Rhodopsin In Membranes Research Articles

Two-dimensional crystallization of bovine rhodopsin

Bovine rhodopsin has been clustered into two-dimensional crystals in highly purified native rod disk membranes and studied with negative staining and transmission electron microscopy. The lattice is P2 1 with dimensions of 8.3 × 7.9 nm and interaxis angles of 86 ± 3 degrees. 110 images of ordered areas were digitized and aligned with computer-correlation methods to calculate an average image with diffraction to the fourth order. The images were computer-filtered and reconstructed to approx. 2 nm resolution. When crystals appeared they covered 20–40% of the surface of the preparation and, since rhodopsin is at least 95% of the protein, there is no doubt that the crystals were due to rhodopsin. There appear to be two rhodopsin dimers per unit cell. Each rhodopsin molecules takes up about 7.5 nm 2 of membrane area and is estimated to be associated with about 12 lipids on each side of the membrane. The membrane area found for bovine rhodopsin supports the rhodopsin origin of rarely seen but more highly ordered two-dimensional crystals found in detergent-treated frog rod membranes (Corles, J.M., McCaslin, D.R. and Soctt, B.L. (1982) Proc. Natl. Acad. Sci. USA 79, 1116–1120). Furthermore, the rhodopsin membrane area is close to that of bacteriorhodopsin and is consistent with a seven transmembrane helix structure proposed for rhodopsin (for references see Dratz E.A. and Hargrave, D.A. (1983) Trends Biochem. Sci. 8, 128–131). Crystallization was accomplished by loweing the pH to 5.5 near the isoelectric point of rhodopsin, raising the salt concentration of 2 M (NH 4) 2SO 4, adding 5% glucose and 0.02% Hibitane (Ayerst), a cationic amphipathic antiseptic that favored crystal growth.

Selectivity in rhodopsin-phospholipid interactions

This series of experiments systematically evaluated the effect of phospholipid headgroup structure on the interaction between rhodopsin and phospholipids. Two types of experiments were reported. First, ESR experiments involving spin-labeled phosphatidylserine, phosphatidic acid, and phosphatidylcholine demonstrated that, in the fluid-isotropic phase of dimyristoylphosphatidylcholine (DMPC)-rhodopsin membranes, the relative order of rhodopsin-induced immobilization was phosphatidic acid > phosphatidylcholine > phosphatidylserine. Second, the effect of rhodopsin incorporation on the dimyristoylphosphatidylserine (DMPS) gel to liquid-crystalline phase transition was analyzed with ESR techniques. A partial, binary phase diagram for the DMPS-rhodopsin system at pH 7.0 was constructed by studying the partitioning of Tempo between polar and hydrophobic domains as a function of temperature and system composition. A main result of this analysis was the finding that rhodopsin broadens and reduces the amplitude of the DMPS phase transition to a much smaller extent than it does the DMPC phase transition. When interpreted in terms of theoretical treatments of integral protein-lipid interactions, this indicates that rhodopsin has a lower affinity for DMPS than DMPC.

Nanosecond laser photolysis of rhodopsin and isorhodopsin.

AbstractKinetic and spectral measurements have been carried out on the primary intermediate in the photolysis of rhodopsin and isorhodopsin, initiated by a 457 nm, 6 ns (FWHM) laser pulse. In rhodopsin the kinetic decay of bathorhodopsin was found to be 140 ± 15 ns at 20°C. The decay of bathorhodopsin to lumirhodopsin has an activation energy of 51 ± 4 kJ/mol (12.2 ± 1 kcal/mol). The decay kinetics of bathorhodopsin were found to be the same for rhodopsin in membrane and detergent solubilized suspensions. The kinetic decay of the batho product in the photolysis of isorhodopsin was found to be the same as rhodopsin.The corrected transient spectrum 50 ns following excitation in rhodopsin has two peaks near 560 and 440 nm. A peak was also observed in isorhodopsin near 550 nm at 50 ns following excitation but no transient was observed in the blue. The 550 nm peak in isorhodopsin has an intensity similar to that in rhodopsin indicating that the quantum yields for the formation of batho products of rhodopsin and isorhodopsin are similar under the irradiation conditions used here. Transient spectra for rhodopsin and isorhodopsin 1 μs following excitation are also different. In isorhodopsin the corrected transient spectrum has a peak at 500 nm, similar to low temperature steady state irradiation spectra. The 1 μs transient spectrum in rhodopsin is more intense than in isorhodopsin and shows a peak at 475 nm.

Fourier transform infrared study of photoreceptor membrane. I. Group assignments based on rhodopsin delipidation and reconstitution

Fourier transform infrared spectroscopy has been used to study the structure of bovine photoreceptor membrane. Rhodopsin appears to contain an extensive alpha-helical structure which is arranged predominantly perpendicular to the membrane plane. Spectra of delipidated rhodopsin and rhodopsin membranes reconstituted from dioleyl-phosphatidylcholine were compared with native photoreceptor membrane from rod outer segments in order to facilitate peak assignments. It is concluded that spectroscopic peaks characteristic of several protein and lipid groups can be assigned. We also find delipidation leads to alteration of the rhodopsin structure which is restored upon reconstitution. Membranes both suspended in 2H 2O and dehydrated were compared in order to detect possible conformational differences. Dehydration does not appear to grossly alter rhodopsin structure, although it may affect delipidated rhodopsin.

