Molecular evolution of proteins involved in vertebrate phototransduction

Vision is one of the most important senses for vertebrates. As a result, vertebrates have evolved a highly organized system of retinal photoreceptors. Light triggers an enzymatic cascade, called the phototransduction cascade, that leads to the hyperpolarization of photoreceptors. It is expected that a systematic comparison of phototransduction cascades of various vertebrates can provide insights into the diversity of vertebrate photoreceptors and into the evolution of vertebrate vision. However, only a few attempts have been made to compare each phototransduction protein participating in this cascade. Here, we determine phylogenetic trees of the vertebrate phototransduction proteins and compare them. It is demonstrated that vertebrate opsin sequences fall into five fundamental subfamilies. It is speculated that this is crucial for the diversity of the spectral sensitivity observed in vertebrate photoreceptors and provides the vertebrates with the molecular tools to discriminate the color of incident light. Other phototransduction proteins can be classified into only a few subfamilies. Cones generally share isoforms of phototransduction proteins that are different from those found in rods. The difference in sensitivity to light between rods and cones is likely due to the difference in the molecular properties of these isoforms. The phototransduction proteins seem to have co-evolved as a system. Switching the expression of these isoforms may characterize individual vertebrate photoreceptors.     

Current issues in invertebrate phototransduction

Investigation of phototransduction in invertebrate photoreceptors has revealed many physiological and biochemical features of fundamental biological importance. Nonetheless, no complete picture of phototransduction has yet emerged. In most known cases, invertebrate phototransduction involves polyphosphoinositide and cyclic GMP (cGMP) intracellular biochemical signaling pathways leading to opening of plasma membrane ion channels. Excitation is Ca²⁺-dependent, as are adaptive feedback processes that regulate sensitivity to light. Transduction takes place in specialized subcellular regions, rich in microvilli and closely apposed to submicrovillar membrane systems. Thus, excitation is a highly localized process. This article focuses on the intracellular biochemical signaling pathways and the ion channels involved in invertebrate phototransduction. The coupling of signaling cascades with channel activation is not understood for any invertebrate species. Although photoreceptors have features that are common to most or all known invertebrate species, each species exhibits unique characteristics. Comparative electrophysiological, biochemical, morphological, and molecular biological approaches to studying phototransduction in these species lead to fundamental insights into cellular signaling. Several current controversies and proposed phototransduction models are evaluated.     

Calcium-sensitive downregulation of the transduction chain in rod photoreceptors of the rat retina

In vertebrate rod outer segments phototransduction is suggested to be modulated by intracellular Ca. We aimed at verifying this hypothesis by recording saturated photosignals in the rat retina after single and double flashes of light and determining the time t(c) to the beginning of the signal recovery. The time course of Ca(i) after a flash was calculated from a change of the spatial Ca(2+) concentration profile recorded in the space between the rods. After single flashes t(c) increased linearly with the logarithm of flash intensity, confirming the assumption that t(c) is determined by deactivation of a single species X* in the phototransduction cascade. The photoresponse was shortened up to 45% if the test flash was preceded by a conditioning preflash. The shortening depended on the reduction of Ca(i) induced by the preflash. The data suggest that the phototransduction gain determining the amount of activated X* is regulated by a Ca(i)-dependent mechanism in a short time period (<800 ms) after the test flash. Lowering of Ca(i) by a preflash reduced the gain up to 20% compared to its value in a dark-adapted rod. The relation between phototransduction gain and Ca(i) revealed a K(1/2) value close to the dark level of Ca(i).     

