Variability in the time course of single photon responses from toad rods: termination of rhodopsin's activity.

We examined the responses of toad rod photoreceptors to single photons of light. To minimize the effects of variability in the early rising phase, we selected sets of responses that closely matched the rise of the mean single photon response. Responses selected in this way showed substantial variations in kinetics, appearing to peel off from a common time course after different delays. Following incorporation of the calcium buffer BAPTA, the time to peeling off was retarded. Our analysis indicates that it is not necessary to invoke a long series of reaction steps to explain the shutoff of rhodopsin activity. Instead, our results suggest that the observed behavior is explicable by the presently known shutoff reactions of activated rhodopsin, modulated by feedback.     

Mechanisms regulating variability of the single photon responses of mammalian rod photoreceptors

Variability in the single photon responses of rod photoreceptors limits the accuracy with which the number and timing of photon absorptions are encoded. We investigated how much single photon responses of mammalian rods fluctuate and what mechanisms control these fluctuations. Mammalian rods, like those of toads, generated responses to single photons with trial-to-trial fluctuations 3-4 times smaller than other familiar signals produced by single molecules. We used the properties of the measured fluctuations to constrain models for how the single photon responses are regulated. Neither feedback control of rhodopsin's activity nor saturation within the transduction cascade were consistent with experiment. The measured responses, however, could be explained by multistep shutoff of rhodopsin or a combination of multistep shutoff and saturation.     

Kinetics of rhodopsin deactivation and its role in regulating recovery and reproducibility of rod photoresponse

The single photon response (SPR) in vertebrate phototransduction is regulated by the dynamics of R* during its lifetime, including the random number of phosphorylations, the catalytic activity and the random sojourn time at each phosphorylation level. Because of this randomness the electrical responses are expected to be inherently variable. However the SPR is highly reproducible. The mechanisms that confer to the SPR such a low variability are not completely understood. The kinetics of rhodopsin deactivation is investigated by a Continuous Time Markov Chain (CTMC) based on the biochemistry of rhodopsin activation and deactivation, interfaced with a spatio-temporal model of phototransduction. The model parameters are extracted from the photoresponse data of both wild type and mutant mice, having variable numbers of phosphorylation sites and, with the same set of parameters, the model reproduces both WT and mutant responses. The sources of variability are dissected into its components, by asking whether a random number of turnoff steps, a random sojourn time between steps, or both, give rise to the known variability. The model shows that only the randomness of the sojourn times in each of the phosphorylated states contributes to the Coefficient of Variation (CV) of the response, whereas the randomness of the number of R* turnoff steps has a negligible effect. These results counter the view that the larger the number of decay steps of R*, the more stable the photoresponse is. Our results indicate that R* shutoff is responsible for the variability of the photoresponse, while the diffusion of the second messengers acts as a variability suppressor.     

Diffusion of the second messengers in the cytoplasm acts as a variability suppressor of the single photon response in vertebrate phototransduction

The single photon response in vertebrate phototransduction is highly reproducible despite a number of random components of the activation cascade, including the random activation site, the random walk of an activated receptor, and its quenching in a random number of steps. Here we use a previously generated and tested spatiotemporal mathematical and computational model to identify possible mechanisms of variability reduction. The model permits one to separate the process into modules, and to analyze their impact separately. We show that the activation cascade is responsible for generation of variability, whereas diffusion of the second messengers is responsible for its suppression. Randomness of the activation site contributes at early times to the coefficient of variation of the photoresponse, whereas the Brownian path of a photoisomerized rhodopsin (Rh*) has a negligible effect. The major driver of variability is the turnoff mechanism of Rh*, which occurs essentially within the first 2-4 phosphorylated states of Rh*. Theoretically increasing the number of steps to quenching does not significantly decrease the corresponding coefficient of variation of the effector, in agreement with the biochemical limitations on the phosphorylated states of the receptor. Diffusion of the second messengers in the cytosol acts as a suppressor of the variability generated by the activation cascade. Calcium feedback has a negligible regulatory effect on the photocurrent variability. A comparative variability analysis has been conducted for the phototransduction in mouse and salamander, including a study of the effects of their anatomical differences such as incisures and photoreceptors geometry on variability generation and suppression.     

