Molecular mechanisms characterizing cone photoresponses

In the vertebrate retina, rods mediate twilight vision and cones mediate daylight vision. Their photoresponse characteristics are different. The light-sensitivity of a cone is 10(2)-10(3) times lower than that of a rod. In addition, the photoresponse time course is much faster in cones. The mechanism characterizing cone photoresponses has not been known mainly because of the difficulty in isolating cones in large quantities to perform biochemistry. Recently, we developed a method to purify cones from carp retina using a density gradient, which made it possible to analyze the differences in the molecular mechanism of phototransduction between rods and cones. The results showed that signal amplification in cones is less effective, which explains the lower light-sensitivity of cones. The results also showed that visual pigment phosphorylation, a quenching mechanism of light-activated visual pigment, is much more rapid in cones than in rods. The rapid phosphorylation in cones is attributed to a very high total kinase activity in cones. Because of this high activity, cone pigment is readily phosphorylated even at very high bleaching levels, which probably explains why cone photoresponses recover quickly. Based on these findings, the molecular mechanisms of the differences in the photoresponse characteristics between rods and cones are outlined.     

Distribution and translocation of photoreceptor Gbetagamma-phosducin system in medaka retina

Unlike other vertebrates, teleosts have rod- and cone-specific phosducins (PD-R and PD-C) in the retina. To evaluate the teleost Gbetagamma-PD systems, we isolated cDNAs encoding medaka Gbeta1 and GbetaC, which selectively expressed rods and cones. Immunohistochemical studies showed that the strong reactivity of GbetaC but not PD-C was detected in cone outer segments. In rod outer segments (ROS), PD-R reactivity was stronger in light-adapted retina than in dark-adapted retina. Western blot analyses of fractions torn from the cryosections showed that the PD-R concentration was low in dark-adapted ROS. It is suggested that PD-R is translocated to ROS and effectively downregulates the phototransduction cascade in light-adapted rods.     

Synaptic inputs from rods and cones to horizontal cells in the tiger salamander retina

Synaptic inputs from rods and cones to horizontal cells (HCs) were studied in the flatmounted isolated retinas of the tiger salamander. Voltage-intensity relations, spectral sensitivities and response waveforms of the rod, the cone, and the HC under dark- and light-adapted conditions were examined. HCs receive mixed inputs from rods and cones in both dark- and light-adapted retinas. The relative rod/cone input in HCs depends on the intensity of stimulus and background illumination, and it varies from HC to HC.     

SYNAPTIC INPUTS FROM RODS AND CONES TO HORIZONTAL CELLS IN THE TIGER SALAMANDER RETINA

Synaptic inputs from rods and cones to horizontal cells (HCs) were studied in the flatmounted isolated retinas of the tiger salamander. Voltage-intensity relations, spectral sensitivities and response waveforms of the rod, the cone, and the HC under dark- and light-adapted conditions were examined. HCs receive mixed inputs from rods and cones in both dark- and light-adapted retinas. The relative rod/cone input in HC_s depends on the intensity of stimulus and background illumination, and it varies from HC to HC.     

Iodopsin, a red-sensitive cone visual pigment in the chicken retina.

