thurmond RL, Wang Y, Brown MF. Conformational energetics of rhodopsin modulated by nonlamellar-forming lipids. Biochemistry 2002 May 21;41(20):6354-68.