Outer Segment Plasma Membrane Research Articles

Membrane assembly in retinal photoreceptors I. Freeze-fracture analysis of cytoplasmic vesicles in relationship to disc assembly.

To study precursor-product relationships between cytoplasmic membranes of the inner segment of photoreceptors and the continually renewed outer disc membrane, we have compared the density and size distribution of intramembrane particles (IMP) in various membrane compartments of freeze-fractured photoreceptor inner and outer segments. Both rod and cone outer segments of Xenopus laevis are characterized by a relatively uniform distribution of approximately 4,400-4,700 IMP/micron2 in P-face (PF) leaflets of disc membranes. A similar distribution of IMP is found in the outer segment plasma membrane, the ciliary plasma membrane, and in the plasma membrane of the inner segment in the immediate periciliary region. In each case the size distribution of IMP can be characterized as unimodal with a mean diameter of approximately 10 nm. PF leaflets of endoplasmic reticulum, Golgi complex, and vesicles near the cilium have IMP with a size distribution like that in the cilium and outer segment, but with an average density of approximately 2,000/micron2. In contrast, IMP are smaller in average size (approximately 7.5 nm) in PF leaflets of inner segment plasma membrane, exclusive of the periciliary rgion. The similarity of size distribution of IMP in inner segment cytoplasmic membranes and those within the plasmalemma of the cilium and outer segment suggest a precursor-product relationship between the two systems. The structure of the vesicle-rich periciliary region and the segregation of IMP with different size distributions in this region suggest that components destined for incorporation into the outer segment exist as preformed membrane packages (vesicles) which fuse with the inner segment plasma membrane in the periciliary region. Subsequently, membrane components may be transferred to forming discs of the outer segment via the ciliary plasma membrane.

Distribution of enzymes involved in nucleotide metabolism in the disk and other membranes.

Abstract— Intact disks and inverted disks were prepared from bovine retinal rods and the distribution in the disk membrane of such enzymes as guanyl cyclase, cyclic nucleotide phosphodiesterase, GTP binding protein (GTPase), 5'‐nucleotidase and rhodopsin kinase was investigated. Guanyl cyclase was not detected in the disk; the enzyme activity was high in a membranous fraction containing the cilium or axoneme and the rod outer segment plasma membrane. Cyclic nucleotide phosphodiesterase, GTP binding protein (GTPase) and rhodopsin kinase were associated on the external surface of disk in the presence of 2 mM Mg2+. The enzymes dissociated from the membrane when Mg2+ was depleted. Thus, magnesium ion seems to regulate the state of these enzymes in the outer segment. 5'‐Nucleotidase activity was low in intact disks but was significantly enhanced after inversion of the disk. The catalytic site of the enzyme, therefore, must be located on the internal (intradiscal) surface. Since the disks are known to be formed by invagination of the plasma membrane, 5'‐nucleotidase, by inference, would have its catalytic site exposed on the external surface of the plasma membrane. Preliminary experiments showed that the capability of light‐activated rhodopsin to activate cyclic nucleotide phosphodiesterase was inhibited by phosphorylation of the pigment. This supports the idea that rhodopsin kinase, cyclic nucleotide phosphodiesterase and GTPase exist as a functional complex on rod membranes.

Ultrastructural localization of lectin binding sites on the surface of retinal photoreceptors and pigment epithelium

The ferritin-lectin complexes of Ricinus communis agglutinin (RCA-120), wheat germ agglutinin (WGA), concanavalin A (con A), soybean agglutinin (SBA) and fucose binding protein (FBP) have been used to study the distribution of sugars along the plasma membranes of frog photoreceptor and pigment epithelial cells. The tissues were fixed prior to labeling in order to prevent the lectin-induced rearrangement of binding sites. The plasma membrane of different regions of the rod and cone photoreceptor cells bound each lectin to a different extent. In all cases, the outer segment plasma membrane bound more of the ferritin-lectin complex than did the inner segment plasma membrane. This might reflect the specific differentiation of the outer segment plasma membranes, related to their role in the visual process. The intensity of binding of each lectin to the pigment epithelium plasma membrane was similar to its binding to the rod outer segment plasma membrane. In cone outer segments, where one side of the disk membrane is accessible to the incubation medium, direct binding of lectin to the disk edges, as well as to the plasma membrane, was seen. Cone outer segments bound more of the α-D-N-acetylgalactosamine and D-galactose specific lectin, SBA, than did rod outer segments. No differences were observed in the binding of ferritin-lectin complexes to rod outer segments from dark or light adapted frogs.

Freeze-fracture evidence for the presence of cholesterol in particle-free patches of basal disks and the plasma membrane of retinal rod outer segments of mice and frogs.

The freeze-fracture technique was used to examine the membranes of the photoreceptors of mice and frogs. Particle-free patches were found in the plasma membrane and basal disk membranes of the outer segments of both mice and frogs housed at room temperature, but not in frogs kept in a cold room. These patches were shown not to be artifacts of cryoprotection or fixation, and they persisted when fresh isolated outer segments were frozen by an ultrarapid method. They were also found to persist in mouse rods when retinas were incubated and subsequently fixed at temperatures up to 80 degrees C. Cholesterol was implicated as a significant component of the patches by the observation that, in the outer segments, pits, induced by treatment with the sterol-specific polyene antibiotic filipin, were present in and confined to the particle-free patches. That these lesions are not inherently limited to particle-free membrane areas was evident in the apical plasma membrane of the photoreceptor inner segments, where particles and pits were intermixed. Treatment with saponin, a surface-active agent which specifically complexes cholesterol, resulted in the disappearance of the particle-free patches. Patches were found in basal disks of both mouse and frog rods but not in older disks nearer the pigment epithelium, which indicates that changes occur in the composition of disk membranes and/or in the molecular ordering of their protein and lipid components during the early phase of their transit from the base towards the apex of the outer segment.

