Structural view of G protein-coupled receptor signaling in the retinal rod outer segment.

Visual phototransduction is the most extensively studied G protein-coupled receptor (GPCR) signaling pathway because of its quantifiable stimulus, non-redundancy of genes, and immense importance in vision. We summarize recent discoveries that have advanced our understanding of rod outer segment (ROS) morphology and the pathological basis of retinal diseases. We have combined recently published cryo-electron tomography (cryo-ET) data on the ROS with structural knowledge on individual proteins to define the precise spatial limitations under which phototransduction occurs. Although hypothetical, the reconstruction of the rod phototransduction system highlights the potential roles of phosphodiesterase 6 (PDE6) and guanylate cyclases (GCs) in maintaining the spacing between ROS discs, suggesting a plausible mechanism by which intrinsic optical signals are generated in the retina.

Structural basis of the partially open central gate in the human CNGA1/CNGB1 channel explained by additional density for calmodulin in cryo-EM map.

The recently reported structure of the human CNGA1/CNGB1 CNG channel in the open state (Xue et al., 2021a) shows that one CNGA1 and one CNGB1 subunit do not open the central hydrophobic gate completely upon cGMP binding. This is different from what has been reported for CNGA homomeric channels (Xue et al., 2021b; Zheng et al., 2020). In seeking to understand how this difference is due to the presence of the CNGB1 subunit, we find that the deposited density map (Xue et al., 2021a) (EMDB 24465) contains an additional density not reported in the images of the original publication. This additional density fits well the structure of calmodulin (CaM), and it unambiguously connects the newly identified D-helix of CNGB1 to one of the CNGA1 helices (A1R) participating in the coiled-coil region. Interestingly, the CNGA1 subunit that engages in the interaction with this additional density is the one that, together with CNGB1, does not open completely the central gate. The sequence of the D-helix of CNGB1 contains a known CaM-binding site of exquisitely high affinity - named CaM2 (Weitz et al., 1998) -, and thus the presence of CaM in that region is not surprising. The mechanism through which CaM reduces currents across the membrane by acting on the native channel (Bauer, 1996; Hsu and Molday, 1993; Weitz et al., 1998) remains unclear. We suggest that the presence of CaM may explain the partially open central gate reported by Xue et al. (2021a). The structure of the open and closed states of the CNGA1/CNGB1 channel may be different with and without CaM present.

Cryo-EM structure of the native rhodopsin dimer in nanodiscs.

Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.

Proteome of Bovine Mitochondria and Rod Outer Segment Disks: Commonalities and Differences.

The retinal rod outer segment (OS) is a stack of disks surrounded by the plasma membrane, housing proteins related to phototransduction, as well as mitochondrial proteins involved in oxidative phosphorylation (OxPhos). This prompted us to compare the proteome of bovine OS disks and mitochondria to assess the significant top gene signatures of each sample. The two proteomes, obtained by LTQ-Orbitrap Velos mass spectrometry, were compared by statistical analyses. In total, 4139 proteins were identified, 2045 of which overlapping in the two sets. Nonhierarchical Spearman's correlogram revealed that the groups were clearly discriminated. Partial least square discriminant plus support vector machine analysis identified the major discriminative proteins, implied in phototransduction and lipid metabolism, respectively. Gene Ontology analysis identified top gene signatures of the disk proteome, enriched in vesiculation, glycolysis, and OxPhos proteins. The tricarboxylic acid cycle and the electron transport proteins were similarly enriched in the two samples, but the latter was up regulated in disks. Data suggest that the mitochondrial OxPhos proteins may represent a true OS proteome component, outside the mitochondrion. This knowledge may help the scientific community in the further studies of retinal physiology and pathology.

The Metabolism of 2-arachidonoylglycerol in Rod Outer Segments Is Modulated by Proteins Involved in the Phototransduction Process.

