Growth factors regulate phototransduction in retinal rods by modulating cyclic nucleotide-gated channels through dephosphorylation of a specific tyrosine residue.

Illumination of vertebrate rod photoreceptors leads to a decrease in the cytoplasmic cGMP concentration and closure of cyclic nucleotide-gated (CNG) channels. Except for Ca(2+), which plays a negative feedback role in adaptation, and 11-cis-retinal, supplied by the retinal pigment epithelium, all of the biochemical machinery of phototransduction is thought to be contained within rod outer segments without involvement of extrinsic regulatory molecules. Here we show that insulin-like growth factor-I (IGF-I), a paracrine factor released from the retinal pigment epithelium, alters phototransduction by rapidly increasing the cGMP sensitivity of CNG channels. The IGF-I-signaling pathway ultimately involves a protein tyrosine phosphatase that catalyzes dephosphorylation of a specific residue in the alpha-subunit of the rod CNG channel protein. IGF-I conjointly accelerates the kinetics and increases the amplitude of the light response, distinct from events that accompany adaptation. These effects of IGF-I could result from the enhancement of the cGMP sensitivity of CNG channels. Hence, in addition to long-term control of development and survival of rods, growth factors regulate phototransduction in the short term by modulating CNG channels.

The cloning of GRK7, a candidate cone opsin kinase, from cone- and rod-dominant mammalian retinas.

PURPOSE: Desensitization in the rod cell of the mammalian retina is initiated when light-activated rhodopsin is phosphorylated by the G protein-coupled receptor kinase (GRK), GRK1, often referred to as rhodopsin kinase. A distinct kinase that specifically phosphorylates cone opsins in a similar manner has not been identified in mammals. To determine the existence of a cone opsin kinase, RNA from the retinas of cone- and rod-dominant mammals was analyzed by PCR. METHODS: RNA prepared from the retinas of two cone-dominant mammals, the thirteen-lined ground squirrel and the eastern chipmunk, and a rod-dominant mammal, the pig, was used to clone a new GRK family member by RT-PCR. The tissue distribution and localization of the kinase in retina were determined by Northern blot hybridization and in situ hybridization. The protein encoded by this cDNA was expressed in human embryonic kidney-293 (HEK-293) cells and compared with bovine GRK1 for its ability to phosphorylate bovine rhodopsin and to undergo autophosphorylation. RESULTS: The cDNA cloned from ground squirrel contains an open reading frame encoding a 548 amino-acid protein. Sequence analysis indicates that this protein is orthologous to GRK7 recently cloned from O. latipes, the medaka fish. Partial cDNA fragments of GRK7 were also cloned from RNA prepared from eastern chipmunk and pig retinas. In situ hybridization demonstrated widespread labeling in the photoreceptor layer of the ground squirrel retina, consistent with expression in cones. Recombinant ground squirrel GRK7 phosphorylates bovine rhodopsin in a light-dependent manner and can be autophosphorylated, similar to bovine GRK1. CONCLUSIONS: These results indicate that cone- and rod-dominant mammals both express GRK7. The presence of this kinase in cones in the ground squirrel and its ability to phosphorylate rhodopsin suggests that it could function in cone cells as a cone opsin kinase.

Rod outer segment maintenance is enhanced in the presence of bFGF, CNTF and GDNF.

We employed a morphological assay of outer segment collapse to determine if growth factors or other supplements directly affect dissociated rod photoreceptors in vitro. The morphological changes in outer segments were correlated with the light responsiveness of rods. Time-lapse video microscopy was used to observe the collapse of rod outer segments from isolated single cells and small clumps of cells. A consistent pattern of outer segment collapse into the inner segment was observed, yielding a convenient assay of the effects of neurotrophic factors on photoreceptor functional maintenance. The functional state of rods, defined as light-responsiveness, was measured with suction electrode recordings and matched with the various stages of outer segment collapse. Ciliary neurotrophic factor (CNTF) and glial cell-line-derived neurotrophic factor (GDNF) at a high concentration, yielded statistically significant improvements in rat outer segment survival times. Basic fibroblast growth factor (bFGF), which rescues photoreceptors in several rodent models of retinal degeneration, produced a significant increase in survival time in the presence of the cofactor heparin. In 4 out of 10 cases using human tisue, bFGF also yielded a significant increase in survival times. When brain-derived neurotrophic factor (BDNF) was applied to rat rods, outer segment survival times did not change. Outer segments collapsed more quickly when either pigment epithelial cell derived factor (PEDF) or sugar N-acetyl D-galactosamine (NAD-gal) were present. Our results show that rod photoreceptors can respond to bFGF, GDNF and CNTF in vitro and provide evidence for a direct effect of these neurotrophic factors on rods. The rapid collapse of isolated photoreceptors in this model provides a convenient means for testing various neurotrophic agents and the induced cellular responses.

Aberrant photon responses in rods of the macaque monkey.

