A reinvestigation of the fatty acid content of bovine, rat and frog retinal rod outer segments

The visual pigment rhodopsin is a prototypical member of the G protein-coupled receptor superfamily. In this study, we have investigated the effect of a series of n-alcohols on the formation of metarhodopsin II (MII), the photoactivated conformation of rhodopsin, which binds and activates transducin. 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.The mechanism of action of alcohols and general anesthetics is generally discussed in terms of two opposing mechanisms. The first involves an alteration of phospholipid bilayer properties by these agents, resulting in a modulation of membrane protein function, while the second is based on the direct interaction with membrane proteins (1, 2). The lipid mechanism was based originally on the observation (3, 4) that anesthetic potency correlated directly with the solubility of an anesthetic in olive oil. Additional support for the lipid mechanism came from the observation that acute exposure of membranes to ethanol resulted in a disordering of the phospholipid acyl chain packing (5). Subsequent experiments demonstrating that the activity of a soluble enzyme, firefly luciferase, could be inhibited by a diverse group of alcohols and general anesthetics (6) have focused attention on the protein binding hypothesis.Many of the recent studies aimed at elucidating the mechanism of action of alcohols and general anesthetics have dealt with the effect of these agents on ligand-gated channels, whose ligand binding site is in a portion of the protein external to the bilayer (1, 7-10). The superfamily of G protein-coupled receptors has received relatively little study. In these receptors, the ligand binding sites are formed by their transmembrane helical segments and lie at the median point of the bilayer. One of the best characterized members of this superfamily is rhodopsin, which triggers the visual transduction pathway in rod cells. A metastable equilibrium between MI 1 and MII (whereis established within milliseconds of photon absorption (11). MII binds and activates the visual G protein, transducin (12, 13). MII formation increases with higher levels of phospholipid acyl chain u...

Section: Resultssupporting

confidence: 70%

Primary Alcohols Modulate the Activation of the G Protein-coupled Receptor Rhodopsin by a Lipid-mediated Mechanism

Mitchell

Lawrence

Litman

1996

“…ROS phospholipids are composed of ϳ45% PC, 42% PE, and 13% PS (17,26), and these phospholipids are enriched in the highly unsaturated long fatty acid docosahexaenoic acid (22:6n-3) (17,21,26). The ROS membrane also contains a substantial amount of cholesterol (12-14%) (46,47).…”

Section: Discussionmentioning

confidence: 99%

Dynamics of Arrestin-Rhodopsin Interactions

Sommer

Smith

Farrens

2006

We report that acidic phospholipids can restore the binding of visual arrestin to purified rhodopsin solubilized in n-dodecyl-␤-Dmaltopyranoside. We used this finding to investigate the interplay between arrestin binding and the status of the retinal chromophore ligand in the receptor binding pocket. Our results showed that arrestin can interact with the late photoproduct Meta III and convert it to a Meta II-like species. Interestingly in these mixed micelles, the release of retinal and arrestin was no longer directly coupled as it is in the native rod disk membrane. For example, up to ϳ50% of the retinal could be released even though arrestin remains bound to the receptor in a long lived complex. We anticipate that this new ability to study these proteins in a defined, purified system will facilitate further structural and dynamic studies of arrestinrhodopsin interactions.The integral membrane protein rhodopsin (Rho) 2 enables the conversion of light to nerve signals in the rod cells, resulting in dim light vision (1-3). In the dark, the chromophore (11-cis-retinal) is linked to Rho at Lys 296 by a protonated Schiff base ( max ϳ 500 nm). Light absorption isomerizes the chromophore to all-trans-retinal. Within milliseconds two photoproducts evolve, Meta I ( max ϳ 480 nm) and Meta II ( max ϳ 380 nm), which are in a pH-and temperature-sensitive equilibrium.The ability of Meta II to bind and activate the G-protein transducin (4) is terminated in several ways. Meta II can decay through hydrolysis of the Schiff base linkage and release of retinal, a process that takes ϳ1 min at physiological temperature and pH (5). Alternatively Meta II can decay to the long lived retinal storage photoproduct Meta III ( max ϳ 470 nm) in which the Schiff base is intact and protonated (6 -9). Finally Rho signaling can be blocked through a series of protein-protein interactions that involves phosphorylation of the C-terminal tail of Rho by Rho kinase and binding of the protein arrestin (10).Earlier we found that these inactivation mechanisms are related. That is, arrestin release and retinal release appear to be directly linked events: both are described by similar activation energies, and arrestin slows the rate of retinal release ϳ2-fold at physiological temperatures. Intriguingly we also found that a fraction of the arrestin remains bound to ROS*-P long after "active" Meta II decay (5).In the present work, we expanded upon these studies and made several surprising discoveries. First, we found that adding phospholipids restores arrestin binding to purified Rho*-P solubilized in n-dodecyl-␤-D-maltopyranoside (DM). Second, we clearly established in the mixed micelle system that arrestin interacts with the post-Meta II photodecay product Meta III. Finally we found that arrestin and retinal release appear to be unlinked in mixed micelles with the acidic phospholipids PS, PI, and PA showing a more pronounced effect than the neutral phospholipids PC and PE ( Fig. 1 shows a scheme of the different lipid head groups). Intriguingly with the ...