The vertebrate retina contains two kinds of visual cells: rods, responsible for twilight (scotopic) vision (black and white discrimination); and cones, responsible for daylight (photopic) vision (color discrimination). Here we attempt to explain some of their functional differences and similarities in terms of their visual pigments. In the chicken retina there are four types of single cones and a double cone; each of the single cones has its own characteristic oil droplet (red, orange, blue, or colorless) and the double cone is composed of a set of principal and accessory members, the former of which has a green-colored oil droplet. Iodopsin, the chicken red-sensitive cone visual pigment, is located at outer segments of both the red single cones and the double cones, while the other single cones and the rod contain their own visual pigments with different absorption spectra. The diversity in absorption spectra among these visual pigments is caused by the difference in interaction between chromophore (11-cis retinal) and protein moiety (opsin). However, the chromophore-binding pocket in iodopsin is similar to that in rhodopsin. The difference in absorption maxima between both pigments could be explained by the difference in distances between the protonated Schiff-bases at the chromophore-binding site and their counter ions in iodopsin and rhodopsin. Furthermore, iodopsin has a unique chloride-binding site whose chloride ion serves for the red-shift of the absorption maximum of iodopsin. Visual pigment bleaches upon absorption of light through several intermediates and finally dissociates into all-trans retinal and opsin. That the sensitivity of cones is lower than rods cannot be explained by the relative photosensitivity of iodopsin to rhodopsin, but may be understood to some extent by the short lifetime of an enzymatically active intermediate (corresponding to metarhodopsin II) produced in the photobleaching process of iodopsin. The rapid formation and decay of the meta II-intermediate of iodopsin compared with metarhodopsin II are not contradictory to the rapid generation and recovery of cone receptor potential compared with rod receptor potential. The rapid recovery of the cone receptor potential may be due to a more effective shutoff mechanism of the visual excitation, including the phosphorylation of iodopsin. The rapid dark adaptation of cones compared with rods has been explained by the rapid regeneration of iodopsin from 11-cis retinal and opsin. One of the reasons for the rapid regeneration and susceptibility to chemicals of iodopsin compared with rhodopsin may be a unique structure near the chromophore-binding site of iodopsin.     

Longitudinal diffusion of a polar tracer in the outer segments of rod photoreceptors from different species

Vertebrate rod photoreceptors are the ultimate light sensors, as they can detect a single photon. In darkness, rods maintain a high concentration of the intracellular messenger cyclic guanosine monophosphate (cGMP), which binds to and keeps open cationic channels on the plasma membrane of the outer segment. Absorption of a photon by the visual pigment of the rod, rhodopsin, initiates a biochemical amplification cascade that leads to a reduction in the concentration of cGMP and closure of the channels, thereby converting the incoming light to an electrical signal. Because the absorption of a photon and the ensuing reactions are localized events, the magnitude of the response of the rod to a single photon depends on the spread of the decrease in the cGMP concentration along the length of the outer segment. The longitudinal diffusion of cGMP depends on the structural parameters of the rod outer segment, specifically the area and the volume available for diffusion. To characterize the effect of rod outer segment cytoarchitecture on diffusion, we have used fluorescence recovery after photobleaching (FRAP) and examined the mobility of a fluorescent polar tracer, calcein, in the rod outer segments from three species with different outer segment structures: frog (Rana pipiens), mouse (Mus musculus domesticus) and gecko (Gekko gekko). We found that the diffusion coefficient is similar for all three species, in the order of 8-17 microm(2) s(-1), in broad agreement with the predictions by Holcman and Korenbrot (Biophys. J. 2004:86;2566-2582) based on the known cytoarchitecture of rod outer segments. Consequently, the results also support their prediction that the longitudinal spread of light excitation in rods is similar across species.     

LECTIN RECEPTORS ON ROD AND CONE MEMBRANES

Abstract— The oligosaccharides of rod and cone membranes were investigated with the aid of fluorescence and ¹²⁵I-labeled lectins. Additionally, the ability of lectins to cause agglutination in rod outer segment (ROS) suspensions was used as an index for the presence of the corresponding lectin receptors. The specificities of lectin-ligand interactions were determined from studies of inhibition by various haptene sugars. The membranes of both rods and cones have receptors for Con A, PNA, RCA-120, RCA-60, SBA and WGA. The affinity of PNA for accessory cones is much higher than for the principal cones. There do not appear to be receptors for UeA and LTA on rods or cones. Additionally, receptors for HPA and DBA were identified on ROS. These results suggest the existence of the following sugar residues:

The binding of Con A and WGA to ROS membrane proteins electrophoresed on SDS-polyacrylamide gels was also investigated. In addition to rhodopsin, these lectins also bind to the 291000-dalton protein, indicating that it is a glycoprotein containing mannose and GlcNAc.     