Recovery from bright light was studied in macaque rods by measuring the membrane current of single outer segments. The recovery phase of some responses displayed a plateau current of about one picoampere lasting for several seconds. The following evidence suggests these "steps" are single photon responses of abnormally long duration. (1) Over a limited range of intensities, step amplitude remained constant and summed linearly with intensity. The collecting area for step generation was about 2.6 x 10(-3) microns2. (2) Step duration varied exponentially with a mean duration of about 6.5 s. (3) Fluctuation analysis of the tail currents was consistent with the idea that a step is evoked by isomerization of a single rhodopsin molecule, and that only 1 in 400 isomerizations leads to a responses with a step-like waveform. (4) With only the distal portion of the outer segment in the electrode, the polarity of the step response reversed when the proximal portion of the outer segment was illuminated, indicating that step generation results from a local change in outer segment conductance near the site of photon absorption. (5) The probability of eliciting a step varied with the wavelength of light in the manner expected from the absorption spectrum of rhodopsin.

Spectra of human L cones.

Variations in the amino acid sequences of the human cone opsins give rise to spectrally variant subtypes of L and M cone pigments even in the population with normal color vision. In vitro mutagenesis studies have shown that a limited number of amino acid substitutions produce shifts in the wavelength sensitivity. Presented here are results comparing electrophysiological measurements of single human cones with the expressed cone pigment gene sequences from the same retina. In a sample of eight long-wavelength sensitive cone (L cone) spectra obtained from five donors the precise spectral sensitivities, measured in situ, of the two most commonly occurring spectral variants were determined. The peak sensitivity of the Lser180 cone was 563 nm while that of the Lala180 cone was 559 nm.

Expression of L cone pigment gene subtypes in females.

Spectral subtypes of L pigment are produced by a serine/alanine dimorphism at amino acid position 180. X-chromosomes that carry genes for the different subtypes occur with about equal frequency in normal men. Females have two X-chromosomes: thus, about 50% of women will inherit genes for both L pigment subtypes, although on different X-chromosomes. In these women, X-inactivation is expected to produce about equal numbers of LS180 and LA180 cones in addition to middle (M) and short (S) wavelength-sensitive cones to total four spectrally distinct cone types. Consistent with this expectation we found nearly equal expression of genes for two spectrally distinct subtypes of L pigment in five of nine female retinas examined.

Visual transduction in human rod photoreceptors.

1. Photocurrents were recorded with suction electrodes from rod photoreceptors of seven humans. 2. Brief flashes of light evoked transient outward currents of up to 20 pA. With increasing light intensity the peak response amplitude increased along an exponential saturation function. A half-saturating peak response was evoked by approximately sixty-five photoisomerizations. 3. Responses to brief dim flashes rose to a peak in about 200 ms. The waveform was roughly like the impulse response of a series of four to five low-pass filters. 4. The rising phases of the responses to flashes of increasing strength were found to fit with a biochemical model of phototransduction with an 'effective delay time' and 'characteristic time' of about 2 and 800 ms, respectively. 5. Spectral sensitivities were obtained over a wavelength range from 380 to 760 nm. The action spectrum, which peaked at 495 nm, followed the template described for photoreceptors in the macaque retina. Variation between rods in the position of the spectrum on the wavelength axis was small. 6. The scotopic luminosity function derived from human psychophysical experiments was found to agree well with the measured rod action spectrum after adjustments were made for lens absorption and photopigment self-screening in the intact eye. 7. Responses to steps of light rose monotonically to a maintained level, showing little or no relaxation. Nevertheless, the relationship between light intensity and steady-state response amplitude was shallower than that expected from simple response saturation. This is consistent with an adaptation mechanism acting on a rapid time scale. 8. Flash sensitivity fell with increasing intensities of background light according to Weber's law. Sensitivity was reduced twofold by lights evoking about 120 photoisomerizations per second. Background lights decreased the time to peak and the integration time of the flash response by up to 20%.

Effects of modified chromophores on the spectral sensitivity of salamander, squirrel and macaque cones.

1. Chemically modified retinal chromophores were used to investigate the mechanisms that produce the characteristic spectral absorptions of cone pigments. Spectral sensitivities of single cones from the salamander, squirrel and macaque retina were determined by electrical recording. The chromophore was then replaced by bleaching the pigment and regenerating it with a retinal analogue. 2. Exposing a bleached cone to 9-cis-retinal for a brief period (less than 20 min) caused its flash sensitivity to recover to about 0.2 of the pre-bleach value. Similar exposure to a locked 6-s-cis, 9-cis analogue gave a recovery to about 0.03 of the pre-bleach value. 3. Unlike the flash sensitivity, the saturating photocurrent amplitude often recovered completely after bleaching and regenerating the pigment. 4. When the 3-dehydroretinal chromophore in the salamander long-wavelength-sensitive (red) cone was replaced with 11-cis-retinal, shortening the conjugated chain in the chromophore, the spectral sensitivity underwent a blue shift of 67 nm. 5. Pigments containing the planar-locked 6-s-cis.9-cis-retinal analogue absorbed at substantially longer wavelength than those containing unmodified 9-cis-retinal. The opsin shift, a measure of the protein's ability to modify the chromophore's absorption was larger for the locked analogue than for 9-cis-retinal. This suggests that the native chromophore assumes a twisted 6-s-cis conformation in these pigments. 6. The spectral sensitivities of red and green macaque cones containing 9-cis-retinal or planar-locked 6-s-cis.9-cis-retinal retained the 30 nm separation characteristic of the native pigments. This suggests that the different absorptions of of the 6-7 carbon bond in the retinal chromophore.

