Chemical shift assignments of calmodulin bound to a C-terminal site (residues 1120-1147) in the ß-subunit of a retinal cyclic nucleotide-gated channel (CNGB1).

Retinal cyclic nucleotide-gated (CNG) channels consist of two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to two separate sites within the cytosolic region of CNGB1: CaM binding to an N-terminal site (human CNGB1 residues 565-587, called CaM1) decreases the open probability of CNG channels at elevated Ca2+ levels in dark-adapted photoreceptors, whereas CaM binding to a separate C-terminal site (CNGB1 residues 1120-1147, called CaM2) may increase channel open probability in light activated photoreceptors. We recently reported NMR chemical shift assignments of Ca2+-saturated CaM bound to the CaM1 site of CNGB1 (BMRB no. 51222). Here, we report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the C-terminal CaM2 site of CNGB1 (BMRB no. 51447).

Chemical shift assignments of calmodulin bound to the ß-subunit of a retinal cyclic nucleotide-gated channel (CNGB1).

Rod cyclic nucleotide-gated (CNG) channels are formed by two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to the cytosolic regulatory domain of CNGB1 and decreases the open probability of CNGA1/CNGB1 channels. The CaM binding site within bovine CNGB1 (residues 679-702) binds tightly to Ca2+-bound CaM, which promotes Ca2+-induced inactivation of CNGA1/CNGB1 channels in retinal rods. We report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the CaM-binding domain of CNGB1 (BMRB no. 51222).

CNG channel structure, function, and gating: a tale of conformational flexibility.

Cyclic nucleotide-gated (CNG) channels are key to the signal transduction machinery of certain sensory modalities both in vertebrate and invertebrate organisms. They translate a chemical change in cyclic nucleotide concentration into an electrical signal that can spread through sensory cells. Despite CNG and voltage-gated potassium channels sharing a remarkable amino acid sequence homology and basic architectural plan, their functional properties are dramatically different. While voltage-gated potassium channels are highly selective and require membrane depolarization to open, CNG channels have low ion selectivity and are not very sensitive to voltage. In the last few years, many high-resolution structures of intact CNG channels have been released. This wealth of new structural information has provided enormous progress toward the understanding of the molecular mechanisms and driving forces underpinning CNG channel activation. In this review, we report on the current understanding and controversies surrounding the gating mechanism in CNG channels, as well as the deep intertwining existing between gating, the ion permeation process, and its modulation by membrane voltage. While the existence of this powerful coupling was recognized many decades ago, its direct structural demonstration, and ties to the CNG channel inherent pore flexibility, is a recent achievement.

Photoreceptor phosphodiesterase (PDE6): activation and inactivation mechanisms during visual transduction in rods and cones.

Rod and cone photoreceptors of the vertebrate retina utilize cGMP as the primary intracellular messenger for the visual signaling pathway that converts a light stimulus into an electrical response. cGMP metabolism in the signal-transducing photoreceptor outer segment reflects the balance of cGMP synthesis (catalyzed by guanylyl cyclase) and degradation (catalyzed by the photoreceptor phosphodiesterase, PDE6). Upon light stimulation, rapid activation of PDE6 by the heterotrimeric G-protein (transducin) triggers a dramatic drop in cGMP levels that lead to cell hyperpolarization. Following cessation of the light stimulus, the lifetime of activated PDE6 is also precisely regulated by additional processes. This review summarizes recent advances in the structural characterization of the rod and cone PDE6 catalytic and regulatory subunits in the context of previous biochemical studies of the enzymological properties and allosteric regulation of PDE6. Emphasis is given to recent advances in understanding the structural and conformational changes underlying the mechanism by which the activated transducin α-subunit binds to-and relieves inhibition of-PDE6 catalysis that is controlled by its intrinsically disordered, inhibitory γ-subunit. The role of the regulator of G-protein signaling 9-1 (RGS9-1) in regulating the lifetime of the transducin-PDE6 is also briefly covered. The therapeutic potential of pharmacological compounds acting as inhibitors or activators targeting PDE6 is discussed in the context of inherited retinal diseases resulting from mutations in rod and cone PDE6 genes as well as other inherited defects that arise from excessive cGMP accumulation in retinal photoreceptor cells.

Supramolecular organization of rhodopsin in rod photoreceptor cell membranes.

Rhodopsin is the light receptor in rod photoreceptor cells that initiates scotopic vision. Studies on the light receptor span well over a century, yet questions about the organization of rhodopsin within the photoreceptor cell membrane still persist and a consensus view on the topic is still elusive. Rhodopsin has been intensely studied for quite some time, and there is a wealth of information to draw from to formulate an organizational picture of the receptor in native membranes. Early experimental evidence in apparent support for a monomeric arrangement of rhodopsin in rod photoreceptor cell membranes is contrasted and reconciled with more recent visual evidence in support of a supramolecular organization of rhodopsin. What is known so far about the determinants of forming a supramolecular structure and possible functional roles for such an organization are also discussed. Many details are still missing on the structural and functional properties of the supramolecular organization of rhodopsin in rod photoreceptor cell membranes. The emerging picture presented here can serve as a springboard towards a more in-depth understanding of the topic.

