This article evaluates each of the reactions known to be involved in visual transduction as a potential site for the regulation of light adaptation. Extensive evidence suggests that calcium acts as a feedback messenger at several different points and recent work suggests a role for cGMP in regulating the primary excitatory pathway. A conclusion is that adaptation is likely to be regulated by multiple and redundant mechanisms. The goal of future experimentation will be to determine the relative importance of each of these.

Using suction electrodes, photocurrent responses to 100-ms saturating flashes were recorded from isolated retinal rods of the larval-stage tiger salamander (Ambystoma tigrinum). The delay period (Te) that preceded recovery of the dark current by a criterion amount (3 pA) was analyzed in relation to the flash intensity (If), and to the corresponding fractional bleach (R*0/Rtot) of the visual pigment; R*0/Rtot was compared with R*s/Rtot the fractional bleach at which the peak level of activated transducin approaches saturation. Over an approximately 8 In unit range of If that included the predicted value of R*s/Rtot, Te increased linearly with In If. Within the linear range, the slope of the function yielded an apparent exponential time constant (TC) of 1.7 ± 0.2 s (mean ± S.D.). Background light reduced the value of Tc measured at a given flash intensity but preserved a range over which Tc increased linearly with In If; the linear-range slope was similar to that measured in the absence of background light. The intensity dependence of Tc resembles that of a delay (Td) seen in light-scattering experiments on bovine retinas, which describes the period of essentially complete activation of transducin following a bright flash; the slope of the function relating Td and In flash intensity is thought to reflect the lifetime of photoactivated visual pigment (R*) (Pepperberg et al., 1988; Kahlert et al., 1990). The present data suggest that the electrophysiological delay has a similar basis in the deactivation kinetics of R*, and that Tc represents TR* the lifetime of R* in the phototransduction process. The results furthermore suggest a preservation of the “dark-adapted” value of TR* within the investigated range of background intensity.

Synaptic transmission from photoreceptors to depolarizing bipolar cells is mediated by the APB glutamate receptor. This receptor apparently is coupled to a G-protein which activates cGMP-phosphodiesterase to modulate cGMP levels and thus a cGMP-gated cation channel. We attempted to localize this system immunocytochemically using antibodies to various components of the rod phototransduction cascade, including Gt (transducin), phosphodiesterase, the cGMP-gated channel, and arrestin. All of these antibodies reacted strongly with rods, but none reacted with bipolar cells. Antibodies to a different G-protein, Go, reacted strongly with rod bipolar cells of three mammalian species (which are depolarizing and APB-sensitive). Also stained were subpopulations of cone bipolar cells but not the major depolarizing type in cat (b1). Go antibody also stained certain salamander bipolar cells. Thus, across a wide range of species, Go is present in retinal bipolar cells, and at least some of these are depolarizing and APB-sensitive.

Opsins, like many other G-protein-coupled receptors, sustain constitutive activity in the absence of ligand. In partially bleached rods and cones, opsin's activity closes cGMP-gated channels and produces a state of “pigment adaptation” with reduced sensitivity to light and accelerated flash response kinetics. The truncated retinal analogue, β-ionone, further desensitizes partially bleached green-sensitive salamander rods, but enables partially bleached red-sensitive cones to recover dark-adapted physiology. Structural differences between rod and cone opsins were proposed to explain the effect. Rods and cones, however, also contain different transducins, raising the possibility that G-protein type determines the photoreceptor-specific effects of β-ionone. To test the two hypotheses, we applied β-ionone to partially bleached blue-sensitive rods and cones of salamander, two cells that couple the same cone-like opsin to either rod or cone transducin, respectively. Immunocytochemistry confirmed that all salamander rods contain one form of transducin, whereas all cones contain another. β-Ionone enhanced pigment adaptation in blue-sensitive rods, but it also did so in blue- and UV-sensitive cones. Furthermore, all recombinant salamander rod and cone opsins, with the exception of the red-sensitive cone opsin, activated rod transducin upon the addition of β-ionone. Thus opsin structure determines the identity of β-ionone as an agonist or an inverse agonist and in that respect distinguishes the red-sensitive cone opsin from all others.