Photocurrents of cone photoreceptors of the golden-mantled ground squirrel.

1. Visual transduction in photoreceptors of the ground squirrel, Citellus lateralis, was studied by recording membrane current from individual cones in small pieces of retina. 2. Brief flashes of light produced transient reductions of the dark current; saturating response amplitudes were up to 67 pA. A flash strength of about 11,000 photons microns-2 at lambda max was required to give a half-saturating response. The stimulus-response relation was well fitted by an exponential saturation curve. Responses below 20% of maximum behaved linearly. 3. The response to a dim flash in most cells had a time to peak of 20-30 ms and resembled the impulse response of a series of five low-pass filters. 4. The variance of the dim-flash response amplitude put an upper limit of 80 fA on the size of the single photon response. Estimates based on the effective collecting area suggest the single photon response to be of the order of 10 fA. 5. Flash responses of squirrel cones usually lacked the undershoot observed in primate cones, although in about 1/3 of the cells a small undershoot developed during recording. 6. Background lights slightly shortened the time to peak of the flash response and reduced the integration time. 7. Spectral sensitivity measurements showed two classes of cones with peak sensitivities at about 520 and 435 nm. Rod sensitivity peaked near 500 nm. Spectral univariance was obeyed by all three classes of cells. 8. The shapes of the spectral sensitivity curves of the rod and both types of cones were similar to each other when plotted on a log wave number scale, but differed significantly from similar plots of monkey and human cone spectra. 9. The kinetics and sensitivity of flash responses of the blue- and green-sensitive cones were indistinguishable.

Spectral sensitivity of primate photoreceptors.

The spectral sensitivities of rods and cones in macaque and human retinas were determined by recording the membrane current from single outer segments. In the macaque retina, the wavelengths of maximum sensitivity were at about 430, 530, and 561 nm for the blue, green, and red cones, respectively, and at 491 nm for the rods. The shapes of the spectra of the three cones were similar when plotted on a log wavenumber scale; the rod spectrum was slightly broader. Spectral sensitivities of the red and green cones from a human retina were virtually identical to those of macaque cones. For comparison with human psychophysical measurements, the rod and cone spectra were adjusted to give the sensitivities expected for light incident on the cornea of the human eye. These functions satisfactorily predicted the scotopic and photopic luminosity functions as well as results from human color-matching experiments. The adjusted spectra of the red and green cones also agreed well with the pi-mechanism of Stiles (1953, 1959).

Spectral sensitivity of human cone photoreceptors.

The brain computes visual colour by analysing the relative excitations of three types of retinal cones. Each cone excitation is governed by a spectral sensitivity function which relates the amplitude of the neural response to wavelength at constant light intensity. The spectral sensitivities of human cones are not well characterized. We report measuring the sensitivities by recording electrical responses of human cones to stimuli of different wavelengths. Spectral sensitivities of 'green' and 'red' cones, determined over the entire visible region, show peaks near 530 and 560 nm respectively, and are remarkably similar to those of the old-world monkey Macaca fascicularis. They satisfactorily predict the photopic luminosity function, a measure of the sensitivity of cone-mediated human vision to light of different wavelengths. The kinetics of the light responses of human cones also appeared similar to those of macaque cones: the time to peak response to a dim flash was 50-100 ms and there was a characteristic undershoot during recovery.

Telodendrites of cone photoreceptors: structure and probable function.

Intracellular dye injection and compartmental modeling were used to analyze the structure and function of telodendrites of cones in the retina of the walleye. After identifying the spectral type of an impaled cone on the basis of its response to red and green light, horseradish peroxidase and/or Lucifer Yellow were injected for 1 to 25 minutes. In 38 of 58 recovered cells, dye spread into the telodendrites; so in many cases, the detailed pattern of the telodendritic arbor could be reconstructed from serial sections. Typically, five telodendritic processes, about 1 micron in diameter and 18 micron in length, radiated from the cone pedicle. A majority of the processes terminated at pedicles of neighboring cones. Some of the Lucifer Yellow injections provided evidence for electrical coupling between cones via telodendrites. Calculations from a compartmental model, based on the measured dimensions of cones and telodendrites, indicate that the signal arising in the inner segment spreads with little loss to the end of a telodendrite, whereas about half of the signal is lost in transmission from telodendrite to inner segment. Assuming that each contact point within the telodendritic network is an electrical synapse of 2,500 M omega, the model shows spatial interaction over a field of some 80 micron, which is comparable to that measured experimentally. Although our anatomical data indicate that orange- and green-sensitive cones may be interconnected via telodendrites, model calculations indicate that such connections do not appreciably distort the intrinsic spectral sensitivity of walleye cones. This outcome agrees with previous experimental results.