Studies on the Stability of the Human Cone Visual Pigments†

The retina of vertebrates contains two kinds of photoreceptor cells, rods and cones, which contain their specific visual pigments that are responsible for scotopic and photopic vision, respectively. In cone photoreceptor cells, there are three types of color pigments: blue, green and red, each with a distinctive absorption maximum. The goal of this investigation was to identify optimal conditions under which these pigments could be obtained and isolated in a stable form, thereby facilitating structural studies using high‐resolution approaches. For this purpose, all three human cone opsins were initially expressed in mammalian cells, reconstituted with 11‐cis retinal, detergent solubilized, purified and their stability compared with rod rhodopsin. As all three pigments showed dramatically reduced stability relative to rhodopsin, site‐directed mutagenesis was used in an attempt to engineer stability into the green cone pigment. The mutations introduced some structural motifs and sites of posttranslational modification present in rhodopsin, as well as amino acid substitutions that have been found to stabilize the rod opsin apo‐protein. We also modified the hydrophobic environment of the green cone pigment by varying the detergent and detergent/lipid composition used during solubilization and purification, and compared them with the retinal reconstituted pigment in membranes. Our results show that these changes do not significantly improve the inherent instability of the human cone pigments, and in some cases, lead to a decrease in stability and protein aggregation. We conclude that further efforts are required to stabilize the human cone pigments in a form suitable for high‐resolution structural studies.     

Biochemical steps in visual transduction: roles for nucleotides and calcium ions

Abstract— Recent studies from this laboratory permit the suggestion of a scheme for describing molecular mechanisms which may regulate excitation and adaptation in amphibian rod photoreceptor cells. The experiments have studied several chemical changes which occur upon illumination of isolated rod outer segments: (1) activation of cyclic GMP phosphodiesterase which is sensitive to calcium concentration, (2) a resulting rapid drop in cyclic GMP levels which has stoichiometry and time course appropriate for the internal transmitter presumed to mediate between photon absorption in the disc membrane system and the permeability decrease in the plasma membrane, (3) a dephosphorylation of two small proteins whose phosphorylation is controlled by cyclic GMP levels, and (4) a slower hydrolysis of GTP which may drive efflux of calcium from the outer segment. It is suggested that the rapid decrease in sodium conductance which follows illumination is caused by the dephosphorylation of the two small proteins, with their dephosphorylation being controlled by the cyclic GMP decrease. In a slower reaction light activates a GTP-dependent extrusion of calcium from the cytoplasmic space. This lowering of internal calcium causes desensitization of the light-sensitive phosphodiesterase enzyme responsible for the cyclic GMP decrease, so that its intensity-response function now resembles that of the light-adapted rod photoreceptor. Thus, changes in plasma membrane conductance are regulated by cyclic GMP, and the sensitivity of the system is controlled by slower calcium movements which set the light-sensitivity of the phosphodiesterase enzyme. Finally, the light-initiated phosphorylation of rhodopsin also appears to play a role, with phosphorylated rhodopsin causing desensitization of the phosphodiesterase enzyme.     

Salamander blue-sensitive cones lost during metamorphosis

The tiger salamander lives in shallow water with bright light in the aquatic phase, and in dim tunnels or caves in the terrestrial phase. In the aquatic phase, there are five types of photoreceptors--two types of rods and three types of cones. Our previous studies showed that the green rods and blue-sensitive cones contain the same visual pigment and have the same absorbance spectra; however, the green rods have a larger photon-catch area and thus have higher light sensitivity than the blue-sensitive cones. Here we show that after metamorphosis, the terrestrial salamander looses the blue-sensitive cones, while the density of the green rods increases. Moreover, the size of the green rod outer segments is increased in the terrestrial phase, compared to that in the aquatic phase. This switch from the blue-sensitive cones to the green rods may represent an adaptation to the dim light environment of the terrestrial phase.     

DISTRIBUTION OF ENZYMES INVOLVED IN NUCLEOTIDE METABOLISM IN THE DISK AND OTHER ROD 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 m M 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.     

THE STRUCTURE OF PHYCOBILISOMES IN MUTANTS OF Synechococcus sp. STRAIN PCC 7942 DEVOID OF SPECIFIC LINKER POLYPEPTIDES

Abstract— The effect of elimination of the 30, 33 and 9 kDa phycobilisome rod-linker polypeptides on rod length was examined by electron microscopy of phycobilisomes isolated from wild-type Synechococcus sp. strain PCC 7942 and from genetically engineered mutants with lesions in the genes encoding the rod-linker polypeptides. The maximum rod length in the absence of the 33 kDa linker polypeptide was two phycocyanin hexamers, whereas rods with up to five hexamers were found in the mutant strain lacking the 30 kDa linker polypeptide. Elimination of the 9 kDa linker polypeptide did not have a significant effect on rod length. Finally, mutants lacking either the 30 or 33 kDa rod-associated linker polypeptides had an increased number of rods that terminated with a phycocyanin trimer. These observations are discussed with respect to the role of the linker polypeptides in the biosynthesis of the rod substructure.     