Biochemistry and physiology of zebrafish photoreceptors.

All vertebrates share a canonical retina with light-sensitive photoreceptors in the outer retina. These photoreceptors are of two kinds: rods and cones, adapted to low and bright light conditions, respectively. They both show a peculiar morphology, with long outer segments, comprised of ordered stacks of disc-shaped membranes. These discs host numerous proteins, many of which contribute to the visual transduction cascade. This pathway converts the light stimulus into a biological signal, ultimately modulating synaptic transmission. Recently, the zebrafish (Danio rerio) has gained popularity for studying the function of vertebrate photoreceptors. In this review, we introduce this model system and its contribution to our understanding of photoreception with a focus on the cone visual transduction cascade.

LRRTM4 is a member of the transsynaptic complex between rod photoreceptors and bipolar cells.

Leucine rich repeat transmembrane (LRRTM) proteins are synaptic adhesion molecules with roles in synapse formation and signaling. LRRTM4 transcripts were previously shown to be enriched in rod bipolar cells (BCs), secondary neurons of the retina that form synapses with rod photoreceptors. Using two different antibodies, LRRTM4 was found to reside primarily at rod BC dendritic tips, where it colocalized with the transduction channel protein, TRPM1. LRRTM4 was not detected at dendritic tips of ON-cone BCs. Following somatic knockout of LRRTM4 in BCs by subretinal injection and electroporation of CRISPR/Cas9, LRRTM4 was abolished or reduced in the dendritic tips of transfected cells. Knockout cells had a normal complement of TRPM1 at their dendritic tips, while GPR179 accumulation was partially reduced. In experiments with heterologously expressed protein, the extracellular domain of LRRTM4 was found to engage in heparan-sulfate dependent binding with pikachurin. These results implicate LRRTM4 in the GPR179-pikachurin-dystroglycan transsynaptic complex at rod synapses.

Photoreceptors in skate are arranged to allow for a broad horizontal field of view.

Studying retinal specializations offers insights into eye functionality and visual ecology. Using light microscopic techniques, including retinal whole-mounts, we investigated photoreceptor densities in the retina of the skate Leucoraja erinacea. We show that photoreceptors are not sized or oriented in the same way, and that they are not evenly distributed across the retina. There was a dorsally located horizontal visual streak with increased photoreceptor density, with additional local maxima in which densities were highest. Photoreceptors were longest and thinnest inside this visual streak, becoming shorter and thicker toward the periphery and toward the ventral retina. Furthermore, in the peripheral retinal parts, photoreceptors (particularly the outer segments) were noticeably tilted with respect to the retinal long axis. In order to understand how photoreceptors are tilted inside the eye, we used computerized tomography (CT) and micro-CT, to obtain geometrical dimensions of the whole skate eye. These CT/micro-CT data provided us with the outlines of the skate eye and the location of the retina and this enabled us to reconstruct how photoreceptors tilt in an intact eye. Findings were analyzed relative to previously published ganglion cell distributions in this species, showing a posteriorly located retinal area with photoreceptor: ganglion cell convergence as low as 39:1. Some peripheral areas showed ratios as high as 391:1. We frame our findings in terms of the animal's anatomy: body and eye shape, specifically the location of the tapetum, as well as the visual demands associated with lifestyle and habitat type. A speculative function in polarization sensitivity is discussed.

Multistep peripherin-2/rds self-assembly drives membrane curvature for outer segment disk architecture and photoreceptor viability.

Rod and cone photoreceptor outer segment (OS) structural integrity is essential for normal vision; disruptions contribute to a broad variety of retinal ciliopathies. OSs possess many hundreds of stacked membranous disks, which capture photons and scaffold the phototransduction cascade. Although the molecular basis of OS structure remains unresolved, recent studies suggest that the photoreceptor-specific tetraspanin, peripherin-2/rds (P/rds), may contribute to the highly curved rim domains at disk edges. Here, we demonstrate that tetrameric P/rds self-assembly is required for generating high-curvature membranes in cellulo, implicating the noncovalent tetramer as a minimal unit of function. P/rds activity was promoted by disulfide-mediated tetramer polymerization, which transformed localized regions of curvature into high-curvature tubules of extended lengths. Transmission electron microscopy visualization of P/rds purified from OS membranes revealed disulfide-linked tetramer chains up to 100 nm long, suggesting that chains maintain membrane curvature continuity over extended distances. We tested this idea in Xenopus laevis photoreceptors, and found that transgenic expression of nonchain-forming P/rds generated abundant high-curvature OS membranes, which were improperly but specifically organized as ectopic incisures and disk rims. These striking phenotypes demonstrate the importance of P/rds tetramer chain formation for the continuity of rim formation during disk morphogenesis. Overall, this study advances understanding of the normal structure and function of P/rds for OS architecture and biogenesis, and clarifies how pathogenic loss-of-function mutations in P/rds cause photoreceptor structural defects to trigger progressive retinal degenerations. It also introduces the possibility that other tetraspanins may generate or sense membrane curvature in support of diverse biological functions.

