Differential Rhodopsin Regeneration in Photoreceptor Membranes is Correlated with Variations in Membrane Properties

Rhodopsin, the major transmembrane protein in both the plasma membrane and the disk membranes of photoreceptor rod outer segments (ROS) forms the apo-protein opsin upon the absorption of light. In vivo the regeneration of rhodopsin is necessary for subsequent receptor activation and for adaptation, in vitro this regeneration can be followed after the addition of 11-cis retinal. In this study we investigated the ability of bleached rhodopsin to regenerate in the compositionally different membrane environments found in photoreceptor rod cells. When 11-cis retinal was added to bleached ROS plasma membrane preparations, rhodopsin did not regenerate within the same time course or to the same extent as bleached rhodopsin in disk membranes. Over 80% of the rhodopsin in newly formed disks regenerated within 90 minutes while only 40% regenerated in older disks. Since disk membrane cholesterol content increases as disks are displaced from the base to the apical tip of the outer segment, we looked at the affect of membrane cholesterol content on the regeneration process. Enrichment or depletion of disk membrane cholesterol did not alter the % rhodopsin that regenerated. Bulk membrane properties measured with a sterol analog, cholestatrienol and a fatty acid analog, cis parinaric acid, showed a more ordered, less fluid, lipid environment within plasma membrane relative to the disks. Collectively these results show that the same membrane receptor, rhodopsin, functions differently as monitored by regeneration in the different lipid environments within photoreceptor rod cells. These differences may be due to the bulk properties of the various membranes.     

Binding and activation of rod outer segment phosphodiesterase and guanosine triphosphate binding protein by disc membranes: influence of reassociation method and divalent cations

Attempts to optimize the recovery of light-stimulated phosphodiesterase activity following reassociation of the hypotonically extractable proteins derived from retinal rod segments with hypotonically stripped disc membranes lead to the following observations: the best reassociations were obtained by mixing proteins and stripped disc membranes under hypotonic conditions and slowly increasing the salt concentration; the binding of G-protein and phosphodiesterase to stripped disc membrane occurs in less than 5 minutes and the recovery of light-stimulated phosphodiesterase activation in response to subsaturating stimulus levels requires 2-3 h to plateau. Stripped disc membranes and proteins were reassociated in 'isotonic' buffers containing KCl/NaCl, KCl/NaCl plus Mg2+, or KCl/NaCl plus Ca2+. Large fractional rhodopsin bleaches produced nearly identical light-stimulated phosphodiesterase activities in each of these samples and in the control rod outer segment membranes. Rod outer segment membranes and reassociated stripped disc membrane samples containing divalent cations showed similar phosphodiesterase activities in response to low fractional rhodopsin bleaches (e.g. less than or equal to 0.1%), however, samples devoid of divalent cations during reassociation required rhodopsin bleaches up to 10-fold larger to elicit comparable phosphodiesterase activities. These results suggest that not all phosphodiesterase and/or G-protein molecules bound to the disc membrane surface are equivalent with regard to their efficiency of activation by bleached rhodopsin and that divalent cations can modulate the distribution of G-protein and/or phosphodiesterase between these populations.     

Ca2+ buffer sites in intact bovine rod outer segments: Introduction to a novel optical probe to measure ionic permeabilities in suspensions of small particles

Summary The nature of the Ca²⁺ buffer sites in intact rod outer segments isolated from bovine retinas (ROS) was investigated. The predominant Ca²⁺ buffer in intact ROS was found to be negatively charged groups confined to the surface of the disk membranes. Accordingly, Ca²⁺ buffering in ROS was strongly influenced by the electrostatic surface potential. The concentration of Ca²⁺ buffer sites was about 30mm, 80% of which were located at the membrane surface in the intradiskal space. A comparison with observations in model systems suggests that phosphatidylserine is the major Ca²⁺ buffer site in ROS. Protons and alkali cations could replace Ca²⁺ as mobile counterions for the fixed negatively charged groups. At physiological ionic strength, the total number of these diffusible, but osmotically inactive, counterions was as large as the number of osmotically active cations in ROS. The surface potential is dependent on the concentration of cations in ROS and can be measured with the optical dye neutral red. Addition of cations to the external solution led to the release of the internally bound dye as the cations crossed the outer membrane. The chemical and spectral properties of the dye enable its use as a real-time indicator of cation transport across the outer envelope of small particles in suspension. In this study, the dye method is illustrated by the use of well-defined ionophores in intact ROS and in liposomes. In the companion paper this method is used to describe the cation permeabilities native to ROS.     