We previously reported that 2-arachidonoylglycerol (2-AG) synthesis by diacylglycerol lipase (DAGL) and lysophosphatidate phosphohydrolase (LPAP) and hydrolysis by monoacylglycerol lipase (MAGL) in rod outer segments (ROS) from bovine retina were differently modified by light applied to the retina. Based on these findings, the aim of the present research was to evaluate whether 2-AG metabolism could be modulated by proteins involved in the visual process. To this end, ROS kept in darkness (DROS) or obtained in darkness and then subjected to light (BROS) were treated with GTPγS and GDPßS, or with low and moderate ionic strength buffers for detaching soluble and peripheral proteins, or soluble proteins, respectively. Only DAGL activity was stimulated by the application of light to the ROS. GTPγS-stimulated DAGL activity in DROS reached similar values to that observed in BROS. The studies using different ionic strength show that (1) the highest decrease in DROS DAGL activity was observed when both phosphodiesterase (PDE) and transducin α (Tα) are totally membrane-associated; (2) the decrease in BROS DAGL activity does not depend on PDE association to membrane, and that (3) MAGL activity decreases, both in DROS and BROS, when PDE is not associated to the membrane. Our results indicate that the bioavailability of 2-AG under light conditions is favored by G protein-stimulated increase in DAGL activity and hindered principally by Tα/PDE association with the ROS membrane, which decreases DAGL activity.

Light regulation of rhodopsin distribution during outer segment renewal in murine rod photoreceptors.

Vision under dim light relies on primary cilia elaborated by rod photoreceptors in the retina. This specialized sensory structure, called the rod outer segment (ROS), comprises hundreds of stacked, membranous discs containing the light-sensitive protein rhodopsin, and the incorporation of new discs into the ROS is essential for maintaining the rod's health and function. ROS renewal appears to be primarily regulated by extrinsic factors (light); however, results vary depending on different model organisms. We generated two independent transgenic mouse lines where rhodopsin's fate is tracked by a fluorescently labeled rhodopsin fusion protein (Rho-Timer) and show that rhodopsin incorporation into nascent ROS discs appears to be regulated by both external lighting cues and autonomous retinal clocks. Live-cell imaging of the ROS isolated from mice exposed to six unique lighting conditions demonstrates that ROS formation occurs in a periodic manner in cyclic light, constant darkness, and artificial light/dark cycles. This alternating bright/weak banding of Rho-Timer along the length of the ROS relates to inhomogeneities in rhodopsin density and potential points of structural weakness. In addition, we reveal that prolonged dim ambient light exposure impacts not only the rhodopsin content of new discs but also that of older discs, suggesting a dynamic interchange of material between new and old discs. Furthermore, we show that rhodopsin incorporation into the ROS is greatly altered in two autosomal recessive retinitis pigmentosa mouse models, potentially contributing to the pathogenesis. Our findings provide insights into how extrinsic (light) and intrinsic (retinal clocks and genetic mutation) factors dynamically regulate mammalian ROS renewal.

Effects of cell size and bicarbonate on single photon response variability in retinal rods.

Accurate photon counting requires that rods generate highly amplified, reproducible single photon responses (SPRs). The SPR is generated within the rod outer segment (ROS), a multilayered structure built from membranous disks that house rhodopsin. Photoisomerization of rhodopsin at the disk rim causes a local depletion of cGMP that closes ion channels in the plasmalemma located nearby with relative rapidity. In contrast, a photoisomerization at the disk center, distant from the plasmalemma, has a delayed impact on the ion channels due to the time required for cGMP redistribution. Radial differences should be greatest in large diameter rods. By affecting membrane guanylate cyclase activity, bicarbonate could impact spatial inhomogeneity in cGMP content. It was previously known that in the absence of bicarbonate, SPRs are larger and faster at the base of a toad ROS (where the ROS attaches to the rest of the cell) than at the distal tip. Given that bicarbonate enters the ROS at the base and diffuses to the tip and that it expedites flash response recovery, there should be an axial concentration gradient for bicarbonate that would accentuate the base-to-tip SPR differences. Seeking to understand how ROS geometry and bicarbonate affect SPR variability, we used mathematical modeling and made electrophysiological recordings of single rods. Modeling predicted and our experiments confirmed minor radial SPR variability in large diameter, salamander rods that was essentially unchanged by bicarbonate. SPRs elicited at the base and tip of salamander rods were similar in the absence of bicarbonate, but when treated with 30 mM bicarbonate, SPRs at the base became slightly faster than those at the tip, verifying the existence of an axial gradient for bicarbonate. The differences were small and unlikely to undermine visual signaling. However, in toad rods with longer ROSs, bicarbonate somehow suppressed the substantial, axial SPR variability that is naturally present in the absence of bicarbonate. Modeling suggested that the axial gradient of bicarbonate might dampen the primary phototransduction cascade at the base of the ROS. This novel effect of bicarbonate solves a mystery as to how toad vision is able to function effectively in extremely dim light.