The influence of arrestin (48K protein) and rhodopsin kinase on visual transduction.

The shutoff of the phototransduction cascade in retinal rods requires the inactivation of light-activated rhodopsin. The underlying mechanisms were studied in functionally intact detached rod outer segments by testing the effect of either sangivamycin, an inhibitor of rhodopsin kinase, or phytic acid, an inhibitor of 48K protein binding to phosphorylated rhodopsin, on light responses recorded in whole-cell voltage clamp. The results suggest that isomerized rhodopsin is inactivated fully by multiple phosphorylation and that the binding of 48K protein accelerates recovery by quenching partially phosphorylated rhodopsin. Higher concentrations of sangivamycin cause changes in the light response that cannot be explained by selective inhibition of rhodopsin kinase and suggest that other protein kinases are needed for normal rod function.     

The dynamics of phosphodiesterase activation in rods and cones

Phototransduction starts with the activation of a rhodopsin (respectively, coneopsin) molecule, located in the outer segment of rod (respectively, cone) photoreceptors. The subsequent amplification pathway proceeds via the G-protein transducin to the activation of phosphodiesterase (PDE), a G-protein coupled effector enzyme. In this article, we study the dynamics of PDE activation by constructing a Markov model that is based on the underlying chemical reactions including multiple rhodopsin phosphorylations. We derive explicit equations for the mean and the variance of activated PDE. Our analysis reveals that a low rhodopsin lifetime variance is neither necessary nor sufficient to achieve reliable PDE activation. The numerical simulations show that during the rising phase the variability of PDE activation is much lower compared to the recovery phase, and this property depends crucially on the transducin activation rates. Furthermore, we find that the dynamics of the activation process greatly differs depending on whether rhodopsin or PDE deactivation limits the recovery of the photoresponse. Finally, our simulations for cones show that only very few PDEs are activated by an excited photopigment, which might explain why in S-cones no single photon response can be observed.     

Multiple steps of phosphorylation of activated rhodopsin can account for the reproducibility of vertebrate rod single-photon responses

Single-photon responses (SPRs) in vertebrate rods are considerably less variable than expected if isomerized rhodopsin (R*) inactivated in a single, memoryless step, and no other variability-reducing mechanisms were available. We present a new stochastic model, the core of which is the successive ratcheting down of R* activity, and a concomitant increase in the probability of quenching of R* by arrestin (Arr), with each phosphorylation of R* (Gibson, S.K., J.H. Parkes, and P.A. Liebman. 2000. Biochemistry. 39:5738-5749.). We evaluated the model by means of Monte-Carlo simulations of dim-flash responses, and compared the response statistics derived from them with those obtained from empirical dim-flash data (Whitlock, G.G., and T.D. Lamb. 1999. Neuron. 23:337-351.). The model accounts for four quantitative measures of SPR reproducibility. It also reproduces qualitative features of rod responses obtained with altered nucleotide levels, and thus contradicts the conclusion that such responses imply that phosphorylation cannot dominate R* inactivation (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.). Moreover, the model is able to reproduce the salient qualitative features of SPRs obtained from mouse rods that had been genetically modified with specific pathways of R* inactivation or Ca2+ feedback disabled. We present a theoretical analysis showing that the variability of the area under the SPR estimates the variability of integrated R* activity, and can provide a valid gauge of the number of R* inactivation steps. We show that there is a heretofore unappreciated tradeoff between variability of SPR amplitude and SPR duration that depends critically on the kinetics of inactivation of R* relative to the net kinetics of the downstream reactions in the cascade. Because of this dependence, neither the variability of SPR amplitude nor duration provides a reliable estimate of the underlying variability of integrated R* activity, and cannot be used to estimate the minimum number of R* inactivation steps. We conclude that multiple phosphorylation-dependent decrements in R* activity (with Arr-quench) can confer the observed reproducibility of rod SPRs; there is no compelling need to invoke a long series of non-phosphorylation dependent state changes in R* (as in Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.). Our analyses, plus data and modeling of others (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.), also argue strongly against either feedback (including Ca2+-feedback) or depletion of any molecular species downstream to R* as the dominant cause of SPR reproducibility.     

