Fluid lipid fraction in rod outer segment membrane

Rod outer segment membrane is analyzed using the spin label technique by means of two probes. The solubility of the first label, 2,2,6,6-tetramethylpiperidin-1-oxyl, is correlated with the membrane fluidity which is measured using a stearic acid spin probe. The two values are compared to the solubility-fluidity relationship which characterizes a model system in which all lipids are in a fluid state. The analysis leads to the conclusion that only two thirds of the membrane lipids are fluid. This conclusion is reinforced by the observation that partial lipid removal leaves rigid lipids associated with the rhodopsin molecules.     

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.     

Membrane fusion in the rod outer segment disk membrane

The rod outer segment disk membrane of bovine retina has been isolated in a predominantly fused state. The physical and chemical properties of the membrane in the fused state are profoundly different from the corresponding properties of the same membrane in the unfused state. Exposure to light induces the transition of the disk membrane from the fused to the unfused state. Evidence is presented which suggests that the fusion-defusion cycle of the disk membrane is a primary event of photoexcitation and nerve stimulation.     

Glycoproteins specific for the retinal rod outer segment plasma membrane

Two ricin-specific glycoproteins have been identified on neuraminidase-treated rod outer segment plasma membranes of bovine retinal photoreceptor cells. Ricin-gold-dextran particles were observed by electron microscopy to densely label the surface of neuraminidase-treated rod outer segments. Western blotting of proteins separated by SDS-gel electrophoresis indicated that two ricin-binding glycoproteins of Mr 230,000 and 110,000 are specific for the plasma membrane and are not found in disk membranes. These glycoproteins can serve as specific probes for the purification of the rod outer segment plasma membrane.     

Third calcium-modulated rod outer segment membrane guanylate cyclase transduction mechanism

Ca²⁺-modulated rod outer segment membrane guanylate cyclase (ROS-GC1) has been cloned and reconstituted to show that it is regulated by two processes: one inhibitory, the other stimulatory. The inhibitory process is consistent with its linkage to phototransduction; the physiology of the stimulatory process is probably linked to neuronal transmission. In both regulatory processes, calcium modulation of the cyclase takes place through the calcium binding proteins; guanylate cyclase activating proteins (GCAP1 and GCAP2) in the case of the phototransduction process and calcium-dependent GCAP (CD-GCAP) in the case of the stimulatory process. The cyclase domains involved in the two processes are located at two different sites on the ROS-GC1 intracellular region. The GCAP1-modulated domain resides within the aa 447-730 segment of ROS-GC1 and the CD-GCAP-modulated domain resides within the aa 731-1054 segment. In the present study the GCAP2-dependent Ca²⁺ modulation of the cyclase activity has been reconstituted using recombinant forms of GCAP2 and ROS-GC1, and its mutants. The results indicate that consistent to phototransduction, GCAP2 at low Ca²⁺ concentration (10 nM) maximally stimulates the cyclase activity of the wild-type and its mutants: ext- (deleted aa 8-408); kin- (deleted aa 447-730) and hybrid consisting of the ext, transmembrane and kin domains of ANF-RGC and the C-terminal domain, aa 731-1054, of ROS-GC1. In all cases, it inhibits the cyclase activity with an IC₅₀ of about 140 nM. A previous study has shown that under identical conditions the kin- and the hybrid mutant are at best only minimally stimulated. Thus, the GCAP1 and GCAP2 signal transduction mechanisms are different, occurring through different modules of ROS-GC1. These findings also demonstrate that the intracellular region of ROS-GC1 is composed of multiple modules, each designed to mediate a particular calcium-specific signalling pathway.     

Light-dependent association of Src with photoreceptor rod outer segment membrane proteins in vivo.

