Absorbance spectra were recorded by microspectrophotometry from 39 different rod and cone types representing amphibians. reptiles, and fishes, with A1- or A2-based visual pigments and lambdamax ranging from 357 to 620 nm. The purpose was to investigate accuracy limits of putative universal templates for visual pigment absorbance spectra, and if possible to amend the templates to overcome the limitations. It was found that (1) the absorbance spectrum of frog rhodopsin extract very precisely parallels that of rod outer segments from the same individual, with only a slight hypsochromic shift in lambdamax, hence templates based on extracts are valid for absorbance in situ: (2) a template based on the bovine rhodopsin extract data of Partridge and De Grip (1991) describes the absorbance of amphibian rod outer segments excellently, contrary to recent electrophysiological results; (3) the lambdamax/lambda invariance of spectral shape fails for A1 pigments with small lambdamax and for A2 pigments with large lambdamax, but the deviations are systematic and can be readily incorporated into, for example, the Lamb (1995) template. We thus propose modified templates for the main "alpha-band" of A1 and A2 pigments and show that these describe both absorbance and spectral sensitivities of photoreceptors over the whole range of lambdamax. Subtraction of the alpha-band from the full absorbance spectrum leaves a "beta-band" described by a lambdamax-dependent Gaussian. We conclude that the idea of universal templates (one for A1- and one for A2-based visual pigments) remains valid and useful at the present level of accuracy of data on photoreceptor absorbance and sensitivity. The sum of our expressions for the alpha- and beta-band gives a good description for visual pigment spectra with lambdamax > 350 nm.

We report a new cellular mechanism of rod photoreceptor adaptation in vivo, which is triggered by daylight levels of illumination. The mechanism involves a massive light-dependent translocation of the photoreceptor-specific G protein, transducin, between the functional compartments of rods. To characterize the mechanism, we developed a novel technique that combines serial tangential cryodissection of the rat retina with Western blot analysis of protein distribution in the sections. Up to 90% of transducin translocates from rod outer segments to other cellular compartments on the time scale of tens of minutes. The reduction in the transducin content of the rod outer segments is accompanied by a corresponding reduction in the amplification of the rod photoresponse, allowing rods to operate in illumination up to 10-fold higher than would otherwise be possible.

Phosducin is a photoreceptor-specific protein known to interact with the ␤␥ subunits of G proteins. In pursuit of the function of phosducin, we tested the hypothesis that it regulates the light-driven translocation of G protein transducin from the outer segments of rod photoreceptors to other compartments of the rod cell. Transducin translocation has been previously shown to contribute to rod adaptation to bright illumination, yet the molecular mechanisms underlying the translocation phenomenon remain unknown. In this study we provide two major lines of evidence in support of the role of phosducin in transducin translocation. First, we have demonstrated that transducin ␤␥ subunits interact with phosducin along their entire intracellular translocation route, as evident from their co-precipitation in serial tangential sections from light-adapted but not darkadapted retinas. Second, we generated a phosducin knockout mouse and found that the degree of lightdriven transducin translocation in the rods of these mice was significantly reduced as compared with that observed in the rods of wild type animals. In knockout animals the translocation of transducin ␤␥ subunits was affected to a larger degree than the translocation of the ␣ subunit. We also found that the amount of phosducin in rods is sufficient to interact with practically all of the transducin present in these cells and that the subcellular distribution of phosducin is consistent with that of a soluble protein evenly distributed throughout the entire rod cytoplasm. Together, these data indicate that phosducin binding to transducin ␤␥ subunits facilitates transducin translocation. We suggest that the mechanism of phosducin action is based on the reduction of transducin affinity to the membranes of rod outer segments, achieved by keeping the transducin ␤␥ subunits apart from the ␣ subunit. This increased solubility of transducin would make it more susceptible to translocation from the outer segments.Vertebrate photoreceptors are highly specialized neurons responsible for the reception and primary processing of visual information. The outer segment compartment of the photoreceptor contains large amounts of the proteins involved in light detection and in the generation of the visual signal (for review, see Refs. 1-4). Photons entering the outer segment are absorbed by rhodopsin, which triggers sequential activation of many molecules of the photoreceptor-specific heterotrimeric G protein, transducin. Transducin activation consists of GTP binding to its ␣ subunit followed by dissociation of the ␣ subunit from the transducin ␤␥ subunits and the subsequent stimulation of the downstream effector, cGMP phosphodiesterase. Signaling persists until GTP is hydrolyzed and transducin subunits re-associate into a heterotrimer. In dark-adapted rods most of the transducin is located in the outer segments. However, prolonged exposures to bright light cause massive transducin translocation from rod outer segments to other subcellular compartments of the rod (5-10). In a recent st...