Freeze-fracture analysis of structure of plasma membrane of photoreceptor cell outer segments

The plasma membrane of the outer segments of photoreceptor cells in the guinea pig retina was analyzed by means of freeze-fracturing. Two fracture faces with greatly different structure were observed. The relationship of these two faces to the edge which was exposed when the fracture passed across the membrane made it possible to establish that they revealed the cytoplasmic and the peripheral surfaces of the plasma membrane. The thickness of the plasma membrane was measured to be about 100 A. The difference in appearance of the cytoplasmic and the peripheral surfaces of the plasma membrane was interpreted to reveal a layered structure with a predominantly lipid layer at the cytoplasmic surface, and a predominantly protein layer at the peripheral surface. A concept describing the molecular structure of the membrane is proposed. The outer segment plasma membrane differs structurally from the outer segment disk membranes. Certain aspects of disk membrane formation as invaginations of the plasma membrane are discussed. The fact that only two fracture faces were observed and no complementary faces was explained by a simple analysis of some effects of the fracturing on the frozen material.

Molecular structure of outer segment disks in photoreceptor cells

The structure of the photoreceptor cell outer segment disks in guinea pig, frog, and gecko retinas were analyzed by means of freeze-fracturing and showed basically identical features. Thanks to a rather accurate locating of the fracture planes based on observations, the observed structure could be interpreted to reveal that the outer segment disk membranes consist of two layers with greatly different physical properties and with each layer measuring about 50 A in thickness. These observations were interpreted on the basis of information obtained earlier by means of polarization optical and x-ray diffraction analysis as well as of what is known about the chemical composition of the photoreceptor cell outer segments. On this basis it was concluded that each disk membrane consists of one lipid bilayer and one monolayer of photopigment molecules dispersed in an aqueous medium of small volume. A particulate structure at the edge of the disk made this part of the disk appear as structurally different from the disk membrane. A hypothetical mechanism is proposed for the transmission of a signal linking energy transduction to the change in permeability of the outer segment plasma membrane caused by absorption of light. The outer segment disk membrane differs structurally extensively from the inner mitochondrial membranes, and the differences were interpreted to reflect the very different functions at a molecular level of these two types of membranes.

An analysis of rod outer segment adaptation based on a simple equivalent circuit.

This model of rod outer segment adaptation is based on the hypothesis that transmitter substance released by bleached rhodopsin closes sodium channels in the outer segment plasma membrane, leading to hyperpolarization of the receptor. The outer segment adaptation processes of the model are associated with the transmitter release, the transmitter background concentration and the plasma membrane leakage. Changes in the three model parameters correspond to the three types of outer segment adaptation process. According to the model the stimulus-response function is in every adaptational state U/Umax = I/(I + IH). The model predicts how each adaptation process affects IH and Umax. Specifically, when the number of liberated transmitter molecules per isomerizing quantum decreases, the amplitude Umax remains constant but IH increases. A short description of this model has been published in a paper reporting experimental results on background adaptation (Hemilä, 1977).

Rhodopsin in the rod outer segment plasma membrane.

Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.

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.

Ionic analysis of photoreceptor membrane currents.

1. Membrane current was derived from simultaneous measurements of interstitial ;dark' voltages and conductivities along the radial axis of frog photoreceptor cells. Membrane current was subsequently resolved into its component ionic currents or fluxes by means of ionic substitution and by the use of inhibitors of active transport.2. The plasma membrane of the frog rod outer segment was found to be permeable to Na(+) and Cl(-), with a ratio of Na(+) to K(+) permeabilities higher than that found in most neuronal cells. A net inward flux of 1.5 x 10(8) Na(+)/sec.rod flows across the outer segment plasma membrane in the dark.3. The proximal portion of the rod receptor, extending from the proximal region of the inner segment to the synaptic terminal, is mainly permeable to K(+), although some degree of Na(+) permeability is also presumed.4. A hyperpolarizing electrogenic Na pump was localized to the base of the outer segment and inner segment of the cell. The pump transfers at least 10(8) charges/sec out of the cell at this level, the pump current dividing and re-entering the cell at both the outer segment and proximal portion of the photoreceptor including the synaptic terminal.5. These findings have been incorporated into an ionic model of the photoreceptor, and its implications for cellular functioning considered.

Dark Current and Photocurrent in Retinal Rods

The interstitial voltages, currents, and resistances of the receptor layer of the isolated rat retina have been investigated with arrays of micropipette electrodes inserted under direct visual observation by infrared microscopy. In darkness a steady current flows inward through the plasma membrane of the rod outer segments. It is balanced by equal outward current distributed along the remainder of each rod. Flashes of light produce a photocurrent which transiently reduces the dark current with a waveform resembling the PII and a-wave components of the electroretinogram. The photocurrent is produced by a local action of light within 12 mum of its point of absorption in the outer segments. The quantum current gain of the photocurrent is greater than 10(6). The electrical space constant of rat rods is greater than 25 mum, so that the electrical effects of the photocurrent are large enough at the rod synapses to permit single absorbed photons to be detected by the visual system. The photocurrent is apparently the primary sensory consequence of light absorption by rhodopsin.