The oligosaccharide moiety of rhodopsin—its structure and cellular location

The sugar chains of bovine rhodopsin released from opsin by hydrazinolysis were reduced with NaB( 3H) 4 and fractionated by paper chromatography. Three oligosaccharides were obtained. The structure of the major ( 3H)-oligosaccharide (ca. 60% of total) was elucidated by sequential exoglycosidase digestion, methylation analysis and endo-β-N-acetyl glucosaminidase D digestion. The structure of the sugar moiety of rhodopsin was thus identified as: ▪ Since the terminal GlcNac serves as galactose acceptor, the location of the sugar moiety of rhodopsin in the disc membrane was studied by incorporation of ( 3H)-galactose (from UDP-( 3H)-galactose) into the disk membrane. After inversion of disks by freeze-thawing, rhodopsin in the membrane incorporated one mole of ( 3H)-galactose per mole purified pigment. Intact disks incorporated lower amounts of ( 3H)-galactose. It was therefore concluded that the sugar moiety of rhodopsin is located on the internal surface of disk membrane. The results of lectin binding studies are consistent with the conclusion. Inverted disks, but not intact disks, bind concanavalin A (specific for α-mannosyl residue) and wheat germ lectin (specific for N-acetylglucosamine). Since intact sealed rod outer segments bind concanavalin A, the sugar moiety of rhodopsin in the plasma membrane is probably exposed on the external surface of the rod.

The initiation of excitation and light adaptation in Limulus ventral photoreceptors.

Two types of experiments indicate that light adaptation and excitation are initiated by the same, rather than different, populations of visual pigment. (a) The criterion action spectra of light adaptation and excitation are the same. (b) Increment-threshold curves were measured with a voltage-clamp technique under conditions of high and low concentration of plasma membrane rhodopsin (Rhpm). SD, the dark-adapted sensitivity, and 1/I2, the inverse of the background irradiance that desensitized by 0.3 log units, underwent the same fractional change when the rhodopsin concentration was changed. Both quantities appear to be linearly related to Rhpm. Reversible reductions in Rhpm were achieved by orange irradiation during a brief increase of extracellular pH from 7.8 to 10. This procedure would be unlikely to produce similar concentration changes in a hypothetical intracellular pigment because the concurrent change in intracellular pH, measured using the dye, phenol red, was only 0.45 pH units. It is thus unlikely that an intracellular pigment initiates light adaptation. On the assumption that light adaptation is mediated by a light-induced release of Ca++ from an intracellular store. the results reported here imply that an intracellular transmitter is needed to couple Rhpm to the intracellular store.

The initiation of excitation and light-adaptation in Limulus ventral photoreceptors

Two types of experiments indicate that light adaptation and excitation are initiated by the same, rather than different, populations of visual pigment. (a) The criterion action spectra of light adaptation and excitation are the same. (b) Increment-threshold curves were measured with a voltage-clamp technique under conditions of high and low concentration of plasma membrane rhodopsin (Rhpm). SD, the dark-adapted sensitivity, and 1/I2, the inverse of the background irradiance that desensitized by 0.3 log units, underwent the same fractional change when the rhodopsin concentration was changed. Both quantities appear to be linearly related to Rhpm. Reversible reductions in Rhpm were achieved by orange irradiation during a brief increase of extracellular pH from 7.8 to 10. This procedure would be unlikely to produce similar concentration changes in a hypothetical intracellular pigment because the concurrent change in intracellular pH, measured using the dye, phenol red, was only 0.45 pH units. It is thus unlikely that an intracellular pigment initiates light adaptation. On the assumption that light adaptation is mediated by a light-induced release of Ca++ from an intracellular store. the results reported here imply that an intracellular transmitter is needed to couple Rhpm to the intracellular store.

Cross-linking of membrane phospholipid and protein using putative monofunctional imidoesters

The reaction of methyl acetimidate or isethionyl acetimidate with mitoplasts at pH 8.5 yields two derivatives of phosphatidylathinolamine. These derivatives are shown to be the mono-amidine derivative and the bis-derivative of phosphatidylethanodamine. The bis-derivative represents one phosphatidylathinolamine cross-linked to another phosphatidylathinolamine. Similar derivatives are formed by the reaction of dipalmitoyl phosphatidylathinolamine with these imidoesters in organic reaction with the exception that much more monoderivative is primarily the mono-derivative. The bis-derivative was not detected. The reaction of bovine rod oester segment discs with methyl acetimidate causes cross-linking of 30% of the membrane rhodopsin as dimers. Putative monofunctional imidoesters cause considerable cross-linking of both phospholipids and protein in cell membranes. Crosslinking can be minimized at pH 9.0.