Docosahexaenoic acid and the brain- what is its role?

Docosahexaenoic acid (DHA) is a 22-carbon omega 3 PUFA highly enriched in the neuronal cell membranes and rod outer segment membranes. When DHA is depleted from these cell membranes it is replaced nearly quantitatively by a 22-carbon omega 6 PUFA, docosapentaenoic acid, which has similar, but less potent, biophysical and physiological properties to DHA. It is speculated that omega 6-docosapentaenoic acid is a buffer to prevent the possible catastrophic effects of DHA depletion on brain and visual function. The primary insult from the loss of DHA from cell membrane glycerophospholipids, and replacement by omega 6-docosapentaenoic acid, is on the flexibility/compression of the membrane lipids which affects the optimal function of integral membrane proteins (receptors, voltage-gated ion channels and enzymes). This leads to effects on second messenger systems, and subsequently affects neurotransmitter concentrations due to 'weakened' signals from the initiating receptors. Remembering there are more than 80 billion neurones and many times more synaptic connections between neurons, a very small loss of "efficiency" in signal due to altered properties of membrane proteins would likely result in meaningful changes in brain and visual function. Additionally, impairment of neurotransmission could be due, in part, to sub-optimal brain energy metabolism (glucose entry into the brain), which is significantly reduced in omega 3 deficiency. Many studies report that dietary omega 3 deficiency results in changes in learning, coping with stress, behavioural changes, and responses in visual function. It is thus concluded that DHA is an essential fatty acid for optimal neuronal function.

A single-cell transcriptome atlas of the adult human retina.

The retina is a specialized neural tissue that senses light and initiates image processing. Although the functional organization of specific retina cells has been well studied, the molecular profile of many cell types remains unclear in humans. To comprehensively profile the human retina, we performed single-cell RNA sequencing on 20,009 cells from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct cell populations representing all known neural retinal cells: rod photoreceptors, cone photoreceptors, Müller glia, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, astrocytes, and microglia. Our data captured molecular profiles for healthy and putative early degenerating rod photoreceptors, and revealed the loss of MALAT1 expression with longer post-mortem time, which potentially suggested a novel role of MALAT1 in rod photoreceptor degeneration. We have demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Müller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to understanding retinal biology and disease.

Identification of PKCα-dependent phosphoproteins in mouse retina.

Adjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKCα), the targets of PKCα phosphorylation in the retina have not been identified. PKCα activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKCα antibody and antibodies to phosphorylated PKC motifs. PKCα activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKCα-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKCα knockout (PKCα-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKCα-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein). Two strongly differentially expressed phosphoproteins, BORG4 and TPBG, were localized to the synaptic layers of the retina, and may play a role in PKCα-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906. SIGNIFICANCE: Retinal rod bipolar cells (RBCs), the second-order neurons of the mammalian rod visual pathway, are able to modulate their sensitivity to remain functional across a wide range of light intensities, from starlight to daylight. Evidence suggests that this modulation requires the serine/threonine kinase, PKCα, though the specific mechanism by which PKCα modulates RBC physiology is unknown. This study examined PKCα phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKCα-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKCα-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKCα-dependent modulation of RBC physiology.

Pushing the limits of photoreception in twilight conditions: The rod-like cone retina of the deep-sea pearlsides.

Most vertebrates have a duplex retina comprising two photoreceptor types, rods for dim-light (scotopic) vision and cones for bright-light (photopic) and color vision. However, deep-sea fishes are only active in dim-light conditions; hence, most species have lost their cones in favor of a simplex retina composed exclusively of rods. Although the pearlsides, Maurolicus spp., have such a pure rod retina, their behavior is at odds with this simplex visual system. Contrary to other deep-sea fishes, pearlsides are mostly active during dusk and dawn close to the surface, where light levels are intermediate (twilight or mesopic) and require the use of both rod and cone photoreceptors. This study elucidates this paradox by demonstrating that the pearlside retina does not have rod photoreceptors only; instead, it is composed almost exclusively of transmuted cone photoreceptors. These transmuted cells combine the morphological characteristics of a rod photoreceptor with a cone opsin and a cone phototransduction cascade to form a unique photoreceptor type, a rod-like cone, specifically tuned to the light conditions of the pearlsides' habitat (blue-shifted light at mesopic intensities). Combining properties of both rods and cones into a single cell type, instead of using two photoreceptor types that do not function at their full potential under mesopic conditions, is likely to be the most efficient and economical solution to optimize visual performance. These results challenge the standing paradigm of the function and evolution of the vertebrate duplex retina and emphasize the need for a more comprehensive evaluation of visual systems in general.