Unknown mechanisms of the GPCR-signaling cascade in vertebrate photoreceptors

Among the GPCR-signaling cascades, phototransduction in vertebrate retinal photoreceptors has been characterized in unprecedented details. It is believed that basic mechanisms of phototransduction and adaptation are reliably and completely established, and phototransduction may serve as a benchmark for understanding other G-protein-coupled systems. In this review, we compare present scheme of phototransduction with other GPCR-cascades in order to reveal their similarities and specific features. We show, based mainly on our physiological and biophysical data, that the existing scheme misses a few important regulations whose molecular basis is unknown. There exists a fast and efficient mechanism that accelerates the turn-off of the activated G-protein (transducin) during light adaptation. A few slowly acting processes result in a long-lasting modification of the cascade's components and regulate the speed of rhodopsin and transducin quenching. Similarly to other GPCR-cascades, one may suggest that there are multiple signalling pathways that start from photoactivated rhodopsin and rely on different secondary messengers (e.g. cAMP vs. cGMP). We also show that rhodopsin in retinal rods may form areas of paracristalline organization, and that the oligomerization might be a mechanism for controlling the amplification of the signalling cascade. The missing mechanisms are by no means minor, and could ensure sensitivity regulation within two orders of magnitude range.     

Induction of the Unfolded Protein Response by Constitutive G-protein Signaling in Rod Photoreceptor Cells

Phototransduction is a G-protein signal transduction cascade that converts photon absorption to a change in current at the plasma membrane. Certain genetic mutations affecting the proteins in the phototransduction cascade cause blinding disorders in humans. Some of these mutations serve as a genetic source of “equivalent light” that activates the cascade, whereas other mutations lead to amplification of the light response. How constitutive phototransduction causes photoreceptor cell death is poorly understood. We showed that persistent G-protein signaling, which occurs in rod arrestin and rhodopsin kinase knock-out mice, caused a rapid and specific induction of the PERK pathway of the unfolded protein response. These changes were not observed in the cGMP-gated channel knock-out rods, an equivalent light condition that mimics light-stimulated channel closure. Thus transducin signaling, but not channel closure, triggers rapid cell death in light damage caused by constitutive phototransduction. Additionally, we show that in the albino light damage model cell death was not associated with increase in global protein ubiquitination or unfolded protein response induction. Taken together, these observations provide novel mechanistic insights into the cell death pathway caused by constitutive phototransduction and identify the unfolded protein response as a potential target for therapeutic intervention.     

Properties of photoreceptor-specific phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Mutations in the norpA gene drastically affect the phototransduction process in Drosophila. To study the biochemical characteristics of the norpA protein and its cellular and subcellular distributions, we have generated antisera against the major gene product of norpA. The antisera recognize an eye-specific protein of 130-kDa relative molecular mass that is present in wild-type head extracts but not in those of strong norpA mutants. The protein is associated with membranes and can be extracted with high salt. Immunohistochemical analysis at the light and electron microscopic levels indicates that the protein is expressed in all adult photoreceptor cells and specifically localized within the rhabdomeres, preferentially adjacent to, but not within, the rhabdomeric membranes. The results of the present study strongly support the previous suggestion that the norpA gene encodes the major phosphoinositol-specific phospholipase C in the photoreceptors. Moreover, insofar as the rhabdomeres are specialized structures for photoreception and phototransduction, specific localization of the norpA protein within these structures, in close association with the membranes, is consistent with the proposal that it has an important role in phototransduction.     

Recoverin improves rod-mediated vision by enhancing signal transmission in the mouse retina

Vision in dim light requires that photons absorbed by rod photoreceptors evoke signals that reliably propagate through the retina. We investigated how a perturbation in rod physiology affects propagation of those signals in the retina and ultimately visual sensitivity. Recoverin is a protein in rods that prolongs phototransduction and enhances visual sensitivity. It is not present in neurons postsynaptic to rods, yet we found that light-evoked responses of rod bipolar and ganglion cells were shortened when measured in recoverin-deficient retinas. Unexpectedly, the effect of recoverin on postsynaptic signals could not be explained by its effect on phototransduction. Instead, it is an effect of recoverin downstream of phototransduction in rods that prolongs signal transmission and enhances visual sensitivity. An important implication of our findings is that the recovery phase of the rod photoresponse does not contribute significantly to visual sensitivity near absolute threshold.     