Effect of cGMP and cations on the permeability of cattle retinal disks

Guanosine 3',5'-monophosphate and Na or Ca ions affect the transmembrane movements of the same pool of intradiskal ions. Extradiskal Na ions activate the efflux of intradiskal Na ions. Extradiskal Ca ions activate the efflux of intradiskal Rb ions. Na and Ca ions activate Na/Ca or Ca/Ca exchange, as previously described. cGMP activates a membrane permeability for all the cations tested, as previously described. The reciprocal relations between cGMP and the other pathways for ion movements through disk membranes are systematically examined. Some analogies between the cGMP-activated permeability of the disk membranes and the light-sensitive conductance of the rod plasma outer membrane are discussed.     

Revised Amino-acid Sequences for Porcine and Human Adrenocorticotrophic Hormone

MEMBRANE protein of bovine rod outer segments has been studied by gel electrophoresis and amino-acid analysis. Membranes were purified in a sucrose density gradient¹ at an ionic strength below 0.001. The isolated material probably consisted of fragmented disk membranes¹. ‘Emulphogene’ solutions of rhodopsin were chromatographed on calcium phosphate²; the results for A₂₇₈: A₄₉₈ were 1.7–1.8, indicating good purity.     

Ultrastructural localization of rhodopsin in the vertebrate retina

Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.     

Differential scanning calorimetry of bovine rhodopsin in rod-outer-segment disk membranes.

Rhodopsin-containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 +/- 0.4 degrees C and a thermal unfolding calorimetric enthalpy change (delta Hcal) of 700 +/- 17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9 +/- 0.3 degrees C and a delta Hcal of 520 +/- 17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4-9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed delta Hcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. Delta Hcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the delta Hcal values while the Tm values remain fairly constant. This shows that the delta Hcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin.     

The isolation of stable cattle rod outer segments with an intact plasma membrane.

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter. Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH. The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.     

The isolation of stable cattle rod outer segments with an intact plasma membrane

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter.Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH.The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.     

Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination

Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. Several control experiments indicated that the labeled proteins are integral membrane proteins and that label is limited to the plasma membrane. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger.     

Calcium-hydrogen exchange in isolated bovine rod outer segments

We have measured Ca-H exchange in rod photoreceptors with different preparations of rod outer segments isolated from bovine retinas (ROS). One preparation contained ROS with an intact plasma membrane (intact ROS), and in the other preparation, the plasma membrane was leaky to small solutes (leaky ROS) and the cytoplasmic space was freely accessible to externally applied solutes. Addition of Ca2+ to Ca2+-depleted ROS (both intact and leaky) resulted in uptake of Ca2+ that was accompanied by the release of protons when catalytic amounts of the ionophore A23187 were present. This ionophore mediates Ca-H exchange transport across ROS membranes and serves to gain access to the intracellular compartment where Ca-H exchange appears to take place. Two protons were ejected for each calcium ion taken up. Conversely, when protons were added to Ca2+-enriched ROS, Ca2+ was released in the presence of A23187. The majority of this Ca-H exchange was observed only when A23187 was present in both intact and leaky ROS. We conclude that Ca-H exchange occurs predominantly in the intradiskal space and at the surface of the disk membrane rather than across the disk membrane. These exchange binding sites can accommodate 10 mol of Ca2+/mol of rhodopsin at physiological pH. We were unable to detect any Ca2+ release when a proton gradient was rapidly established across the disk membrane in the absence of A23187. These results are discussed in relation to the hypothesis that protons produced by the light-induced hydrolysis of cGMP cause the release of Ca2+ into the cytoplasm of rod photoreceptor cells.     

Rhodopsin is spatially heterogeneously distributed in rod outer segment disk membranes

The visual photoreception takes place in the retina, where specialized rod and cone photoreceptor cells are located. The rod outer segments contain a stack of 500-2,000 sealed membrane disks. Rhodopsin is the visual pigment located in rod outer segment disks, it is a member of the G-protein-coupled receptor (GPCR) superfamily, an important group of membrane proteins responsible for the majority of physiological responses to stimuli such as light, hormones, peptides, etc. Alongside rhodopsin, peripherin/Rom proteins located in the disk rims are thought to be responsible for disk morphology. Here we describe the supramolecular structure of rod outer segment disk membranes and the spatial organization of rhodopsin and peripherin/Rom molecules. Using atomic force microscopy operated in physiological buffer solution, we found that rhodopsin is loosely packed in the central region of the disks, in average about 26?000 molecules covering approximately one third of the disk surface. Peripherin/Rom proteins form dense assemblies in the rim region. A protein-free lipid bilayer girdle separates the rhodopsin and peripherin/Rom domains. The described supramolecular assembly of rhodospin, peripherin/Rom and lipids in native rod outer segment disks is consistent with the functional requirements of photoreception.     

