Mutations in the gene encoding Cav1.4, CACNA1F, are associated with visual disorders including X-linked incomplete congenital stationary night blindness type 2 (CSNB2). In mice lacking Cav1.4 channels, there are defects in the development of “ribbon” synapses formed between photoreceptors (PRs) and second-order neurons. However, many CSNB2 mutations disrupt the function rather than expression of Cav1.4 channels. Whether defects in PR synapse development due to altered Cav1.4 function are common features contributing to the pathogenesis of CSNB2 is unknown. To resolve this issue, we profiled changes in the subcellular distribution of Cav1.4 channels and synapse morphology during development in wild-type (WT) mice and mouse models of CSNB2. Using Cav1.4-selective antibodies, we found that Cav1.4 channels associate with ribbon precursors early in development and are concentrated at both rod and cone PR synapses in the mature retina. In mouse models of CSNB2 in which the voltage-dependence of Cav1.4 activation is either enhanced (Cav1.4I756T) or inhibited (CaBP4 KO), the initial stages of PR synaptic ribbon formation are largely unaffected. However, after postnatal day 13, many PR ribbons retain the immature morphology. This synaptic abnormality corresponds in severity to the defect in synaptic transmission in the adult mutant mice, suggesting that lack of sufficient mature synapses contributes to vision impairment in Cav1.4I756T and CaBP4 KO mice. Our results demonstrate the importance of proper Cav1.4 function for efficient PR synapse maturation, and that dysregulation of Cav1.4 channels in CSNB2 may have synaptopathic consequences.

αδ-4 is an auxiliary subunit of voltage-gated Ca1.4 L-type channels that regulate the development and mature exocytotic function of the photoreceptor ribbon synapse. In humans, mutations in the gene encoding αδ-4 cause heterogeneous forms of vision impairment in humans, the underlying pathogenic mechanisms of which remain unclear. To investigate the retinal function of αδ-4, we used genome editing to generate an αδ-4 knock-out (αδ-4 KO) mouse. In male and female αδ-4 KO mice, rod spherules lack ribbons and other synaptic hallmarks early in development. Although the molecular organization of cone synapses is less affected than rod synapses, horizontal and cone bipolar processes extend abnormally in the outer nuclear layer in αδ-4 KO retina. In reconstructions of αδ-4 KO cone pedicles by serial block face scanning electron microscopy, ribbons appear normal, except that less than one-third show the expected triadic organization of processes at ribbon sites. The severity of the synaptic defects in αδ-4 KO mice correlates with a progressive loss of Ca1.4 channels, first in terminals of rods and later cones. Despite the absence of b-waves in electroretinograms, visually guided behavior is evident in αδ-4 KO mice and better under photopic than scotopic conditions. We conclude that αδ-4 plays an essential role in maintaining the structural and functional integrity of rod and cone synapses, the disruption of which may contribute to visual impairment in humans with mutations. In the retina, visual information is first communicated by the synapse formed between photoreceptors and second-order neurons. The mechanisms that regulate the structural integrity of this synapse are poorly understood. Here we demonstrate a role for αδ-4, a subunit of voltage-gated Ca channels, in organizing the structure and function of photoreceptor synapses. We find that presynaptic Ca channels are progressively lost and that rod and cone synapses are disrupted in mice that lack αδ-4. Our results suggest that alterations in presynaptic Ca signaling and photoreceptor synapse structure may contribute to vision impairment in humans with mutations in the gene encoding αδ-4.

Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation. The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood. We have probed these mechanisms by examining light-dependent localizations of the transducin-␣ subunit (Gt␣) in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant Gt␣Q200L. An illumination threshold for the Gt␣ movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST. Transgenic Gt␣Q200L mice have significantly diminished levels of proteins involved in cGMP metabolism in rods, most notably the PDE6 catalytic subunits, and severely reduced sensitivity to light. Similarly to the native Gt␣, the Gt␣Q200L mutant is localized to the IS/ST compartment in light-adapted transgenic mice. However, the return of Gt␣Q200L to the OS during dark adaptation is markedly slower than normal. Thus, the light-dependent translocations of transducin are controlled by the GTP-hydrolysis on Gt␣, and apparently, do not require Gt␣ interaction with RGS9 and PDE6.Heterotrimeric GTP-binding proteins (G proteins) propagate a variety of hormonal and sensory signals from specific cell surface receptors to intracellular effectors (1-3). The visual transduction cascade in vertebrate photoreceptors has served for many years as a paradigm for G protein signaling. In rod photoreceptor cells, illuminated rhodopsin stimulates GTP-GDP exchange on the retinal G protein, transducin (Gt), 2 resulting in dissociation of Gt␣GTP from Gt␤␥ and rhodopsin. Gt␣ in the active GTP-bound conformation stimulates the effector enzyme, cGMP phosphodiesterase (PDE6), by displacing the inhibitory ␥-subunits (P␥) from the PDE6 catalytic core (PDE6␣␤). cGMP hydrolysis by active PDE6 results in closure of cGMP gated channels in the plasma membrane (4, 5). The turn-off phase of the visual signal is determined by reactions controlling the lifetimes of photoexcited rhodopsin (R*) and activated transducin. The catalytic function of R* is blocked by the rhodopsin-kinase mediated phosphorylation and the binding of arrestin to phosphorylated R* (6 -8). The lifetime of Gt␣GTP is controlled by intrinsic GTPase activity. Hydrolysis of GTP switches the Gt␣ molecule to the inactive GDP-bound conformation and allows reinhibition of PDE␣␤ by P␥. RGS9-1, a photoreceptor-specific member of the RGS (regulators of G protein signaling) family, in the complex with G␤5L acts as a GTPase-activating protein for transducin and thus is a major regulator of the turn-off kinetics of the visual signal (9 -11). The RGS9-1/G␤5L complex is anchored to disc membranes through the interaction with R9AP (RGS-9-1-anchor protein) that enhances the complex GAP acti...

Mutations in the CACNA1F gene encoding the Cav1.4 Ca2+ channel are associated with X-linked congenital stationary night blindness type 2 (CSNB2). Despite the increasing knowledge about the functional behavior of mutated channels in heterologous systems, the pathophysiological mechanisms that result in vision impairment remain to be elucidated. This work provides a thorough functional characterization of the novel IT mouse line that harbors the gain-of-function mutation I745T reported in a New Zealand CSNB2 family.1 Electroretinographic recordings in IT mice permitted a direct comparison with human data. Our data supported the hypothesis that a hyperpolarizing shift in the voltage-dependence of channel activation—as seen in the IT gain-of-function mutant2—may reduce the dynamic range of photoreceptor activity. Morphologically, the retinal outer nuclear layer in adult IT mutants was reduced in size and cone outer segments appeared shorter. The organization of the outer plexiform layer was disrupted, and synaptic structures of photoreceptors had a variable, partly immature, appearance. The associated visual deficiency was substantiated in behavioral paradigms. The IT mouse line serves as a specific model for the functional phenotype of human CSNB2 patients with gain-of-function mutations and may help to further understand the dysfunction in CSNB.

PDE6 (phosphodiesterase-6) is the effector molecule in the vertebrate phototransduction cascade. Progress in understanding the structure and function of PDE6 has been hindered by lack of an expression system of the enzyme. Here we report ectopic expression and analysis of compartmentalization and membrane dynamics of the enhanced green fluorescent protein (EGFP) fusion protein of human cone PDE6C in rods of transgenic Xenopus laevis. EGFP-PDE6C is correctly targeted to the rod outer segments in transgenic Xenopus, where it displayed a characteristic striated pattern of EGFP fluorescence. Immunofluorescence labeling indicated significant and light-independent co-localization of EGFP-PDE6C with the disc rim marker peripherin-2 and endogenous frog PDE6. The diffusion of EGFP-PDE6C on disc membranes investigated with fluorescence recovery after photobleaching was markedly slower than theoretically predicted. The enzymatic characteristics of immunoprecipitated recombinant PDE6C were similar to known properties of the native bovine PDE6C. PDE6C was potently inhibited by the cone-and rod-specific PDE6 ␥-subunits. Thus, transgenic Xenopus laevis is a unique expression system for PDE6 well suited for analysis of the mechanisms of visual diseases linked to PDE6 mutations.Phosphodiesterases of cyclic nucleotides (PDEs) 2 are essential enzymes controlling cellular levels of cAMP and cGMP. Eleven families of PDEs have been identified in mammals on the grounds of sequence homology, substrate selectivity, and regulation (1). Photoreceptor-specific PDEs in rods and cones comprise the sixth PDE family (PDE6) and serve as the effector enzymes in the vertebrate phototransduction cascade (1-5). Rod PDE6 is composed of homologous catalytic ␣-subunit (PDE6A) and ␤-subunit (PDE6B) and two copies of a small inhibitory ␥-subunit (P␥) (3). Cone PDE6 is a catalytic dimer of two identical ␣Ј-subunits (PDE6C) (3). A cone-specific inhibitory P␥-subunit is highly homologous to the rod P␥ (6). In rod photoreceptors, PDE6 is located in the specialized compartments called rod outer segments (ROS), where it associates with disc membranes. The membrane attachment of PDE6 is mediated by farnesylation of the PDE6A C terminus and geranylgeranylation of the PDE6B C terminus (7). In cones, geranylgeranylated PDE6C resides on infoldings of the cone outer segment plasma membrane (8). Following photoexcitation of rod or cone photoreceptor cells, PDE6 is activated by the GTPbound transducin ␣-subunit (G␣ t GTP) that relieves the P␥ inhibition of the enzyme. cGMP hydrolysis by active PDE6 leads to a cellular response due to a closure of cGMP-gated channels in the photoreceptor plasma membrane (2-5).Although PDE6 plays a prominent role in vertebrate vision, the structure-function relationships of PDE6 are poorly understood in comparison with other key phototransduction proteins. The lack of an expression system for PDE6 has become a major impediment for PDE6 research. Importantly, an expression system for PDE6 is required to elucidate the mechanisms of visua...

