Rhodopsin In Photoreceptor Membranes Research Articles

Dark-adaptation in the eyes of a lake and a sea population of opossum shrimp (Mysis relicta): retinoid isomer dynamics, rhodopsin regeneration, and recovery of light sensitivity

We have studied dark-adaptation at three levels in the eyes of the crustacean Mysis relicta over 2–3 weeks after exposing initially dark-adapted animals to strong white light: regeneration of 11-cis retinal through the retinoid cycle (by HPLC), restoration of native rhodopsin in photoreceptor membranes (by MSP), and recovery of eye photosensitivity (by ERG). We compare two model populations (“Sea”, Sp, and “Lake”, Lp) inhabiting, respectively, a low light and an extremely dark environment. 11-cis retinal reached 60–70% of the pre-exposure levels after 2 weeks in darkness in both populations. The only significant Lp/Sp difference in the retinoid cycle was that Lp had much higher levels of retinol, both basal and light-released. In Sp, rhodopsin restoration and eye photoresponse recovery parallelled 11-cis retinal regeneration. In Lp, however, even after 3 weeks only ca. 25% of the rhabdoms studied had incorporated new rhodopsin, and eye photosensitivity showed only incipient recovery from severe depression. The absorbance spectra of the majority of the Lp rhabdoms stayed constant around 490–500 nm, consistent with metarhodopsin II dominance. We conclude that sensitivity recovery of Sp eyes was rate-limited by the regeneration of 11-cis retinal, whilst that of Lp eyes was limited by inertia in photoreceptor membrane turnover.

Small-angle neutron and X-ray scattering analysis of the supramolecular organization of rhodopsin in photoreceptor membrane

The supramolecular organization of the visual pigment rhodopsin in the photoreceptor membrane remains contentious. Specifically, whether this G protein-coupled receptor functions as a monomer or dimer remains unknown, as does the presence or absence of ordered packing of rhodopsin molecules in the photoreceptor membrane. Completely opposite opinions have been expressed on both issues. Herein, using small-angle neutron and X-ray scattering approaches, we performed a comparative analysis of the structural characteristics of the photoreceptor membrane samples in buffer, both in the outer segment of photoreceptor cells, and in the free photoreceptor disks. The average distance between the centers of two neighboring rhodopsin molecules was found to be ~5.8 nm in both cases. The results indicate an unusually high packing density of rhodopsin molecules in the photoreceptor membrane, but molecules appear to be randomly distributed in the membrane without any regular ordering.

Interaction of GTP-binding protein Gq with photoactivated rhodopsin in the photoreceptor membranes of crayfish

Interaction of G-protein with photoactivated rhodopsin (Rh ∗) in crayfish photoreceptor membranes was investigated by immunoprecipitation using an antibody against rhodopsin. Two kinds of protein were co-precipitated with rhodopsin. One is an α subunit of class-q G-protein (42 kDa, CGqα) which showed light-induced, dose-dependent binding to rhodopsin, and the other is an actin-like protein (44 kDa) with light-independent binding. Most of the CGqα was available for binding to Rh ∗ but was dissociated from Rh ∗ in the presence of GTPγS. These findings demonstrate that, in the crayfish photoreceptor, a Gq class of G-protein is activated by Rh ∗.

Purification of squid rhodopsin and reassembly into lipid bilayers

Rhodopsin from squid photoreceptor membranes was solubilized in octyl glucoside and purified to a single band on SDS-polyacrylamide gels of M r 46 000. Purified rhodopsin was recombined with phospholipids to form vesicles by detergent dialysis. Spectroscopic analysis of the rhodopsin-lipid vesicles showed that the interconversion between acid and basic metarhodopsin had a pK of 8. Furthermore, rhodopsin in the vesicles could be photoregenerated from metarhodopsin in solutions of either neutral or alkaline pH. These two spectroscopic properties are comparable to those for rhodopsin in photoreceptor membranes. The results indicate that the native conformation of rhodopsin is preserved during purification and after recombination with phospholipids into vesicles. This preparation is, therefore, an active starting point for functional reconstitution studies.

Investigation of the organization of rhodopsin in the sheep photoreceptor membrane by using cross-linking reagents.

The organization of rhodopsin in the photoreceptor membrane of sheep rod outer segments was investigated by using a variety of bifunctional reagents. Of the nine reagents used, seven gave oligomeric opsin species, whereas two, copper phenanthroline and dithiobisphenyl azide, failed to cross-link the protein. In general, the cross-linked species obtained showed diminishing yields from dimer to tetramer, together with some higher-molecular-weight aggregates. It is proposed that the patterns of cross-linking arise as a result of collision complexes and best describe a monomeric organization for native rhodopsin. No significant differences between the patterns obtained with dark-adapted bleached or regenerated protein states were observed. This interpretation is discussed in relation to the postulated mechanism of action of rhodopsin.

