Clint L. Makino - US grants

The goal of these studies is to elucidate factors that determine the spectral sensitivities of individual photoreceptor cells. In general, a photoreceptor's spectral sensitivity will depend upon the type of visual pigment expressed and the absorption properties of the pigment. Neither of these aspects is well understood. A visual pigment's spectral tuning is produced by perturbations of the chromophore by its specific opsin. The first aim of this proposal is to find out how and where opsin perturbs the chromophore to tune its absorption. My approach will be to characterize the spectral sensitivities of isolated rods and cones of salamander and goldfish electrophysiologically before and after replacing their native chromophore with chemically modified retinal analogs. Using a series of dihydroretinal analogs, we will localize the chromophoric sites that interact with opsin. The second aim is to find out whether a cell can express multiple opsins and whether all opsins form functional pigments. Although an individual photoreceptor is thought to express only one type of opsin, there is evidence to suggest that some photoreceptors violate this rule. We will test for the presence of two functional opsins in guinea pig cones and three in salamander cones by measuring the cell's spectral sensitivity before and after exposure to an intense bleaching light. The spectral composition of the bleaching light will be carefully chosen so that one pigment type is bleached preferentially. Additional evidence that multiple opsins are expressed will be obtained using immunohistochemical and molecular biological probes. The third aim is to investigate the ability of photoreceptors to change the type of opsin they express. We will measure the spectral sensitivities of cones in salamander and winter flounder at different ages to see if their pigment content changes as a function of time. The results of these studies could have developmental significance for the regulation of cone density and the formation of cone-specific neural circuits in the retina. The long term goal of these studies is to understand how the retina encodes visual information carried by wavelength and to explore the plasticity of the underlying mechanisms.

DESCRIPTION (provided by applicant): Rod photoreceptors approach the ultimate in sensitivity to light but are also capable of adjusting that sensitivity over a wide range of ambient lighting conditions. This process of light adaptation is important because it extends the dynamic range over which rods are capable of encoding visual information. The goal of these studies is to understand the molecular bases for the changes in rod photoreceptor sensitivity that take place in the presence of steady illumination. Light adaptation is known to involve Ca+2 feedback onto the phototransduction cascade accelerating rhodopsin shutoff, increasing cGMP production and opening ion channels at lower levels of cGMP. At least some of these feedback mechanisms operate rapidly and cause the response to steps of light to partially recover or droop. This droop is essentially complete in a few seconds and helps to keep the rod from saturating so that it can continue to signal changes in light intensity. From suction electrode recordings on single rods of amphibians, we have obtained evidence that there is a component of light adaptation that operates on a slower time scale. The time course and magnitude of desensitization of the slow component will be measured. The dependency of the slow component upon Ca+2 feedback will be determined. Then three putative mechanisms will be tested: transducin's lifetime shortens as cGMP dissociates from noncatalytic sites on PDE, transducin availability decreases because phosducin prevents its recycling, the cGMP-gated channel opens at lower concentrations of cGMP due to a change in its apparent affinity for cGMP. The three mechanisms are not mutually exclusive. Interestingly, light adaptation in amphibian rods far surpasses that in mammalian rods. Two hypotheses will be explored. First, biochemical methods will be used to test whether the decrease in transducin's lifetime during light adaptation in amphibian rods due to the dissociation of cGMP from noncatalytic sites on phosphodiesterase fails to occur in mammalian rods, because cGMP binds much more tightly to these sites on mammalian PDE. Second, rare, aberrant, prolonged single photon responses occur in mammalian rods, but have not been seen in amphibian rods. The cumulative effects of aberrant responses during exposures to steps of light may drive mammalian rods into saturation prematurely. Suction electrode recording will be used to assess the impact of aberrant responses on light adaptation in wild type mouse rods and in rods where the frequency of aberrant responses is higher than normal. Aberrant responses occur with higher frequency in some human retinal diseases and severely limit the intensity range of scotopic vision.