In rod photoreceptors, several phototransduction components display light-dependent translocation between cellular compartments. Notably, the G protein transducin translocates from rod outer segments to inner segments/spherules in bright light, but the functional consequences of translocation remain unclear. We generated transgenic mice where light-induced transducin translocation is impaired. These mice exhibited slow photoreceptor degeneration, which was prevented if they were dark-reared. Physiological recordings showed that control and transgenic rods and rod bipolar cells displayed similar sensitivity in darkness. After bright light exposure, control rods were more strongly desensitized than transgenic rods. However, in rod bipolar cells, this effect was reversed; transgenic rod bipolar cells were more strongly desensitized than control. This sensitivity reversal indicates that transducin translocation in rods enhances signaling to rod bipolar cells. The enhancement could not be explained by modulation of inner segment conductances or the voltage sensitivity of the synaptic Ca 2+ current, suggesting interactions of transducin with the synaptic machinery.retina | adaptation | presynaptic modulation | SNARE complex | palmitoylation

Photoreceptor rod and cone phosphodiesterases comprise the sixth family of cyclic nucleotide phosphodiesterases (PDE6). PDE6s have uniquely evolved as effector enzymes in the vertebrate phototransduction cascade. To understand the evolution of the PDE6 family, we have examined PDE6 in lamprey, an ancient vertebrate group. A single PDE6 catalytic subunit transcript was found in the sea lamprey Petromyzon marinus cDNA library. The lamprey PDE6 sequence showed a high degree of homology with mammalian PDE6 and equally distant relationships with the rod and cone enzymes. In contrast, two different PDE6 inhibitory Pgamma subunits, a cone-type Pgamma1 and a mixed cone/rod-type Pgamma2, have been identified in the lamprey retina. Immunofluorescence analysis demonstrated that Pgamma1 and Pgamma2 are expressed in the long and short photoreceptors of sea lamprey, respectively. The catalytic PDE6 subunit was present in the photoreceptors of both types and colocalized with the Pgamma subunits. Recombinant Pgamma1 and Pgamma2 potently inhibited trypsin-activated lamprey and bovine PDE6 enzymes. Our results point to a high degree of conservation of PDE6 genes during the vertebrate evolution. The apparent duplication of the Pgamma gene in the stem of vertebrate lineage may have been an essential component of the evolution of scotopic vision in early vertebrates.

The Nougaret form of dominant stationary night blindness is linked to a G38D mutation in the rod transducin-␣ subunit (T␣). In this study, we have examined the mechanism of Nougaret night blindness using transgenic mice expressing T␣G38D. The biochemical, electrophysiological, and vision-dependent behavioral analyses of the mouse model revealed a unique phenotype of reduced rod sensitivity, impaired activation, and slowed recovery of the phototransduction cascade. Two key deficiencies in T␣G38D function, its poor ability to activate PDE6 (cGMP phosphodiesterase) and decreased GTPase activity, are found to be the major mechanisms altering visual signaling in transgenic mice. Despite these defects, rod-mediated sensitivity in heterozygous mice is not decreased to the extent seen in heterozygous Nougaret patients.