G-protein-coupled enzyme cascades have intrinsic properties that improve signal localization and fidelity

G-protein-coupled enzyme cascades are used by eukaryotic cells to detect external signals and transduce them into intracellular messages that contain biological information relevant to the cell's function. Since G-protein-coupled receptors that are designed to detect different kinds of external signals can generate the same kind of intracellular response, effective signaling requires that there are mechanisms to increase signal specificity and fidelity. Here we examine the kinetic equations for the initial three stages in a generic G-protein-coupled cascade and show that the physical properties of the transduction pathway result in two intrinsic features that benefit signaling. 1), The response to a single activated receptor is naturally confined to a localized spatial domain, which could improve signal specificity by reducing cross talk. 2), The peak of the response generated by such a signaling domain is limited. This saturation effect reduces trial-to-trial variability and increases signaling fidelity by limiting the response to receptors that remain active for longer than average. We suggest that this mechanism for reducing response fluctuations may be a contributing factor in making the single photon responses of vertebrate retinal rods so remarkably reproducible.     

Cyclic GMP and photoreceptor function.

A single photon can be detected by a rod photoreceptor cell. The absorption of light by rhodopsin triggers a cascade of reactions that amplifies the photon signal and results in ion channel closure with hyperpolarization of the rod photoreceptor cell. Light-induced conformational changes in rhodopsin facilitate the binding of a guanosine nucleotide-binding protein, transducin, which then undergoes a GTP-GDP exchange reaction and dissociation of the transducin complex. A subunit of transducin then activates a phosphodiesterase complex that hydrolyzes cyclic GMP. In darkness, cyclic GMP binds to cation channels of the photoreceptor plasma membrane, maintaining them in an open configuration. The light-induced reduction in cyclic GMP concentration dissociates the bound cyclic GMP, resulting in channel closure and hyperpolarization. Down-regulation of the cascade involves other proteins that block the interaction of transducin with rhodopsin and another protein that may interfere with transducin recycling. Cone photoreceptors possess a light-activated cascade that follows the rod format, but it is composed of proteins that are homologous to those of rod photoreceptors. Phototransduction in invertebrate photoreceptors uses rhodopsin to activate a cascade that uses phosphoinositides and calcium ion to regulate membrane polarization.     

Arrestin and its splice variant Arr1-370A (p44). Mechanism and biological role of their interaction with rhodopsin

Deactivation of G-protein-coupled receptors relies on a timely blockade by arrestin. However, under dim light conditions, virtually all arrestin is in the rod inner segment, and the splice variant p(44) (Arr(1-370A)) is the stop protein responsible for receptor deactivation. Using size exclusion chromatography and biophysical assays for membrane-bound protein-protein interaction, membrane binding, and G-protein activation, we have investigated the interactions of Arr(1-370A) and proteolytically truncated Arr(3-367) with rhodopsin. We find that these short arrestins do not only interact with the phosphorylated active receptor but also with inactive phosphorylated rhodopsin or opsin in membranes or solution. Because of the latter interaction they are not soluble (like arrestin) but membrane-bound in the dark. Upon photoexcitation, Arr(3-367) and Arr(1-370A) interact with prephosphorylated rhodopsin faster than arrestin and start to quench G(t) activation on a subsecond time scale. The data indicate that in the course of rhodopsin deactivation, Arr(1-370A) is handed over from inactive to active phosphorylated rhodopsin. This mechanism could provide a new aspect of receptor shutoff in the single photon operating range of the rod cell.     