Phosducin (Pd) is a 28-kD phosphoprotein whose expression in retina appears limited to photoreceptor cells. Pd binds to the β,γ subunits of transducin (Gt). Their binding affinity is markedly diminished by Pd phosphorylation. While Pd has long been regarded as a candidate for the regulation of Gt, the molecular details of Pd function remain unclear. This gap in understanding is due in part to a lack of precise information concerning the total amount and subcellular localization of rod Pd. While earlier studies suggested that Pd was a rod outer segment (ROS) protein, recent findings have demonstrated that Pd is distributed throughout the rod. In this report, the subcellular distribution and amounts of rat Pd are quantified with immunogold electron microscopy. After light or dark adaptation, retinal tissues were fixed in situ and prepared for ultrathin sectioning and immunogold labeling. Pd concentrations were analyzed over the entire length of the rod. The highest Pd labeling densities were found in the rod synapse. Less intense Pd staining was observed in the ellipsoid and myoid regions, while minimal labeling densities were found in the ROS and the rod nucleus. In contrast with rod Gt, no evidence was found for light-dependent movement of Pd between inner and outer segments. There is a relative paucity of Pd in the ROS as compared with the large amounts of Gt found there. This does not support the earlier idea that Pd could modulate Gt activity by controlling its concentration. On the other hand, the presence of Pd in the nucleus is consistent with its possible role as a regulator of transcription. The functions of Pd in the ellipsoid and myoid regions remain unclear. The highest concentration of Pd was found at the rod synapse, consistent with a suggested role for Pd in the regulation of synaptic function.

The present study has examined the spatial and temporal expression patterns of various proteins associated with the structure and function of mature photoreceptor outer segments in the developing ferret's retina using immunocytochemistry and RT-PCR. One set of proteins, including rod opsin, arrestin, and recoverin, was detected progressively in photoreceptors as they became postmitotic, being expressed well before the differentiation of outer segments. A second set of proteins, including β- and γ-transducin, cGMP-phosphodiesterase, phosducin, rhodopsin kinase, rod cGMP-gated cation channel protein, and peripherin, displayed a contrasting temporal onset and pattern of spatial emergence. These latter proteins first became detectable either shortly before or coincident with outer segment formation, and were expressed simultaneously in both older and younger photoreceptor cells. A third set, the short wavelength-sensitive (SWS) and medium wavelength-sensitive (MWS) cone opsin proteins, was the last to be detected, but materialized in a spatio-temporal pattern reminiscent of the neurogenetic gradient of the cones. These different spatial and temporal patterns indicate that cellular maturation must play a primary role in regulating the onset of expression of some of these proteins, while extrinsic signals must act to coordinate the expression of other proteins across photoreceptors of different ages.

The mammalian retina is known to contain two distinct transducins that interact with their respective rod and cone pigments. However, there are no reports of a nonmammalian species having two distinct transducins. In the present study, we report the cloning and cellular localization of two transducin α subunits (Gαt) from the tiger salamander. Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of Gαt. When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone transducins. The salamander Gαt-1 has 91.2–93.7% amino acid sequence identity to mammalian rod Gαt subunits and 79.7–80.9% to mammalian cone Gαts. The salamander Gαt-2 has 86.2–87.9% sequence identity to mammalian cone Gαts and 78.9–80.9% to mammalian rod Gαts at the amino acid level. The Gαt-1 cDNA encodes 350 amino acids while the Gαt-2 cDNA encodes 354 residues, which is typical for rod and cone Gαts, respectively, and we thus identified the Gαt-1 as rod and Gαt-2 as cone Gαt. Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits. Genomic Southern blot analysis showed that rod and cone Gαt subunits are both encoded by single-copy genes. Northern blot analysis identified retina-specific transcripts of 3.0 kb for rod Gαt and 2.6 kb for cone Gαt. Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod Gαt is localized to rods, predominantly in the outer segments; similarly, cone Gαt is localized to cone outer segments. The results confirm that the two sequences encode rod and cone transducins and demonstrate that this lower vertebrate contains two distinct transducins that are localized specifically to rod and cone photoreceptors.