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PubMed1990

Assembly of functional rhodopsin requires a disulfide bond between cysteine residues 110 and 187.

Cysteine residues 110 and 187 are essential for the formation of the correct bovine rhodopsin structure (Karnik, S. S., Sakmar, T. P., Chen, H.-B., and Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8459-8463). We now show that the sulfhydryl groups of these 2 cysteine residues interact to form a disulfide bond. Rhodopsin mutants containing cysteine----serine substitutions were prepared as follows. In one mutant, CysVII, all the 10 cysteine residues of rhodopsin were replaced by serines. A second mutant, CysVIII, contained only C110 and C185; a third mutant, CysIX, contained only C185 and C187 while the fourth mutant, CysX, contained only C110 and C187. Only mutant CysX formed functional rhodopsin. Mutants CysVIII and CysIX reacted with [3H]iodoacetic acid showing the presence of free sulfhydryl groups while mutant CysX was inert to this reagent. CysX reacted with cyanide ion to form a thiocyanate derivative showing the presence of a disulfide bond. The C110-C187 disulfide bond is buried in rhodopsin because reactions with disulfide reducing agents and cyanide ion require prior treatment with denaturants.

Amino Acid Sequence↗
PubMed1990

Localization of iodopsin in the chick retina during in vivo and in vitro cone differentiation.

Using highly specific antibodies against a chick red-sensitive cone pigment, iodopsin, we investigated the localization of iodopsin in the developing and mature chick retina. The chick retina contains several different photoreceptor types, including a rod, a double cone with a principal and accessory cone, and four different types of single cones. Immunocytochemical observations revealed that outer segments (OS) of one of the single cones (type 1) and both cells of the double cone were strongly immunoreactive to anti-iodopsin antibodies. The Golgi regions and small vesicular structures in the inner segments (IS) of these cells also were intensely stained, indicating a continuous synthesis of iodopsin and its addition to the newly formed cone OS. In the differentiating cones of the developing but immature chick retina, iodopsin immunoreactivity was found at the plasma membranes of both the IS and the terminals (pedicles). This suggests that unidirectional transport of iodopsin to the outer segment may be established during cone differentiation. Immunostaining in the outer plexiform layer (OPL) produced two bands, suggesting that the pedicles of the double cones and type 1 single cones terminate at different positions in this layer. Application of the antibodies to a cell culture system of the chick retina revealed that cells immunoreactive to anti-iodopsin differ slightly in morphology from those reactive with anti-rhodopsin. Since antibodies to iodopsin and rhodopsin stained different types of photoreceptors in the intact chick retina, it will be possible to analyze cell lineage of rods and cones in vitro by use of these antibodies.

Animals↗
PubMed1989

[Studies on the differentiation of the retina receptor cell of toad (Bufo raddei Strauch)].

The development of the retinal receptor cell in the young tadpoles (Bufo raddei Strauch), from the stage 20 to the stage 25, was studied by TEM and immunohistochemical method. The morphological differentiation of the photoreceptor cell may be described as follows. The time and the degree of differentiation of the cells in the tadpole retina is asynchronous between central (posterior pole) and peripheral parts of the tadpole retina, namely, they are earlier and higher in the central than in the peripheral. The cells of the outer nuclear layer are undifferentiated at the stage 20. The cells in the posterior part of the retina elongate at the beginning of the stage 21 (Plate I, Fig. 1). This is the first sign of differentiation in the photoreceptor cell. A small hillock-like process forms the inner segment at the scleral pole of the receptor cell. The inner segment is rich in mitochondria, rough-surfaced cytomembrane, free ribosomes, and vesicles. One or two large lipid droplets are also found in the inner segment (Plate I Fig. 2-3). Later on, the connecting structure develops at the tip of the inner segment. The newly formed filaments and the plasma membrane form the outer segment. Its membrane forms some evaginations oriented perpendicularly to the longitudinal axis of the receptor cell. In this way, disks of the outer segment are formed (Plate I Fig. 4-5). The length of the outer segment gradually increases with the number of disks increasing at the base. At the same time, an axon process of receptor cell, extending vitreal, develops synapses with dendrites of the bipolar cell in the outer plexiform layer. At the beginning (the stage 22), the synaptic structure is an immature form that lacks synaptic ribbons and vesicles (Plate II Fig. 8). Later on, ribbons and vesicles are observed in the further developed synaptic structure (Plate II Fig. 9). The toad rhodopsin was prepared by a method of Dewey et al. (1969) and Papermaster & Dreyer (1974) with slight modification. A specific immune serum against the toad rhodopsin was produced in rabbits. Using the indirect Coon's antibody technique, the localization of the rhodopsin antibody and the time when the antibody was seen in the retina of the early developing tadpoles was traced.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

[The effect of anions on spectral properties of iodopsin and native cones in the frog retina (microspectrophotometric study)].

Absorption spectra of single outer segments of the frog Rana temporaria photoreceptors were registered. Effects of nitrate and chloride ions on spectral properties of cone and rod pigments were compared. These pigments proved to differ in structure of the native photoreceptor membrane and, therefore, in effect of hydrophile environment on the chromophore centrum. Substitution of chloride by nitrate ions led to the hypochromic shift of the cone absorption spectrum (20-25 nm) but does not affect the spectrum on case of rod pigment. The ionochromic behaviour of cone pigments resembles that of the light-sensitive halobacterium protein halorhodopsin, in native membrane. We suppose that the effect of anions on the chromophore centrum may be the cause of bathochromic shifts of absorption spectra of longwave-length retinal-containing pigments.

Animals↗
PubMed1984

Regeneration of rhodopsin and isorhodopsin in rod outer segment preparations: absence of effect of solvent parameters.