Photopigment quenching is Ca2+ dependent and controls response duration in salamander L-cone photoreceptors

The time scale of the photoresponse in photoreceptor cells is set by the slowest of the steps that quench the light-induced activity of the phototransduction cascade. In vertebrate photoreceptor cells, this rate-limiting reaction is thought to be either shutoff of catalytic activity in the photopigment or shutoff of the pigment''s effector, the transducin-GTP–phosphodiesterase complex. In suction pipette recordings from isolated salamander L-cones, we found that preventing changes in internal [Ca²⁺] delayed the recovery of the light response and prolonged the dominant time constant for recovery. Evidence that the Ca²⁺-sensitive step involved the pigment itself was provided by the observation that removal of Cl⁻ from the pigment''s anion-binding site accelerated the dominant time constant for response recovery. Collectively, these observations indicate that in L-cones, unlike amphibian rods where the dominant time constant is insensitive to [Ca²⁺], pigment quenching rate limits recovery and provides an additional mechanism for modulating the cone response during light adaptation.     

Mathematical and computational modelling of spatio-temporal signalling in rod phototransduction

Rod photoreceptors are activated by light through activation of a cascade that includes the G protein-coupled receptor rhodopsin, the G protein transducin, its effector cyclic guanosine monophosphate (cGMP) phosphodiesterase and the second messengers cGMP and Ca2+. Signalling is localised to the particular rod outer segment disc, which is activated by absorption of a single photon. Modelling of this cascade has previously been performed mostly by assumption of a well-stirred cytoplasm. We recently published the first fully spatially resolved model that captures the local nature of light activation. The model reduces the complex geometry of the cell to a simpler one using the mathematical theories of homogenisation and concentrated capacity. The model shows that, upon activation of a single rhodopsin, changes of the second messengers cGMP and Ca2+ are local about the particular activated disc. In the current work, the homogenised model is computationally compared with the full, non-homogenised one, set in the original geometry of the rod outer segment. It is found to have an accuracy of 0.03% compared with the full model in computing the integral response and a 5200-fold reduction in computation time. The model can reconstruct the radial time-profiles of cGMP and Ca2+ in the interdiscal spaces adjacent to the activated discs. Cellular electrical responses are localised near the activation sites, and multiple photons sufficiently far apart produce essentially independent responses. This leads to a computational analysis of the notion and estimate of 'spread' and the optimum distribution of activated sites that maximises the response. Biological insights arising from the spatio-temporal model include a quantification of how variability in the response to dim light is affected by the distance between the outer segment discs capturing photons. The model is thus a simulation tool for biologists to predict the effect of various factors influencing the timing, spread and control mechanisms of this G protein-coupled, receptor-mediated cascade. It permits ease of simulation experiments across a range of conditions, for example, clamping the concentration of calcium, with results matching analogous experimental results. In addition, the model accommodates differing geometries of rod outer segments from different vertebrate species. Thus it represents a building block towards a predictive model of visual transduction.     

Identification of a rhodopsin photoreceptor in Euglena gracilis.

Visual pigments are a class of receptor proteins that absorb light and trigger sensory signals. Retinal-containing proteins are used in nature as photoreceptors mainly in animals vision. Mammalian rhodopsin is the best studied example of a light sensor which couples photon absorption to a cascade of biochemical reactions amplifying the input signal. A surprising discovery was to find rhodopsin also in Archaebacteria and in unicellular eukaryotes. On the basis of absorption microspectroscopic measurements and of inhibition experiments on pigment biosynthetic pathways, we have recently suggested that a rhodopsin could be the functional receptor of the visual process in Euglena gracilis, a flagellate which can use light directly to promote photosynthetic reactions, or as an incident flux of information to adjust its swimming orientation. We here report purification and identification of all-trans-retinal by column chromatography, HPLC and GC-MS in E. gracilis; these findings indicate with absolute certainty that rhodopsin is the photoreceptor molecule of this microorganism.     

Molecular origin of continuous dark noise in rod photoreceptors.