Maximizing the Rod Outer Segment Yield in Retinas Extracted from Cattle Eyes.

The retina is a thin neuronal multilayer responsible for the detection of visual information. The first step in visual transduction occurs in the photoreceptor outer segment. The studies on photoreception and visual biochemistry have often utilized rod outer segments (OS) or OS disks purified from mammalian eyes. Literature reports several OS and disk purification procedures that rarely specify the procedure utilized to collect the retina from the eye. Some reports suggest the use of scissors, while others do not mention the issue as they declare to utilize frozen retinas. Because the OS are deeply embedded in the retinal pigmented epithelium (RPE), the detachment of the retina by a harsh pull-out can cause the fracture of the photoreceptor cilium. Here, we present a protocol maximizing OS yield. Eye semi-cups, obtained by hemisecting the eyeball and discarding the anterior chamber structures and the vitreous, are filled with Mammalian Ringer. After 10-15 min of incubation, the retinas spontaneously detach with their wealth of OS almost intact. The impressive ability of the present protocol to minimize the number of OS stuck inside the RPE, and therefore lost, compared with the classic procedure, is shown by confocal laser scanning microscopy analysis of samples stained ex vivo with a dye (MitoTracker deep red) that stains both retinal mitochondria and OS. Total protein assay of OS disks purified by either procedure also shows a 300% total protein yield improvement. The advantage of the protocol presented is its higher yield of photoreceptor OS for subsequent purification procedures, while maintaining the physiological features of the retina.

Dynamic lipid turnover in photoreceptors and retinal pigment epithelium throughout life.

The retinal pigment epithelium-photoreceptor interphase is renewed each day in a stunning display of cellular interdependence. While photoreceptors use photosensitive pigments to convert light into electrical signals, the RPE supports photoreceptors in their function by phagocytizing shed photoreceptor tips, regulating the blood retina barrier, and modulating inflammatory responses, as well as regenerating the 11-cis-retinal chromophore via the classical visual cycle. These processes involve multiple protein complexes, tightly regulated ligand-receptors interactions, and a plethora of lipids and protein-lipids interactions. The role of lipids in maintaining a healthy interplay between the RPE and photoreceptors has not been fully delineated. In recent years, novel technologies have resulted in major advancements in understanding several facets of this interplay, including the involvement of lipids in phagocytosis and phagolysosome function, nutrient recycling, and the metabolic dependence between the two cell types. In this review, we aim to integrate the complex role of lipids in photoreceptor and RPE function, emphasizing the dynamic exchange between the cells as well as discuss how these processes are affected in aging and retinal diseases.

The Flavone Cirsiliol from Salvia x jamensis Binds the F1 Moiety of ATP Synthase, Modulating Free Radical Production.

Several studies have shown that mammalian retinal rod outer segments (OS) are peculiar structures devoid of mitochondria, characterized by ectopic expression of the molecular machinery for oxidative phosphorylation. Such ectopic aerobic metabolism would provide the chemical energy for the phototransduction taking place in the OS. Natural polyphenols include a large variety of molecules having pleiotropic effects, ranging from anti-inflammatory to antioxidant and others. Our goal in the present study was to investigate the potential of the flavonoid cirsiliol, a trihydroxy-6,7-dimethoxyflavone extracted from Salvia x jamensis, in modulating reactive oxygen species production by the ectopic oxidative phosphorylation taking place in the OS. Our molecular docking analysis identified cirsiliol binding sites inside the F1 moiety of the nanomotor F1Fo-ATP synthase. The experimental approach was based on luminometry, spectrophotometry and cytofluorimetry to evaluate ATP synthesis, respiratory chain complex activity and H2O2 production, respectively. The results showed significant dose-dependent inhibition of ATP production by cirsiliol. Moreover, cirsiliol was effective in reducing the free radical production by the OS exposed to ambient light. We report a considerable protective effect of cirsiliol on the structural stability of rod OS, suggesting it may be considered a promising compound against oxidative stress.