Regeneration of rhodopsin and isorhodopsin from bleached bovine rod outer segment suspensions and 11- or 9-cis retinal occurs at similar rates (second-order rate constants of 1000 M-1s-1 and 900 M-1s-1, respectively) and to a similar extent (maximal regeneration at pH 5.5-7.0). Formation of both pigments follows pseudo-first order kinetics in an excess of retinal at pH 7.0. These results suggest that the mechanisms of regeneration are similar for the 11-cis and 9-cis retinal isomers. Solvent ionic strength, buffer concentration, viscosity, and polarity have no significant effect on the rate of pigment formation, providing further evidence that the retinal binding site in bleached bovine rod outer segments is well protected from the hydrophilic environment.

Animals↗
PubMed1994

Molecular and biochemical analyses of iodopsin in rd chick retina.

PURPOSE: The results of previous immunocytochemical and electrophysiological studies of retinas of rd (retinal degeneration) chicks suggest that the iodopsin cone visual pigment may be defective in this mutant. The goal of this study was to determine if the primary structure and synthesis of this protein is normal in this animal model of inherited retinal degeneration. METHODS: Northern cDNA sequence and western analyses were used to study rd/rd iodopsin. cDNAs encoding rd/rd iodopsin were obtained by screening an rd/rd cDNA retinal expression library and by reverse transcription PCR. Western blots were probed with either R4 or COS-1, two different monoclonal antibodies that have been shown to specifically recognize chicken iodopsin. RESULTS: Hybridization of the +/+, +/rd, and rd/rd poly(A)+ RNA with an iodopsin cDNA probe revealed the presence of a single 1.5 kb band in each of the samples, all of which were labeled with equal intensity. No significant differences were found between the published nucleic acid sequences for normal chicken iodopsin cDNA and that determined for rd/rd iodopsin cDNA. Antibody-dependent differences in the staining intensity of the 34 kDa band containing iodopsin were observed on western blots of +/+, +/rd, and rd/rd retinal protein. R4 stained the 34 kDa band in each sample with equal intensity. COS-1 labeling of the 34 kDa band in the rd/rd sample was less intense than that observed in the +/+ and +/rd samples. CONCLUSIONS: Based on the results of the cDNA sequence and northern blot experiments, the authors conclude that the gene encoding iodopsin and transcription of this gene are normal in the rd mutant. The results of the western blot analyses of rd/rd iodopsin suggest that post-translational processing of iodopsin may be abnormal in this mutant.

Animals↗
PubMed1994

Insertional mutagenesis as a probe of rhodopsin's topography, stability, and activity.

This paper reports a study of rhodopsin's structure and function using insertional mutagenesis with a flexible epitope. Sixteen rhodopsin derivatives were constructed, each of which carried a 12-amino acid epitope derived from the c-Myc protein flanked by penta-glycine linkers. For eight of the insertion mutants, the membrane sideness of the epitope insert was determined by immunostaining of intact or permeabilized cells. The results confirm the sidedness of each of the six helix connecting loops and the amino and carboxyl termini as postulated by the current seven-helix models of G-protein-coupled receptors and provide the first experimental evidence for the existence of the third extracellular loop. In general, inserts that were either closer to the amino terminus or on the extracellular face were more likely to disrupt folding and/or stability than were inserts near the carboxyl terminus or on the cytosolic face. Epitope insertion at positions 139 or 239, in the second and third cytosolic loops, respectively, failed to activate transducin, whereas an insertion at position 333 in the carboxyl-terminal tail was fully functional. The experimental approach described here should prove generally useful for elucidating structural and functional properties of both membrane and globular proteins.

Amino Acid Sequence↗
PubMed1993

[A comparative study of the detection of abnormal visual pigment genes by PCR and hybridization].

Since color vision abnormalities always involved the exon 5 of visual pigment genes, 2 oligonucleotide primers were designed to amplify the exon 5 of the red and green pigment genes. After electrophoresis of PCR products digested with RsaI or Sau 3A, the DNA fragments from the exon 5 of the red and green pigment genes were separated by virtue of their different restriction endonuclease sites. In the meantime, we analyzed the exon 5 related fragments by Southern blot hybridization with probe from the 3' end of the fourth intron of the visual pigment genes, yielding consistent results. The PCR technique may be of value in prenatal evaluation and genetic counseling.

Base Sequence↗
PubMed1993

Process outgrowth and synaptic varicosity formation by adult photoreceptors in vitro.

To assess the regenerative capability of the photoreceptor synapse, we have isolated and cultured photoreceptors from the mature salamander retina. Both rod and cone photoreceptors were able to regenerate processes within 3 d of plating. Cells extended numerous actin-containing filopodia as well as a few neuritic processes. The neurites contained microtubules and formed synaptic vesicle-filled varicosities, as shown by immunostaining for tubulin and synaptic vesicle proteins and by electron microscopy. Furthermore, regenerated varicosities were capable of depolarization-induced vesicle labeling, suggesting that they can recycle synaptic vesicles and release neurotransmitter by synaptic vesicle exocytosis. Differences were observed between rod and cone cell synaptic regeneration in vitro, which resembled structural differences between their synaptic terminals in situ: rod cells formed multiple synaptic vesicle-filled varicosities along neurites at a distance from the soma, whereas cone cells tended to accumulate synaptic vesicles within the soma. The regeneration of neurites and synaptic vesicle-filled varicosities was abolished by microtubule depolymerizing agents, suggesting a role for microtubule-based vesicle transport in the formation of varicosities. Finally, process outgrowth and varicosity formation were independent of cell-cell contact and, indeed, proceeded in the complete absence of other cells. These findings suggest not only that differentiated photoreceptors are capable of synaptic renewal but that the regeneration of presynaptic-like terminals is an intrinsic ability of rod and cone cells.

Animals↗