Kinetics and saturation of light-induced near-infrared scattering changes in isolated bovine rod outer segments

The axial and radial shrinkage of bovine rod outer segments, monitored by near-infrared scattering changes (P-signal), is investigated in dependence on the intensity of the activating flash. Suspensions of axially oriented and randomly oriented rod outer segments were measured. In the latter case, axial and radial effects are superimposed to another. The following results are obtained:

1. The axial signal (P _a, Τ≈10 ms) and the radial signal (P _r, Τ=40–100 ms), simultaneously measured on axially oriented rod outer segments, are similarly saturated with a half-saturation at a rhodopsin turnover of 3.5%.
2. For the saturation of the signal amplitude, measured on randomly oriented rod outer segments, a good fit is obtained by: $$\begin{gathered} P\left( \varrho \right) \sim 1 - e\beta \varrho , \hfill \\ \varrho : relative rhodopsin turnover by the flash; \hfill \\ \beta is found in the range 23 \leqslant \beta \leqslant 27 in all measurements \hfill \\ \end{gathered} $$
3. The kinetics of the signal, also measured on the isotropic sample, depends on the rhodopsin turnover, the apparent time constant becoming faster with increasing turnover. The distortion of the signal cannot be fitted by a sum of exponentials with a fixed set of time constants.

The signals from the isotropic sample are fitted by a phenomenological model. It introduces three first order processes concatenated in series; the first step is assumed as a rhodopsin transition inducing the two further processes. The distortion of the signals with increasing? is then described assuming a?-dependent quenching of this induction, according to the measured amplitude saturation. The time constants remain thereby unchanged. The fit yields the values ln 2/k=4, 11, and 45 ms with mean square deviations of 20%.     

Formation,structure, and spectrophotometry of air-water interface films containing rhodopsin

Summary Air-water interface films of cattle rhodopsin and defined lipids are formed without the use of organic solvents by a method in which vesicle membranes consisting of egg phosphatidyl choline and purified rhodopsin are osmotically shocked at the interface. Lipid and protein molecules organize as insoluble films at the interface. The structure of these films varies with the lipid to protein mole ratio of the source vesicle membranes. Electron microscopic observations reveal that films formed with membranes of 1501 mole ratio consist of nonoverlapping, randomly distributed vesicle membrane fragments separated by a lipid monolayer. These membrane fragments exist as single sheets on the water surface and occupy approximately 35% of this surface. Essentially all the rhodopsin molecules at the interface are spectroscopically intact and are contained within the membrane fragments. The visible absorption spectrum of the interface films is identical to that of suspensions of rod disc membranes. Moreover, flash illumination of rhodopsin in air-dried multilayers formed from the interface films results in the formation of a stable MetarhodopsinI intermediate (_max480 nm) which can be fully bleached by increasing the relative humidity of the multilayers or can be photoconverted into rhodopsin and, presumably, isorhodopsin. Furthermore, rhodopsin is chemically regenerable at the air-water interface. Bleached rhodopsin can generate dark rhodopsin at the interface in the presence of 11-cis retinal in the aqueous subphase. Thus, the spectroscopic structure and the chemical regenerability function of rhodopsin in these interface films are indistinguishable from those exhibited by the protein in intact rod disc membranes.     

Light-induced interfacial potentials in photoreceptor membranes

A rapid change in an interfacial electric potential of isolated bovine rod outer segment disk membranes occurs upon illumination. This potential change, which has been detected by the use of spin-labeled hydrophobic ions, apparently occurs within a low dielectric boundary region of the membrane near the external (cytoplasmic) surface and is positive with respect to the aqueous exterior of the disk. The magnitude of the potential change is pH-and temperature-dependent and appears with a first-order half-time of approximately 7 ms at 21 degrees C. A simple model in which one positive charge per bleached rhodopsin is translocated from the cytoplasmic aqueous space into the membrane low dielectric boundary region readily accounts for all experimental observations. The great similarity of the boundary potential change to the R2 phase of the early receptor potential suggests that the two have the same molecular origin.