First 3D-Structural Data of Full-Length Guanylyl Cyclase 1 in Rod-Outer-Segment Preparations of Bovine Retina by Cross-Linking/Mass Spectrometry.

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.

Determinants shaping the nanoscale architecture of the mouse rod outer segment.

The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ROS architecture is characterized by a stack of identically shaped and tightly packed membrane disks loaded with the visual receptor rhodopsin. A wide range of genetic aberrations have been reported to compromise ROS ultrastructure, impairing photoreceptor viability and function. Yet, the structural basis giving rise to the remarkably precise arrangement of ROS membrane stacks and the molecular mechanisms underlying genetically inherited diseases remain elusive. Here, cryo-electron tomography (cryo-ET) performed on native ROS at molecular resolution provides insights into key structural determinants of ROS membrane architecture. Our data confirm the existence of two previously observed molecular connectors/spacers which likely contribute to the nanometer-scale precise stacking of the ROS disks. We further provide evidence that the extreme radius of curvature at the disk rims is enforced by a continuous supramolecular assembly composed of peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) oligomers. We suggest that together these molecular assemblies constitute the structural basis of the highly specialized ROS functional architecture. Our Cryo-ET data provide novel quantitative and structural information on the molecular architecture in ROS and substantiate previous results on proposed mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.

A hybrid stochastic/deterministic model of single photon response and light adaptation in mouse rods.

The phototransduction cascade is paradigmatic for signaling pathways initiated by G protein-coupled receptors and is characterized by a fine regulation of photoreceptor sensitivity and electrical response to a broad range of light stimuli. Here, we present a biochemically comprehensive model of phototransduction in mouse rods based on a hybrid stochastic and deterministic mathematical framework, and a quantitatively accurate description of the rod impedance in the dark. The latter, combined with novel patch clamp recordings from rod outer segments, enables the interconversion of dim flash responses between photovoltage and photocurrent and thus direct comparison with the simulations. The model reproduces the salient features of the experimental photoresponses at very dim and bright stimuli, for both normal photoreceptors and those with genetically modified cascade components. Our modelling approach recapitulates a number of recent findings in vertebrate phototransduction. First, our results are in line with the recently established requirement of dimeric activation of PDE6 by transducin and further show that such conditions can be fulfilled at the expense of a significant excess of G protein activated by rhodopsin. Secondly, simulations suggest a crucial role of the recoverin-mediated Ca2+-feedback on rhodopsin kinase in accelerating the shutoff, when light flashes are delivered in the presence of a light background. Finally, stochastic simulations suggest that transient complexes between dark rhodopsin and transducin formed prior to light stimulation increase the reproducibility of single photon responses. Current limitations of the model are likely associated with the yet unknown mechanisms governing the shutoff of the cascade.

Differential expression of the five redox complexes in the retinal mitochondria or rod outer segment disks is consistent with their different functionality.

PURPOSE: The retinal rod outer segment (OS) disk membranes, devoid of mitochondria, conducts oxidative phosphorylation (OxPhos). This study aimed at identifying which proteins expressed in the retinal rod OS disks determined the considerable adenosine-5'-triphosphate production and oxygen consumption observed in comparison with retinal mitochondria. PROCEDURES: Characterization was conducted by immunogold transmission electron microscopy on retinal sections. OxPhos was studied by oximetry and luminometry. The proteomes of OS disks and mitochondria purified from bovine retinas were studied by mass spectrometry. Statistical and bioinformatic analyses were conducted by univariate, multivariate, and machine learning methods. RESULTS: Weighted gene coexpression network analysis identified two protein expression profile modules functionally associated with either retinal mitochondria or disk samples, in function of a strikingly different ability of each sample to utilized diverse substrate for F1Fo-ATP synthase. The OS disk proteins correlated better than mitochondria with the tricarboxylic acids cycle and OxPhos proteins. CONCLUSIONS: The differential enrichment of the expression profile of the OxPhos proteins in the disks versus mitochondria suggests that these proteins may represent a true proteome component of the former, with different functionality. These findings may shed new light on the pathogenesis of rod-driven retinal degenerative diseases.