Noise in the rod photoreceptors limits the ability of the dark-adapted visual system to detect dim lights. We investigated the molecular mechanism of the continuous component of the electrical dark noise in toad rods. Membrane current was recorded from intact, isolated rods or truncated, internally dialyzed rod outer segments. The continuous noise was separated from noise due to thermal activation of rhodopsin and to transitions in the cGMP-activated channels. Selectively disabling different elements of the phototransduction cascade allowed examination of their contributions to the continuous noise. These experiments indicate that the noise is generated by spontaneous activation of cGMP phosphodiesterase (PDE) through a process that does not involve transducin. The addition of recombinant gamma, the inhibitory subunit of PDE, did not suppress the noise, indicating that endogenous gamma does not completely dissociate from the catalytic subunit of PDE during spontaneous activation. Quantitative analysis of the noise provided estimates of the rate constants for spontaneous PDE activation and deactivation and the catalytic activity of a single PDE molecule in situ.     

The effect of proteolytic removal of the C-terminal fragment of rhodopsin on its ability to activate visual cascade

The role of the C-terminal domain of rhodopsin in the activation of transducin was studied. The treatment of photoreceptor membranes with trypsin, thermolysin, and Asp-N-endoprotease led to the respective rhodopsin species devoid of 9, 12-, or 19-aa C-terminal fragments. It was shown that the removal of 9-aa fragment by trypsin does not affect the catalytic activity of the receptor, whereas the thermolysin-induced truncation of the rhodopsin C-terminus by 12 aa about 1.5-fold enhances its activity. The Asp-N-endoprotease-assisted removal of 19 aa (i.e., the shortening by seven more C-terminal aa) virtually unchanges the rhodopsin catalytic activity compared to the preparation truncated with thermolysin. These results suggest that the part of the rhodopsin C-terminal fragment between the sites of its cleavage by trypsin and thermolysin (Val337-Ser338-Lys339) inhibits the signal transduction from rhodopsin to the next component of visual cascade. The English version of the paper.     

A modified approach to the measurement problem: objective reduction in the retinal molecule prior to conformational change

A new analysis of the measurement problem reveals the possibility that collapse of the wavefunction may now take place just before photoisomerization of the rhodopsin molecule in the retinal rods. It is known that when a photon is initially absorbed by the retinal molecule which, along with opsin comprises the rhodopsin molecule, an electron in the highest pi orbital is immediately excited to a pi* orbital. This means that a measurement or transfer of information takes place at the quantum level before the retinal molecule commences the conformational change from cis to trans. This could have profound implications for resolving some of the foundational issues confronting quantum mechanics.     

Leukemia inhibitory factor coordinates the down-regulation of the visual cycle in the retina and retinal-pigmented epithelium

Leukemia inhibitory factor (LIF), an interleukin-6 family neurocytokine, is up-regulated in response to different types of retinal stress and has neuroprotective activity through activation of the gp130 receptor/STAT3 pathway. We observed that LIF induces rapid, robust, and sustained activation of STAT3 in both the retina and retinal pigmented epithelium (RPE). Here, we tested whether LIF-induced STAT3 activation within the RPE can down-regulate RPE65, the central enzyme in the visual cycle that provides the 11-cis-retinal chromophore to photoreceptors in vivo. We generated conditional knock-out mice to specifically delete STAT3 or gp130 in RPE, retina, or both RPE and retina. After intravitreal injection of LIF, we analyzed the expression levels of visual cycle genes and proteins, isomerase activity of RPE65, levels of rhodopsin protein, and the rates of dark adaptation and rhodopsin regeneration. We found that RPE65 protein levels and isomerase activity were reduced and recovery of bleachable rhodopsin was delayed in LIF-injected eyes. In mice with functional gp130/STAT3 signaling in the retina, rhodopsin protein was also reduced by LIF. However, the LIF-induced down-regulation of RPE65 required a functional gp130/STAT3 cascade intrinsic to RPE. Our data demonstrate that a single cytokine, LIF, can simultaneously and independently affect both RPE and photoreceptors through the same signaling cascade to reduce the generation and utilization of 11-cis-retinal.     