Ca(2+)-binding proteins in the retina: structure, function, and the etiology of human visual diseases

The complex sensation of vision begins with the relatively simple photoisomerization of the visual pigment chromophore 11-cis-retinal to its all-trans configuration. This event initiates a series of biochemical reactions that are collectively referred to as phototransduction, which ultimately lead to a change in the electrochemical signaling of the photoreceptor cell. To operate in a wide range of light intensities, however, the phototransduction pathway must allow for adjustments to background light. These take place through physiological adaptation processes that rely primarily on Ca(2+) ions. While Ca(2+) may modulate some activities directly, it is more often the case that Ca(2+)-binding proteins mediate between transient changes in the concentration of Ca(2+) and the adaptation processes that are associated with phototransduction. Recently, combined genetic, physiological, and biochemical analyses have yielded new insights about the properties and functions of many phototransduction-specific components, including some novel Ca(2+)-binding proteins. Understanding these Ca(2+)-binding proteins will provide a more complete picture of visual transduction, including the mechanisms associated with adaptation, and of related degenerative diseases.     

G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor

We have shown previously that phosphoinositide 3-kinase in the retina is activated in vivo through light-induced tyrosine phosphorylation of the insulin receptor (IR). The light effect is localized to photoreceptor neurons and is independent of insulin secretion (Rajala, R. V., McClellan, M. E., Ash, J. D., and Anderson, R. E. (2002) J. Biol. Chem. 277, 43319-43326). These results suggest that there exists a cross-talk between phototransduction and other signal transduction pathways. In this study, we examined the stage of phototransduction that is coupled to the activation of the IR. We studied IR phosphorylation in mice lacking the rod-specific alpha-subunit of transducin to determine if phototransduction events are required for IR activation. To confirm that light-induced tyrosine phosphorylation of the IR is signaled through bleachable rhodopsin, we examined IR activation in retinas from RPE65(-/-) mice that are deficient in opsin chromophore. We observed that IR phosphorylation requires the photobleaching of rhodopsin but not transducin signaling. To determine whether the light-dependent activation of IR is mediated through the rod or cone transduction pathway, we studied the IR activation in mice lacking opsin, a mouse model of pure cone function. No light-dependent activation of the IR was found in the retinas of these mice. We provide evidence for the existence of a light-mediated IR pathway in the retina that is different from the known insulin-mediated pathway in nonneuronal tissues. These results suggest that IR phosphorylation in rod photoreceptors is signaled through the G-protein-coupled receptor rhodopsin. This is the first study demonstrating that rhodopsin can initiate signaling pathway(s) in addition to its classical phototransduction.     

cAMP controls rod photoreceptor sensitivity via multiple targets in the phototransduction cascade

In early studies, both cyclic AMP (cAMP) and cGMP were considered as potential secondary messengers regulating the conductivity of the vertebrate photoreceptor plasma membrane. Later discovery of the cGMP specificity of cyclic nucleotide–gated channels has shifted attention to cGMP as the only secondary messenger in the phototransduction cascade, and cAMP is not considered in modern schemes of phototransduction. Here, we report evidence that cAMP may also be involved in regulation of the phototransduction cascade. Using a suction pipette technique, we recorded light responses of isolated solitary rods from the frog retina in normal solution and in the medium containing 2 µM of adenylate cyclase activator forskolin. Under forskolin action, flash sensitivity rose more than twofold because of a retarded photoresponse turn-off. The same concentration of forskolin lead to a 2.5-fold increase in the rod outer segment cAMP, which is close to earlier reported natural day/night cAMP variations. Detailed analysis of cAMP action on the phototransduction cascade suggests that several targets are affected by cAMP increase: (a) basal dark phosphodiesterase (PDE) activity decreases; (b) at the same intensity of light background, steady background-induced PDE activity increases; (c) at light backgrounds, guanylate cyclase activity at a given fraction of open channels is reduced; and (d) the magnitude of the Ca²⁺ exchanger current rises 1.6-fold, which would correspond to a 1.6-fold elevation of [Ca²⁺]_in. Analysis by a complete model of rod phototransduction suggests that an increase of [Ca²⁺]_in might also explain effects (b) and (c). The mechanism(s) by which cAMP could regulate [Ca²⁺]_in and PDE basal activity is unclear. We suggest that these regulations may have adaptive significance and improve the performance of the visual system when it switches between day and night light conditions.     