Rotational motion of rhodopsin in the visual receptor membrane as studied by saturation transfer spectroscopy

The saturation transfer spectrum of a nitroxide spin probe rigidly bound to rhodopsin in frog rod outer segment membrane has been measured. The rotational correlation time at 20°C was obtained as 20, 3.6, and 13 μs from the peak height ratio in the low field, central, and high field part of the spectrum, respectively. The difference in the obtained values suggests an anisotropic rotation of rhodopsin in the membrane. Glutaraldehyde treatment restricted the rotational motion. The correlation time became 45, 9.6, and 45 μs as estimated from the low field, central, and high field part of the spectrum, respectively. Comparison with the data by Cone (1972) Nat. New Biol. 236, 39–43) suggests oscillatory rotation within a limited angle. Photobleaching affected the spectrum slightly but definitely.

Rhodopsin in bilayer membranes.

Visual excitation is initiated with the absorption of light by the membrane-bound visual pigment rhodopsin. The effect of light on rhodopsin is now quite generally believed to be a photoisomerization of the chromophore, retinaldehyde, from the 1 1-cis to the all-rransconliguration(Hubbard & Kropf, 1957; Wald, 1968). This is followed by changes in the shape of the protein, and these lead to conductance changes in the retinal photoreceptor cell membrane (Hagins, 1972). Such changes could result from a simple mechanism based on rhodopsin functioning as a light-activated ion-translocator. We have obtained evidence in favour of this proposal by incorporating rhodopsin into vesicular and planar bilayer membranes and demonstrating that light increases the membrane conductance in a pattern consistent with the formation of a transmembrane channel (Montal, 1975; M. Montal, A. Darszon & H. W. Trissl, unpublished work). The rhodopsin/lipid vesicles were formed and loaded with radioactive markers by sonication. Exposure of the vesicles to light increased the permeability to Na+, Cs+, Caz+, glycerol and glucose, but not to C1- and sucrose. These results indicate that light induces the formation of a cation-selective wide permeability pathway with an apparent cut-off diameter of about 0.8-1 .Onm (8-lo&. Planar bilayers containing rhodopsin were formed by apposing two rhodopsin-lipid monolayers. Such monolayers are formed from an ether-soluble complex of rhodopsin and lipid (a proteolipid) that exhibits in the organic phase spectral characteristics similar to those of native rhodopsin in retinal rod discs. Exposure of the bilayer to light increased the membrane conductance. A distinct latency period between illumination and onset of conductance changes is characteristic: it is shorter the higher the rhodopsin content in the proteolipid extract and can vary from 1 s up to several hundred seconds. At high resolution the conductance of the rhodopsin membranes can be seen to fluctuate in discrete steps. Three main transition amplitudes are well identified, corresponding to conductances of 2 x and 6.1 xlO-'OR-' in symmetric media containing either 0.2~-NaCl or O.~M-KCI. By contrast, a conductance transition of 8 x 10-10i2-1 is most frequent in symmetric 0.2~-CaCl, solutions. Illumination does not change the fluctuation pattern, it merely increases the number of conductance events. The conductance transitions are ohmic; by contrast, the macroscopic membrane conductance is not ohmic but voltage-dependent: at voltages greater than 25mV the initially high conductance relaxes to lower steady-state values. The conductance events are reminiscence of those induced in lipid bilayers by molecules presently thought to act by forming transmembrane channels (Hladky et al., 1974). Further, concentrationpotential measurements indicate that the bilayer conductance is cation-selective. The results obtained with vesicles and planar bilayers are in remarkable agreement and establish that the effect of rhodopsin bleaching in bilayer membranes is to induce the formation of a transmembrane channel of about 0.8-1.0nm diameter. Since the light-induced permeability response can be elicited at 3C, metarhodopsin I1 may be the first conducting form of rhodopsin. Although the data are not sufficient to enable us to propose a specific molecular picture, three models have been considered, namely a unimolecular channel, a channel made of a fixed aggregate (e.g. a tetramer) or an association-dissociation equilibrium process. On the basis of these findings a model of vertebrate visual phototransduction that considers CaZ+ as the transmitter (Hagins, 1972) can be proposed, as follows. In the dark, the channel is closed and the Caz+-dependent adenosine triphosphatase would pump Ca2+ into the discs; on bleaching, the channel would form and allow Caz+ to diffuse down its concentration gradient. The efflux of CaZ+ would change the disc membrane 4 x

Chemical labeling and freeze-fracture studies on the localization of rhodopsin in the rod outer segment disk membrane

The molecular organization of the bovine rod outer segment (ROS) disk was investigated with respect to localization of the visual pigment rhodopsin in the membrane. The study made use of preparations of intact disks and ROS fragments. We used the membrane impermeable reagent sodium isethionyl acetimidate hydrochloride and membrane permeable reagent ethyl acetimidate hydrochloride to label membrane amino groups. We found that half the rhodopsin amino groups were accessible to the membrane impermeable reagent; therefore, rhodopsin must protrude into the extradiskal space. A complementary experiment using fluorescein isothiocyanate confirmed these results. Freeze-fracture electron microscopy showed that the disk membrane is asymmetric and that the sidedness of our disk preparation is uniform.