Piecing together the timetable for visual transduction with transgenic animals

Transgenic mice bearing null or functional mutations are being used to define the roles of specific elements in phototransduction and also to time the molecular interactions. Genetic manipulation of the collision frequency between rhodopsin and transducin molecules identified this parameter as rate-limiting for the photoresponse onset. Genetic interference with rhodopsin phosphorylation and arrestin binding, transducin shut-off and calcium feedback has revealed their respective roles in shaping the response waveform. The timetable for all of these molecular events determines the amplitude, kinetics and reproducibility of the photoresponse.     

Molecular design of an amplification cascade in vision

The photoexcitation of rhodopsin triggers a cascade that results in the hydrolysis of a large number of molecules of cyclic GMP. The molecular mechanism of this amplification cascade has been delineated. Transducin, a multisubunit perpheral membrane protein, is the information-carrying intermediate in the activation of the cyclic GMP phosphodiesterase. Photoexcited rhodopsin (R*) castalyzes the exchange of GRP for GDP bound to the α-subunit of transducin (T). About 500 molecules of T_α-GTP are formed per absorbed photon at low light levels. T_α-GTP, rekeased from the β- and γ-subunits of transducin, then activates the phosphodiesterase by relieving an inhibitory constraint imposed by its small sununit. Each actived phosphodiesterase molecule hydrolyzes more than 100 cyclic GMP/s, giving an overall gain of more than 500,000. Photoexcited rhodopsin triggers the activation of a molecule of transducin in a millisecond, which is sufficiently rapid to enable this cascade to participate in visual excitation. Hydrolysis of GTP bound to T_α seves to restore the system to the dark state. Transducin, like the G proteins of the adenylate cyclase casecade, can be specifically ADP-ribosylated by cholera toxin and pertussis toxin. In both cascades, labling by pertussis toxin blocks the capacity of transducin to interact with the excited receptor, whereas labeling by cholera toxin inhibits the hydrolysis of bound GTP, leading to persistent activation. Moreover, the moleculaar design of the hormone-triggered cyclic AMP cascade is similar to that of the light-triggered cyclic GMP cascade. It seems likely that transducin, the stimulatory G protein, the inhibitor G protein, and the ras protein are members of the same family of signal amplifiers. The study of the cyclic nucleotide cascade of vision is providing rewarding views of recurring motifs of signal amplification in nature.     

Light causes phosphorylation of nonactivated visual pigments in intact mouse rod photoreceptor cells

Phosphorylation of G-protein-coupled receptors (GPCRs) is a required step in signal deactivation. Rhodopsin, a prototypical GPCR, exhibits high gain phosphorylation in vitro whereby a hundred-fold molar excess of phosphates are incorporated into the rhodopsin pool per molecule of activated rhodopsin. The extent by which high gain phosphorylation occurs in the intact mammalian photoreceptor cell, and the molecular mechanism underlying this reaction in vivo, is not known. Trans-phosphorylation is a mechanism proposed for high gain phosphorylation, whereby rhodopsin kinase, upon phosphorylating the activated receptor, continues to phosphorylate nearby nonactivated rhodopsin. We used two different transgenic mouse models to test whether trans-phosphorylation occurs in the intact photoreceptor cell. The first transgenic model expressed a murine cone pigment, S-opsin, together with the endogenous rhodopsin in the rod cell. We showed that selective stimulation of rhodopsin also led to phosphorylation of S-opsin. The second mouse model expressed the constitutively active human opsin mutant K296E. K296E, in the arrestin-/- background, also led to phosphorylation of endogenous mouse rhodopsin in the dark-adapted retina. Both mouse models provide strong support of trans-phosphorylation as an underlying mechanism of high gain phosphorylation, and provide evidence that a substantial fraction of nonactivated visual pigments becomes phosphorylated through this mechanism. Because activated, phosphorylated receptors exhibit decreased catalytic activity, our results suggest that dephosphorylation would be an important step in the full recovery of visual sensitivity during dark adaptation. These results may also have implications for other GPCR signaling pathways.