Breaking Parallel Orientation of Rods via a Dendritic Architecture toward Diverse Supramolecular Structures

Self‐assembled nanostructures of rod‐like molecules are commonly limited to nematic or layered smectic structures dominated by the parallel arrangement of the rod‐like components. Distinct self‐assembly behavior of four categories of dendritic rods constructed by placing a tri(hydroxy) group at the apex of dendritic oligo‐fluorenes is observed. Designed hydrogen bonding and dendritic architecture break the parallel arrangement of the rods, resulting in molecules with specific (fan‐like or cone‐like) shapes. While the fan‐shaped molecules tend to form hexagonal packing cylindrical phases, the cone‐shaped molecules could form spherical motifs to pack into various ordered structures, including the Frank–Kasper A15 phase and dodecagonal quasicrystal. This study provides a model system to engineer diverse supramolecular structures by rod‐like molecules and sheds new light into the mechanisms of the formation of unconventional spherical packing structures in soft matter.     

LIGHT-SENSITIVITY MODULATING PROTEIN IN FROG RODS

Cyclic GMP is the second messenger in the phototransduction mechanism in rod photoreceptors. Light-induced activation of cGMP phosphodiesterase (PDE), the hydrolyzing enzyme of cGMP, reduces cytoplasmic cGMP concentration to close the cGMP-activated channel and thereby causes a hyperpolarizing light response. Ca2+ concentration decreases during light-adaptation and this decrease is thought to be at least one of the underlying mechanisms of light-adaptation. Our previous electrophysiological work suggested that PDE in frog rod photoreceptors is regulated by this Ca2+ concentration decrease. In the present work, we isolated a protein that binds to disk membranes at high Ca2+ concentrations. In the presence of this protein (a 26 kDa protein), PDE light sensitivity becomes high at high Ca2+ concentrations. The effect was observed at physiological ranges of Ca2+ concentrations. Thus we could explain high light-sensitivity of photoreceptors under the dark-adapted condition. According to its function, we termed the 26 kDa protein 'sensitivity-modulating protein' or 'S-modulin'. During the purification we noticed that there are additional mechanisms present that may contribute to light-adaptation in frog rod photoreceptors.     

Patch-clamp recording of human retinal photoreceptors and bipolar cells

Photoreceptors and retinal bipolar cells are considered as nonspiking neurons; however, we recently showed that human rod photoreceptors can generate sodium action potentials in response to membrane depolarization from membrane potentials of -60 or -70 mV (Kawai et al., Neuron 30 [2001] 451). We performed patch-clamp recording of human cone photoreceptors and retinal bipolar cells to examine whether functional voltage-gated sodium channels are expressed in these cells as well as rod photoreceptors. Under current-clamp conditions, the injection of depolarizing current steps into a cone photoreceptor-induced marked action potentials. These action potentials were blocked by 1 microM tetrodotoxin, a voltage-gated sodium channel blocker. Under voltage-clamp conditions, depolarizing voltage steps-induced a fast transient inward current in several bipolar cells (n = 4/78). This current was activated from -70 to + 20 mV (maximal at -10 mV) and inactivated within 5 ms. The 10-90% rise time of this current was shorter than another inward current (less than one-hundredth). These results indicate that human cones and bipolar cells express voltage-gated sodium channels as rod photoreceptors. Sodium channels may serve to amplify the release of a neurotransmitter and to accelerate the light-dark change in photosignals.     