“…Retinas were suspended in ice-cold Kuhn's buffer (0.05 M monobasic potassium phosphate, 0.015 M dibasic potassium phosphate, 1 mM Mg(OAc) 2 , 1 mM dithiothreitol, 0.1 mM EDTA, pH 7.0, sparged with argon). ROS were sheared from the retinas by mechanical shaking (35), purified with discontinuous sucrose gradients (27.0%, 28.7%, 34.0%, and 38.0%), and stored in liquid nitrogen. The purity of the rod outer segments was determined by measuring the ratio of total protein absorbing at 280 nm to photoactivatable rhodopsin.…”

Section: Methodsmentioning

confidence: 99%

Equilibrium between Metarhodopsin-I and Metarhodopsin-II Is Dependent on the Conformation of the Third Cytoplasmic Loop

Piscitelli

Angel

Bailey

et al. 2006

Rhodopsin is a G-protein-coupled receptor (GPCR) that is the light detector in the rod cells of the eye. Rhodopsin is the best understood member of the large GPCR superfamily and is the only GPCR for which atomic resolution structures have been determined. However, these structures are for the inactive, dark-adapted form. Characterization of the conformational changes in rhodopsin caused by light-induced activation is of wide importance, because the metarhodopsin-II photoproduct is analogous to the agonist-occupied conformation of other GPCRs, and metarhodopsin-I may be similar to antagonist-occupied GPCR conformations. In this work we characterize the interaction of antibody K42-41L with the metarhodopsin photoproducts. K42-41L is shown to inhibit formation of metarhodopsin-II while it stabilizes the metarhodopsin-I state. Thus, K42-41L recognizes an epitope accessible in dark-adapted rhodopsin and metarhodopsin-I that is lost upon formation of metarhodopsin-II. Previous work has shown that the peptide TGALQERSK is able to mimic the K42-41L epitope, and we have now determined the structure of the K42-41L-peptide complex. The structure demonstrates a central role for elements of the rhodopsin C3 loop, particularly Gln 238 and Glu 239 , in the interaction with K42-41L. Geometric constraints taken from the antibody-bound peptide were used to model the epitope on the rhodopsin surface. The resulting model suggests that K42-41L locks the C3 loop into an extended conformation that is intermediate between two compact conformations seen in crystal structures of dark-adapted rhodopsin. Together, the structural and functional data strongly suggest that the equilibrium between metarhodopsin-I and metarhodopsin-II is dependent upon the conformation of the C3 loop. The biological implications of this model and its possible relations to dimeric and multimeric complexes of rhodopsin are discussed.Rhodopsin is a G-protein-coupled receptor (GPCR) 2 characterized by seven transmembrane helices (H1 through H7) (1). It is an efficient photoreceptor, with the chromophoric retinal cofactor undergoing photoisomerization from 11-cis to all-trans upon activation. The retinal isomerization propagates conformational changes through the lightexcited rhodopsin protein to a meta-stable active state, metarhodopsin-II, which is in equilibrium with the inactive metarhodopsin-I precursor (see Fig. 1A). Metarhodopsin-II binds to the heterotrimeric G-protein transducin (Gt) and triggers GDP release followed by GTP uptake (2, 3).A large fraction of existing drugs and drugs under development target GPCRs (4). The rhodopsin system is considered a prototypical model for the GPCR superfamily, and recent work has produced new insights on the biochemistry of rhodopsin (1, 2). However, many questions remain unanswered, particularly with respect to the mechanism of receptor and G-protein activation.Metarhodopsin-II is characterized by its specific ability to bind and activate multiple copies of the heterotrimeric G-protein transducin (Gt ␣␤␥ ) in rapid s...