Inhibitory Action of Antidiabetic Drugs on the Free Radical Production by the Rod Outer Segment Ectopic Aerobic Metabolism.

Rod outer segments (OS) express the FoF1-ATP synthase and the respiratory chain, conducting an ectopic aerobic metabolism that produces free radicals in vitro. Diabetic retinopathy, a leading cause of vision loss, is associated with oxidative stress in the outer retina. Since metformin and glibenclamide, two anti-type 2 diabetes drugs, target the respiratory complexes, we studied the effect of these two drugs, individually or in association, on the free radical production in purified bovine rod OS. ATP synthesis, oxygen consumption, and oxidative stress production were assayed by luminometry, oximetry and flow cytometry, respectively. The expression of FoF1-ATP synthase was studied by immunogold electron microscopy. Metformin had a hormetic effect on the OS complex I and ATP synthetic activities, being stimulatory at concentrations below 1 mM, and inhibitory above. Glibenclamide inhibited complexes I and III, as well as ATP production in a concentration-dependent manner. Maximal concentrations of both drugs inhibited the ROI production by the light-exposed OS. Data, consistent with the delaying effect of these drugs on the onset of diabetic retinopathy, suggest that a combination of the two drugs at the beginning of the treatment might reduce the oxidative stress production helping the endogenous antioxidant defences in avoiding retinal damage.

Sclareol modulates free radical production in the retinal rod outer segment by inhibiting the ectopic f1fo-atp synthase.

We have previously shown that the retinal rod outer segments (OS) produce reactive oxygen species in the function of illumination in vitro, establishing a relationship among the extra-mitochondrial oxidative phosphorylation and phototransduction. This source of oxidative stress in the OS can be modulated by polyphenols, acting as inhibitors of F1Fo-ATP synthase. The present study aimed at exploring whether sclareol, a diterpene, interacts with F1Fo-ATP synthase mitigating the light-induced free radical production in the rod OS. Characterization of bovine retinal sections was conducted by immunogold analysis. Reactive oxygen intermediates production, oxygen consumption, the activity of the four respiratory complexes and ATP synthesis were evaluated in purified bovine rod OS. Molecular docking analyses were also conducted. Sclareol reduced free radical production by light-exposed rod OS. Such antioxidant effect was associated with an inhibition of the respiratory complexes and oxygen consumption (OCR), in coupled conditions. Sclareol also inhibited the rod OS ATP synthetic ability. Since the inhibitor effect on respiratory complexes and OCR is not observed in uncoupled conditions, it is supposed that the modulating effect of sclareol on the ectopic oxidative phosphorylation in the rod OS targets specifically the F1Fo-ATP synthase. This hypothesis is confirmed by the in silico molecular docking analyses, which shows that sclareol binds the F1 moiety of ATP synthase with high affinity. In conclusion, a beneficial effect of sclareol can be envisaged as a modulator of oxidative stress in the photoreceptor, a risk factor for the degenerative retinal diseases, suggestive of its potential beneficial action also in vivo.

Retinal proteomic evaluation of rats following streptozotocin­injection using shotgun proteomics.

It is important to elucidate how retinal stimulation leads to retinal protection and dysfunction. The current study aimed to identify factors that are up­ and downregulated in the retinas of streptozotocin (STZ)­induced diabetic rats with acute retinal dysfunction. Retinal function was measured and changes in protein expressions were determined using electroretinograms (ERGs) and liquid chromatography/mass spectroscopy­based shotgun proteomics, respectively. The results revealed that the plasma glucose levels of STZ rats were markedly higher when compared with normal rats. Furthermore, levels of a­waves, b­waves and oscillatory potential amplitudes on ERGs in STZ rats were decreased compared with healthy animals. With use of shotgun proteomics, 391 proteins were identified in the retinas of normal rats and 541 proteins were found in the retinas of STZ rats. Of the 560 proteins identified in rat retinas, 372 (66.4%) were present in both normal and STZ rats. Of these, 19 (3.39%) were unique to normal rats and 169 (30.1%) were unique to STZ rats. Gene Ontology analysis was performed on the candidate proteins that were differentially regulated in the retinas of STZ rats and focused on those classified as 'protein binding', which serve important roles in retinal neurodegeneration. The results revealed an excessive expression of retinol­binding protein 1 (RBP1) and a negative expression of rod outer segment membrane protein 1 (Rom-1) in the retinas of STZ rats. Therefore, retinal function may be decreased with STZ­induced injury, and expressions of Rom­1 and RBP1 may be altered in the retinas of STZ rats.