[unreadable] DESCRIPTION (provided by applicant): The sensitivity and response kinetics of a vertebrate photoreceptor depend on the intrinsic properties of the visual pigment molecule, such as its spectral sensitivity, as well as environmental factors that affect visual pigment function, such as the lateral mobility of rhodopsin in the disk membrane. The goal of this application is to explore the impact of two environmental factors on the photoresponse: rhodopsin packing density and type of transducin present. The first three components involved in the activation of phototransduction: rhodopsin, transducin and phosphodiesterase, interact on the disk membrane surface. Rhodopsin kinase and RGS9, two key proteins in the shutoff of phototransduction are also attached to the membrane. In normal rods, the rate-limiting steps in the activation and recovery of the photoresponse are limited by the rates of molecular collisions on the disk membrane surface. These rates are slowed by the prohibitive effect of the high rhodopsin packing density on membrane protein mobility, because a 50% reduction in the rhodopsin packing density accelerated the rising and falling phases of the photoresponse. The effect of a further decrease in rhodopsin packing density on the photoresponse cannot be predicted reliably, because the identity of the rate-limiting step could change. The magnitude of the effect of an increase in packing density cannot be predicted either, because the change in protein mobility with packing density is not known in vivo. These conditions will be tested in rods of transgenic mice that express sub- and supranormal amounts of rhodopsin in their disk membranes. Phototransduction will be evaluated from suction electrode recordings of the electrical responses of the rods to flashes and steps of light. [unreadable] [unreadable] Rods and cones differ in their sensitivity and response kinetics. They appear to have similar pigment packing densities but in general, the identities of their pigments and transducins are different. The effects of pigment and transducin type on photoresponse sensitivity and kinetics will be tested in rods and cones of salamander, where green rods and blue-sensitive cones couple the same visual pigment to different transducins and UV-sensitive cones contain multiple pigments coupled to the same transducin.

[unreadable] DESCRIPTION (provided by applicant): Many G protein coupled receptors, visual opsins included, weakly activate G protein in the absence of ligand. Opsins are unique in their binding of an inverse agonist at rest, 11-cis retinal, which is transformed into an agonist by light. The interactions between rod opsin and its 11-cis retinal chromophore have been well studied for their roles in pigment stability, activation and spectral tuning. Recently, it has been proposed that opsin possesses two additional retinoid binding sites that enable retinoids to approach and exit the pigment-forming chromophore binding pocket. In this study, we will provide further evidence for multiple ligand binding sites on opsin. More importantly, we hypothesize that opsins bind other ligands besides retinoids and that the functions of the binding sites extend beyond visual cycle. Using single cell physiology, single cell microspectrophotometry and spectroscopy on rhodopsin in solution, we propose experiments to test three functions. The light-regulated channel is susceptible to potent inhibition by retinoids. One function of the alternate binding sites may be to allow rhodopsin and opsin to serve as buffers that protect the cGMP-gated channel from retinoid inhibition after exposure to bright light, when vast amounts of retinoids are exchanged with the pigment epithelium. A second function of retinoid binding to alternate sites is to modulate the catalytic activity of rhodopsin and opsin. This function may play roles in conferring reproducibility to the single photon response, in setting the absolute sensitivity and response kinetics of the receptor. A third function may be to enable compounds to absorb light and transfer the energy to rhodopsin, causing the latter to photoisomerize and give rise to an electrical response as if by direct absorption. Thus spectral sensitivity may be altered without a change in opsin protein expression. Each of these functions may depend upon opsin type. A strong link has been established between phototransduction cascade activity and retinal disease raising the possibility that mutations affecting the alternate ligand binding sites or that affect ligand processing may be responsible for or contribute to certain retinal pathologies. Then pharmaceuticals that target the alternate site may be used for the treatment of a retinal disease or for the enhancement of vision. Therefore, this proposal will be highly significant to our understanding of the structure and function of visual opsins and opens new directions for vision research. [unreadable] [unreadable] [unreadable]