Retinal rods signal the activation of a single receptor molecule by a photon. To ensure efficient photon capture, rods maintain about 109 copies of rhodopsin densely packed into membranous disks. But a high packing density of rhodopsin may impede other steps in phototransduction that take place on the disk membrane, by restricting the lateral movement of, and hence the rate of encounters between, the molecules involved. Although it has been suggested that lateral diffusion of proteins on the membrane sets the rate of onset of the photoresponse, it was later argued that the subsequent processing of the complexes was the main determinant of this rate. The effects of protein density on response shut-off have not been reported. Here we show that a roughly 50% reduction in protein crowding achieved by the hemizygous knockout of rhodopsin in transgenic mice accelerates the rising phases and recoveries of flash responses by about 1.7-fold in vivo. Thus, in rods the rates of both response onset and recovery are set by the diffusional encounter frequency between proteins on the disk membrane.

Scanning electron microscopy, immunocytochemistry, and single cell microspectrophotometry were employed to characterize the photoreceptors and visual pigments in the retina of the garter snake, Thamnophis sirtalis. The photoreceptor population was found to be comprised entirely of cones, of which four distinct types were identified. About 45.5% of the photoreceptors are double cones consisting of a large principal member joined near the outer segment with a much smaller accessory member. About 40% of the photoreceptors are large single cones, and about 14.5% are small single cones forming two subtypes. The outer segments of the large single cones and both the principal and accessory members of the doubles contain the same visual pigment, one with peak absorbance near 554 nm. The small single cones contain either a visual pigment with peak absorbance near 482 nm or one with peak absorbance near 360 nm. Two classes of small single cones could be distinguished also by immunocytochemistry and scanning electron microscopy. The small single cones with the 360-nm pigment provide the garter snake with selective sensitivity to light in the near ultraviolet region of the spectrum. This ultraviolet sensitivity might be important in localization of pheromone trails.

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SUMMARY1. The dark current and responses to dim flashes were recorded with the suction pipette technique from single rods in pieces of bull-frog retina taken from either the dorsal porphyropsin or the ventral rhodopsin field.2. The composition of visual pigment in the rods was determined by microspectrophotometry. Rods from the dorsal pieces contained 70-88 % porphyropsin523 mixed with rhodopsin502. The ventral rods contained almost pure rhodopsin, any possible admixture of porphyropsin being below the level of detectability (less than 5°%).3. In most cells, the responses to dim flashes were well fitted by a four-stage linear filter model, with no systematic differences in the response kinetics of porphyropsin and rhodopsin rods. The amplitude of saturated responses varied between 8 and 55 pA and that of responses to single isomerizations between 0 4 and 3-5 pA.4. In porphyropsin rods, discrete events similar to the response to one photoisomerization were clearly seen in complete darkness. The dark current amplitude histogram was fitted by a convolution of the probability densities for the Gaussian continuous noise component and the averaged dim-flash response waveform. This allows estimation of the frequency and amplitude of discrete events and the standard deviation of the continuous component. The mean frequency of discrete dark events thus obtained from six porphyropsin cells was 0 057 rod-' s-1 at 18°C.5. In rhodopsin rods, the dark current amplitude histogram appeared completely symmetrical, indicating that the frequency of discrete events must be lower than 0 005 rod-' s-I (except in one rod where it was 0-006 events rod-' s-). Per molecule of rhodopsin, the events are then at least 5 times rarer than reported for toad rhodopsin rods at the same temperature.6. The low rate of isomerization-like 'dark' events in bull-frog rhodopsin rods shows, firstly, that results cannot be generalized across species even for rhodopsins which appear spectrally identical. Secondly, it suggests that these events need not (in

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50–70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10–40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.