In vivo light exposure results in tyrosine phosphorylation of several rod outer segment (ROS) proteins (Ghalayini, A. J., Guo, X. X., Koutz, C. A, and Anderson, R. E. (1998) Exp. Eye Res. 66, 817-821). We now report the presence of Src in ROS and its increased association with bleached ROS membranes. Immunoprecipitation with anti-phosphotyrosine revealed that tyrosine kinase activity recovered from light-adapted ROS membranes was twice that recovered from dark-adapted ROS. Other experiments revealed the presence of both bleached rhodopsin and arrestin in immunoprecipitates of LROS, suggesting the formation of a multimeric complex containing Src, arrestin, and bleached rhodopsin. Additionally, when immobilized Src homology domains 2 and 3 (SH2 and SH3, respectively) were used to study the association of Src with ROS membranes, only bleached opsin and arrestin were found to associate with the SH2 domain of Src. These data strongly suggest that Src through its SH2 domain interacts with bleached rhodopsin and arrestin either directly or indirectly. Similar results were also obtained when dark-adapted and light-adapted retinas were used instead of ROS membranes. Our data strongly suggest that light exposure in vivo activates Src and promotes its association through its SH2 domain with a complex containing bleached rhodopsin and arrestin. A hypothesis for the functional significance of this phenomenon is presented.     

Disaturated and dipolyunsaturated phospholipids in the bovine retinal rod outer segment disk membrane.

Thin-layer chromatography was used to separate the major phospholipid headgroup classes of the rod outer segment disk membrane into subfractions which differ markedly in fatty acid composition. At least 18% of the rod outer segment phosphatidylcholine must contain two saturated fatty acids. Furthermore, two unsaturated fatty acids are found in at least 43% of the phosphatidylserine, 24% of the phosphatidylcholine, and 24% of the phosphatidylethanolamine. The unsaturated acids are predominantly polyunsaturated in all cases. A similar separation, but with less resolution, was achieved with silicic acid column chromatography. The temperature dependence of the polarization of the fluorescence of trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid) showed that the thermal behavior of aqueous dispersions of the phosphatidylcholine subfractions was consistent with their fatty acid compositions.     

Protein kinase C-mediated phosphorylation of retinal rod outer segment membrane proteins

We have previously reported that the purified GDP-bound alpha-subunit of the GTP-binding protein transducin (TD), present in outer segments of retinal rod cells (ROS), serves as a high affinity substrate (Km = 1 microM) for protein kinase C (PKC) [Zick et al. (1986) Proc. natn. Acad. Sci., U.S.A. 83, 9294-9297]. In the present study we demonstrate that TD-alpha undergoes phosphorylation by PKC when present in its native form in intact ROS membranes. This phosphorylation is inhibited by GTP-gamma-S which activates TD, suggesting that it is only the inactive conformation of TD-alpha that serves as a substrate for PKC. Indeed, both vanadate and AlF4, that confer an active conformation on TD-alpha-GDP, inhibit PKC-mediated phosphorylation of purified TD-alpha-GDP. We demonstrate that the purified beta subunit of TD also serves as an in vitro substrate for PKC. Moreover, following their phosphorylation, both TD-alpha and beta form high affinity complexes with PKC. This is evident from the findings that PKC coprecipitates with both the alpha and beta subunits of TD when the latter are immunoprecipitated by their respective antibodies. PKC phosphorylates additional ROS proteins of 36, 48 and 92 kDa, tentatively identified as rhodopsin, arrestin and the cGMP-phosphodiesterase. Taken together our results strongly suggest that phosphorylation of TD is of physiological relevance and that through phosphorylation of endogenous ROS proteins, PKC could play a key role in regulating phototransduction.     

Inactivation of cGMP-dependent conductance of rod outer segment plasma membrane induced by cGMP.

Integral cGMP-dependent currents as well as activity of single cGMP-activated channels in plasma membrane of rod outer segment (ROS) were recorded using the patch-clamp method. The dependence of integral currents on cGMP concentration is shown to be bell-shaped. Decrease in cGMP-dependent conductance at high cGMP concentration results from a decrease in channel opening probability. Thus, cGMP in high concentrations inactivates cGMP-dependent conductance.     