Biochemical aspects of the visual process: XXXIX. Sulfhydryl group reactivity as a probe of transient protein conformational changes during rhodopsin photolysis

1. 1.|The number of sulfhydryl groups available to 5,5′-dithiobis-(2-nitrobenzoic acid) during the photolysis of bovine rhodopsin in photoreceptor membranes has been determined. Under most conditions two sulfhydryl groups react per mole of rhodopsin, in agreement with earlier observations. However, an additional sulfhydryl group is detected if during illumination the chromophore is reductively linked to the protein with NaBH 4. This group does not arise from the cleavage of a disulfide bridge. 2. 2.|This phenomenon is transient and reduction with NaBH 4 at increasing time intervals after illumination results in a gradual disappearance of the additional sulfhydryl group. 3. 3.|In parallel, the reaction rate of the rhodopsin sulfhydryl groups towards 5,5′-dithiobis-(2-nitrobenzoic acid) is greatly increased in this reductively stabilized photointermediate. 4. 4.|It is suggested that this stabilized photointermediate represents a transitional state between metarhodopsin I and metarhodopsin II and then our observations indicate that sulfhydryl group reactivity transiently increases during the photolysis of rhodopsin. 5. 5.|In conclusion, the availability of an additional sulfhydryl group to 5,5′-dithiobis-(2-nitrobenzoic acid) is though to reflect transient protein conformational changes and/or changes in protein-lipid interactions during the photolysis of rhodopsin. Their maximal expression during the metarhodopsin I to metarhodopsin II transition suggests that these changes may play an essential role in the process of visual excitation.

Organization of rhodopsin in photoreceptor membranes. 2. Transmembrane organization of bovine rhodopsin: evidence from proteolysis and lactoperoxidase-catalyzed iodination of native and reconstituted membranes.

Proteolysis of reconstituted membranes with papain and thermolysin reveals the existence of two rhodopsin populations: one susceptible to proteolysis and the other protected. The susceptible population corresponds to rhodopsin molecules with the same orientation as rhodopsin in the native membrane, while the protected population corresponds to "inverted" rhodopsin molecules only found in reconstituted membranes. Using an iodination enhancement probe, we demonstrate that lactoperoxidase catalyzes iodination of rhodopsin exclusively on the external surface of these sealed reconstituted vesicles. Furthermore, we find that both rhodopsin populations in reconstituted membranes (normal and inverted) are readily iodinated by lactoperoxidase, providing definitive evidence that the rhodopsin polypeptide spans the membrane thickness. Additional conclusions from these experiments are discussed in terms of a model for the folding of the rhodopsin polypeptide in the membrane.

Organization of rhodopsin in photoreceptor membranes. 1. Proteolysis of bovine rhodopsin in native membranes and the distribution of sulfhydryl groups in the fragments.

Papain and thermolysin are shown to cleave bovine rhodopsin in native membranes in two temporally distinct steps at room temperature. The final product of the proteolysis consists of two membrane-bound fragments of molecular weights 27 000 (Rh27) and 12 500 (Rh12). The molecular weights are not changed by reduction with dithiothreitol. The two fragments remain closely associated in both the membrane and nondenaturing detergents before and after bleaching and can be selectively cross-linked with carbodiimides. The sulfhydryl chemistry of the cleaved protein in nearly indistinguishable from native rhodopsin, and of the total of six sulfhydryl groups, two are located on Rh12 and four on Rh27. In the membrane-bound protein, two sulfhydryl groups are accessible for modification, one on Rh12 and the other on Rh27. The sulfhydryl on Rh12 is particularly reactive and may be selectively labeled with maleimides. Continuous irradiation with white light induces additional sulfhydryl reactivity on Rh27.

A proposed model for rhodopsin in photoreceptor membranes.

Transient electric birefringence studies have been made on bovine rhodopsin solubilized in the detergent lauryldimethylamine oxide from glutaraldehyde fixed rod outer segment (ROS) membranes. It was found that fixation caused no appreciable differences in the measured relaxation times when compared with unfixed ROS. On the basis of these findings a model for the orientation of rhodopsin in photoreceptor membranes is proposed which accounts for translational diffusion and two modes of rotational diffusion. The proposed model is related to a number of experimentally determined biophysical properties reported in the literature.

Thermal stability of squid rhodopsin in photoreceptor membrane and their triton extracts

Kinetic parameters of the thermal decay of squid rhodopsin in isolated photoreceptor membranes and in a 1% Triton X-100 solution have been determined. It has been shown that the treatment with Triton X-100 considerably decreases thermal stability of rhodopsin. Addition of hydroxylamine (0.5 m) to Triton X-100 solutions leads to further decrease of rhodopsin thermostability. When Triton extracts are exposed to light, bleaching of visual pigments occurs at room temperature.