Continuum transport model of Ogston sieving in patterned nanofilter arrays for separation of rod-like biomolecules

This article proposes a simple computational transport model of rod-like short dsDNA molecules through a microfabricated nanofilter array. Using a nanochannel consisting of alternate deep wells and shallow slits, it is demonstrated that the complex partitioning of rod-like DNA molecules of different sizes over the nanofilter array can be well described by continuum transport theory with the orientational entropy and anisotropic transport parameters properly quantified. In this model, orientational entropy of the rod-like DNA is calculated from the equilibrium distribution of rigid cylindrical rod near the solid wall. The flux caused by entropic differences is derived from the interaction between the DNA rods and the solid channel wall during rotational diffusion. In addition to its role as an entropic barrier, the confinement of the DNA in the shallow channels also induces large changes in the effective electrophoretic mobility for longer molecules in the presence of EOF. In addition to the partitioning/selectivity of DNA molecules by the nanofilter, this model can also be used to estimate the dispersion of separated peaks. It allows for fast optimization of nanofilter separation devices, without the need of stochastic modeling techniques that are usually required.     

PHOTORECEPTOR CELL TYPES IN THE RETINA OF VARIOUS VERTEBRATE SPECIES: IMMUNOCYTOCHEMISTRY WITH ANTIBODIES AGAINST RHODOPSIN AND IODOPSIN

Types of photoreceptor cells in the retinas of 36 species of vertebrates (5 classes, 14 orders) were investigated immunocytochemically with monoclonal antibodies against chicken iodopsin (Io-mAb) and antiserum against bovine rhodopsin (Rh-As). In mammals, Rh-As labeled the outer segments of some photoreceptor cells in striped squirrels (a diurnal mammal) and those of most photoreceptor cells in mice (a nocturnal mammal), while Io-mAb labeled any photoreceptor cells in either of them. In all species of birds studied, Io-mAb labeled the principal and accessory members of double cones and single cones with a red oil droplet. Rh-As labeled single cones with a yellow or clear oil droplet in addition to rods. In turtles, both Rh-As and Io-mAb labeled single cones with a red or clear oil droplet and the principal (with a yellow oil droplet) and accessory members of double cones. This suggests that the visual pigments in these cones of turtles have common epitopes with bovine rhodopsin and chicken iodopsin. In Japanese grass lizards, single cones with a yellow oil droplet and double cones were immunoreactive to both Rh-As and Io-mAb. In snakes, rods and cones could not be distinguished but both positively and negatively stained cells were observed by the use of each antibody. In geckos, however, all photoreceptor cells were immunonegative to Io-mAb. In all species studied in amphibians, Rh-As labeled rods but not cones. Neither rods nor cones reacted with Io-mAb. In fishes, almost all species studied had well developed cones, and some of these cones were labeled by Rh-As. However, Io-mAb labeled the outer segments of some cones only in loaches. Rh-As labeled photoreceptor cells in all species of fishes studied. Thus, Rh-As recognized the outer segments of rods in all species studied from fishes to mammals, whereas the epitope recognized by Io-mAb is conserved in some species of fishes, most species of reptiles and all species of birds studied.     

LIGHT-INDUCED INFRARED LIGHT SCATTERINGSIGNAL IN THE RETINA: EFFECT OF PHOSPHODIESTERASE INHIBITORS

Visible light changes IR light scatter through a toad retina. This signal presents three components: at low light intensity (100-400 bleached rhodopsins/rod) an early decrease in IR light scatter, of small amplitude, with time to peak of 1-6 s; at intermediate light intensity (1200-16,000 bleached rhodopsins/rod) a slow increase in IR light scatter, with time to peak of 10-30 s; at high light intensity (50,000-160,000 bleached rhodopsins/rod) a last increase in IR light scatter, with time to peak of 1 min. Light sensitivity, amplitude and time to peak of the last two components are increased by inhibitors (3-isobutyl-1-methyl-xanthine and papaverine) of the cyclic 3'5' guanosine monophosphate phosphodiesterase.     

Self-assembled monolayers of clamped oligo(phenylene-ethynylene-butadiynylene)s

Rigid rod oligo(phenylene-ethynylene-butadiynylene)s (oPEBs), "half-rings" of two rigid rods connected via a molecular clamp unit, and shape-persistent macrocycles (cyclic "half-ring dimers") are synthesized and their self-assembled monolayers (SAMs) are investigated by scanning tunneling microscopy (STM) at the interface of 1,2,4-trichlorobenzene (TCB)/highly oriented pyrolytic graphite (HOPG). The results are important for the design of molecular building blocks for two-dimensional nanoscale architectures on solid surfaces.