The Endocannabinoid System Is Present in Rod Outer Segments from Retina and Is Modulated by Light.

The aim of the present research was to evaluate if the endocannabinoid system (enzymes and receptors) could be modulated by light in rod outer segment (ROS) from bovine retina. First, we analyzed endocannabinoid 2-arachidonoylglycerol (2-AG) metabolism in purified ROS obtained from dark-adapted (DROS) or light-adapted (LROS) retinas. To this end, diacylglycerol lipase (DAGL), monoacylglycerol lipase (MAGL), and lysophosphatidate phosphohydrolase (LPAP) enzymatic activities were analyzed using radioactive substrates. The protein content of these enzymes and of the receptors to which cannabinoids bind was determined by immunoblotting under light stimulus. Our results indicate that whereas DAGL and MAGL activities were stimulated in retinas exposed to light, no changes were observed in LPAP activity. Interestingly, the protein content of the main enzymes involved in 2-AG metabolism, phospholipase C ß1 (PLCß1), and DAGLα (synthesis), and MAGL (hydrolysis), was also modified by light. PLCß1 content was increased, while that of lipases was decreased. On the other hand, light produced an increase in the cannabinoid receptors CB1 and CB2 and a decrease in GPR55 protein levels. Taken together, our results indicate that the endocannabinoid system (enzymes and receptors) depends on the illumination state of the retina, suggesting that proteins related to phototransduction phenomena could be involved in the effects observed.

Modulation of the rod outer segment aerobic metabolism diminishes the production of radicals due to light absorption.

Oxidative stress is a primary risk factor for both inflammatory and degenerative retinopathies. Our previous data on blue light-irradiated retinas demonstrated an oxidative stress higher in the rod outer segment (OS) than in the inner limb, leading to impairment of the rod OS extra-mitochondrial aerobic metabolism. Here the oxidative metabolism and Reactive Oxygen Intermediates (ROI) production was evaluated in purified bovine rod OS in function of exposure to different illumination conditions. A dose response was observed to varying light intensities and duration in terms of both ROI production and ATP synthesis. Pretreatment with resveratrol, inhibitor of F1Fo-ATP synthase, or metformin, inhibitor of the respiratory complex I, significantly diminished the ROI production. Metformin also diminished the rod OS Complex I activity and reduced the maximal OS response to light in ATP production. Data show for the first time the relationship existing in the rod OS between its -aerobic- metabolism, light absorption, and ROI production. A beneficial effect was exerted by metformin and resveratrol, in modulating the ROI production in the illuminated rod OS, suggestive of their beneficial action also in vivo. Data shed new light on preventative interventions for cone loss secondary to rod damage due to oxidative stress.

Molecular Details of Retinal Guanylyl Cyclase 1/GCAP-2 Interaction.

The rod outer segment guanylyl cyclase 1 (ROS-GC1) is an essential component of photo-transduction in the retina. In the light-induced signal cascade, membrane-bound ROS-GC1 restores cGMP levels in the dark in a calcium-dependent manner. With decreasing calcium concentration in the intracellular compartment, ROS-GC1 is activated via the intracellular site by guanylyl cyclase-activating proteins (GCAP-1/-2). Presently, the exact activation mechanism is elusive. To obtain structural insights into the ROS-GC1 regulation by GCAP-2, chemical cross-linking/mass spectrometry studies using GCAP-2 and three ROS-GC1 peptides were performed in the presence and absence of calcium. The majority of cross-links were identified with the C-terminal lobe of GCAP-2 and a peptide comprising parts of ROS-GC1's catalytic domain and C-terminal extension. Consistently with the cross-linking results, surface plasmon resonance and fluorescence measurements confirmed specific binding of this ROS-GC peptide to GCAP-2 with a dissociation constant in the low micromolar range. These results imply that a region of the catalytic domain of ROS-GC1 can participate in the interaction with GCAP-2. Additional binding surfaces upstream of the catalytic domain, in particular the juxtamembrane domain, can currently not be excluded.