Disaturated and dipolyunsaturated phospholipids in the bovine retinal rod outer segment disk membrane

Thin-layer chromatography was used to separate the major phospholipid headgroup classes of the rod outer segment disk membrane into subfractions which differ markedly in fatty acid composition. At least 18% of the rod outer segment phosphatidylcholine must contain two saturated fatty acids. Furthermore, two unsaturated fatty acids are found in at least 43% of the phosphatidylserine, 24% of the phosphatidylcholine, and 24% of the phosphatidylethanolamine. The unsaturated acids are predominantly polyunsaturated in all cases. A similar separation, but with less resolution, was achieved with silicic acid column chromatography.The temperature dependence of the polarization of the fluorescence of trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid) showed that the thermal behavior of aqueous dispersions of the phosphatidylcholine subfractions was consistent with their fatty acid compositions.     

Distinct states of lipid mobility in bovine rod outer segment membranes. Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0 degrees C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24 degrees C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0 degrees C and a considerable temperature dependence. This component which is not resolved at high temperatures (24--35 degrees C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62--64 G at 0 degrees C, with very little temperature dependence (61--62 G at 35 degrees C), and is attributed to protein aggregation.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

Molecular mechanisms of photoreception. V. Reconstitution of a sodium channel on an artificial lipid membrane modified by bovine rod outer segment components

An attempt is made to reconstitute the Na+-conductivity elements of rod outer segment (ROS) plasma membrane on the artificial lipid membrane (ALM). ALM were modified by preparation with bovine ROS plasma membrane fragments. Discrete fluctuations of the ALM conductivity were observed at addition of this preparation to the ALM bathing solutions to a final concentration of 0.1--1.0 microgram/ml. The magnitude of these fluctuations was about 25 pS at 140 mM NaCl. The modified ALM possessed preferentially the Na+-conductivity, which was at least five times as great as that for K+ or Li+. The modified ALM showed practically no conductivity for Cl-. The Na+-channels were voltage-dependent. The Na+-channels were "sensitive to visible light" at some experimental conditions. The optimal conditions were obtained for reconstitution of Na+-channel on the ALM. The temporal and termal stabilities of the modified preparation were investigated. It was shown that the frequency of the modified ALM conductivity fluctuations are dependent on temperature and on lipids composition of ALM. The data obtained in our work are in a good agreement with the results of electrophysiological studies of photoreceptor cells. It may be indicated, that the investigated ALM contains the Na+-channel of the ROS plasma membranes. It is suggested that the reconstituted system will be useful for studying the principles of regulation of ROS plasma membrane sodium conductivity and the nature of a mediator in a photoreceptor transduction mechanism.     

X-ray diffraction and electron microscope study of phase separation in rod outer segment photoreceptor membrane multilayers.

Phase separation in artificially stacked multilayers of isolated bovine retinal rod outer segment (ROS) membranes has been examined via x-ray diffraction and electron microscopy. Specimens were prepared by isopotential spin drying followed with partial hydration by equilibration against moist gas streams. Upon dehydration, the multilamellar membrane phase assumes a binary phase composition consisting of concentrated protein-containing lamellae interspersed with microdomains of hexagonally packed tubes of lipid in a HII configuration. The HII lattice is geometrically coupled to the lamellar phase with one set of hexagonal crystal planes co-planar to the local membrane lamellae. The hexagonal microdomains bear a striking resemblance to the "paracrystalline inclusions" observed in fast-frozen, intact frog ROS (Corless and Costello. 1981. Exp. Eye Res. 32:217). The lamellar lattice is characterized by an unusually small degree of disorder. Sharp lamellar diffraction with a 120 A unit cell is observed (at near total dehydration) to a resolution of 6 A. A model consistent with the data is that a multilamellar array of ROS disks is stable as long as the external disk surfaces are kept sufficiently far apart. If the distance between the membranes is allowed to shrink below a certain critical value, the disk lipids spontaneously convert to a nonbilayer phase. This suggests that the structure of the ROS is stabilized by an internal framework that acts to keep the disks apart from one another and from the plasmalemma. Thus, the necessity of avoiding phase separations may provide a rationale for the peculiar morphology of the ROS.