A novel culture method reveals unique neural stem/progenitors in mature porcine iris tissues that differentiate into neuronal and rod photoreceptor-like cells.

Iris neural stem/progenitor cells from mature porcine eyes were investigated using a new protocol for tissue culture, which consists of dispase treatment and Matrigel embedding. We used a number of culture conditions and found an intense differentiation of neuronal cells from both the iris pigmented epithelial (IPE) cells and the stroma tissue cells. Rod photoreceptor-like cells were also observed but mostly in a later stage of culture. Neuronal differentiation does not require any additives such as fetal bovine serum or FGF2, although FGF2 and IGF2 appeared to promote neural differentiation in the IPE cultures. Furthermore, the stroma-derived cells were able to be maintained in vitro indefinitely. The evolutionary similarity between humans and domestic pigs highlight the potential for this methodology in the modeling of human diseases and characterizing human ocular stem cells.

Cell Type-Specific Epigenomic Analysis Reveals a Uniquely Closed Chromatin Architecture in Mouse Rod Photoreceptors.

Rod photoreceptors are specialized neurons that mediate vision in dim light and are the predominant photoreceptor type in nocturnal mammals. The rods of nocturnal mammals are unique among vertebrate cell types in having an 'inverted' nuclear architecture, with a dense mass of heterochromatin in the center of the nucleus rather than dispersed clumps at the periphery. To test if this unique nuclear architecture is correlated with a unique epigenomic landscape, we performed ATAC-seq on mouse rods and their most closely related cell type, cone photoreceptors. We find that thousands of loci are selectively closed in rods relative to cones as well as >60 additional cell types. Furthermore, we find that the open chromatin profile of photoreceptors lacking the rod master regulator Nrl is nearly indistinguishable from that of native cones, indicating that Nrl is required for selective chromatin closure in rods. Finally, we identified distinct enrichments of transcription factor binding sites in rods and cones, revealing key differences in the cis-regulatory grammar of these cell types. Taken together, these data provide insight into the development and maintenance of photoreceptor identity, and highlight rods as an attractive system for studying the relationship between nuclear organization and local changes in gene regulation.

Histone Deacetylase 1 Is Essential for Rod Photoreceptor Differentiation by Regulating Acetylation at Histone H3 Lysine 9 and Histone H4 Lysine 12 in the Mouse Retina.

Histone acetylation has a regulatory role in gene expression and is necessary for proper tissue development. To investigate the specific roles of histone deacetylases (HDACs) in rod differentiation in neonatal mouse retinas, we used a pharmacological approach that showed that inhibition of class I but not class IIa HDACs caused the same phenotypic changes seen with broad spectrum HDAC inhibitors, most notably a block in the differentiation of rod photoreceptors. Inhibition of HDAC1 resulted in increase of acetylation of lysine 9 of histone 3 (H3K9) and lysine 12 of histone 4 (H4K12) but not lysine 27 of histone 3 (H3K27) and led to maintained expression of progenitor-specific genes such as Vsx2 and Hes1 with concomitant block of expression of rod-specific genes. ChiP experiments confirmed these changes in the promoters of a group of progenitor genes. Based on our results, we suggest that HDAC1-specific inhibition prevents progenitor cells of the retina from exiting the cell cycle and differentiating. HDAC1 may be an essential epigenetic regulator of the transition from progenitor cells to terminally differentiated photoreceptors.

Can the Stark-Einstein law resolve the measurement problem from an animate perspective?

Analysis of the Stark-Einstein law as it applies to the retinal molecule, which is part of the rhodopsin molecule within the rod cells of the retina, reveals that it may provide the solution to the measurement problem from an animate perspective. That it represents a natural boundary where the Schrödinger equation or wave function automatically goes from linear to nonlinear while remaining in a deterministic state. It will be possible in the near future to subject this theory to empirical tests as has been previously proposed. This analysis provides a contrast to the many decades well studied and debated inanimate measurement problem and would represent an addition to the Stark-Einstein law involving information carried by the photon.

Imaging Ca2+ dynamics in cone photoreceptor axon terminals of the mouse retina.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca(2+)), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca(2+) imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca(2+) biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections ("slices") of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca(2+) level. The protocol also allows "in-slice measurement" of absolute Ca(2+) concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca(2+) signaling as well as the potential involvement of Ca(2+) in photoreceptor death and retinal degeneration.