Molecular physiology of visual pigment rhodopsin

The key physiological functions of the rhodopsin molecule are reviewed. Molecular mechanisms of visual pigments spectral tuning, photoisomerization of the 11-cis-retinal chromophore that triggers the phototransduction process, formation of physiologically active state of rhodopsin as a G-protein-coupled receptor, rhodopsin visual cycle, and consequences of its impairment are evaluated. Visual pigment rhodopsin performs several functions, providing spectral sensitivity of photoreceptor cells, phototransduction processes and light and dark adaptation. Genetically determined defects of visual pigment molecule and proteins involved into mechanisms of phototransduction and adaptation or into mechanism of visual cycle are directly linked to pathogenesis of different forms of degenerative retina diseases. Understanding the molecular mechanisms of these physiological processes uncovers the way to direct investigation of pathogenesis of these severe eye diseases.     

Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina

Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption. Alternatively, production can be set by a signal that anticipates demand. In this report we investigate the hypothesis that signaling through phototransduction controls production of energy in mouse retinas. We found that respiration in mouse retinas is not coupled tightly to ATP consumption. By analyzing metabolic flux in mouse retinas, we also found that phototransduction slows metabolic flux through glycolysis and through intermediates of the citric acid cycle. We also evaluated the relative contributions of regulation of the activities of α-ketoglutarate dehydrogenase and the aspartate-glutamate carrier 1. In addition, a comprehensive analysis of the retinal metabolome showed that phototransduction also influences steady-state concentrations of 5′-GMP, ribose-5-phosphate, ketone bodies, and purines.     

Additivity in murine circadian phototransduction

Additivity in the circadian phototransduction system of the mouse has not been tested directly. Because of this, accurate prediction of circadian phase shifts elicited by polychromatic light stimuli cannot be derived from the results of studies using monochromatic light stimuli. This limitation also makes it impossible to deduce the relative contributions of the photoreceptive mechanisms (rods, cones and melanopsin-containing retinal ganglion cells) underlying circadian phototransduction in the mouse. Using nearly monochromatic light stimuli of different spectral composition, and combinations thereof, we demonstrated that murine circadian phototransduction exhibits additivity. Based on the locomotor activity phase shifts elicited by these stimuli, we developed the first quantitative assessment of the relative contributions of conventional and novel photoreceptive mechanisms for circadian functioning in the mouse.     

New insights into Drosophila vision

Studies of the Drosophila visual system have provided valuable insights into the function and regulation of phototransduction signaling pathways. Much of this work has stemmed from or relied upon the genetic tools offered by the Drosophila system. In this issue of Neuron, Wang and colleagues and Acharya and colleagues have further exploited the Drosophila genetic system to characterize two new phototransduction players.     

Light adaptation in Drosophila photoreceptors: II. Rising temperature increases the bandwidth of reliable signaling

It is known that an increase in both the mean light intensity and temperature can speed up photoreceptor signals, but it is not known whether a simultaneous increase of these physical factors enhances information capacity or leads to coding errors. We studied the voltage responses of light-adapted Drosophila photoreceptors in vivo from 15 to 30 degrees C, and found that an increase in temperature accelerated both the phototransduction cascade and photoreceptor membrane dynamics, broadening the bandwidth of reliable signaling with an effective Q(10) for information capacity of 6.5. The increased fidelity and reliability of the voltage responses was a result of four factors: (1) an increased rate of elementary response, i.e., quantum bump production; (2) a temperature-dependent acceleration of the early phototransduction reactions causing a quicker and narrower dispersion of bump latencies; (3) a relatively temperature-insensitive light-adapted bump waveform; and (4) a decrease in the time constant of the light-adapted photoreceptor membrane, whose filtering matched the dynamic properties of the phototransduction noise. Because faster neural processing allows faster behavioral responses, this improved performance of Drosophila photoreceptors suggests that a suitably high body temperature offers significant advantages in visual performance.