The inhibitory interaction of phosphodiesterase-6 (PDE6) with its c-subunit (Pc) is pivotal in vertebrate phototransduction. Here, crystal structures of a chimaeric PDE5/ PDE6 catalytic domain (PDE5/6cd) complexed with sildenafil or 3-isobutyl-1-methylxanthine and the Pc-inhibitory peptide Pc 70À87 have been determined at 2.9 and 3.0 Å , respectively. These structures show the determinants and the mechanism of the PDE6 inhibition by Pc and suggest the conformational change of Pc on transducin activation. Two variable H-and M-loops of PDE5/6cd form a distinct interface that contributes to the Pc-binding site. This allows the Pc C-terminus to fit into the opening of the catalytic pocket, blocking cGMP access to the active site. Our analysis suggests that disruption of the H-M loop interface and Pc-binding site is a molecular cause of retinal degeneration in atrd3 mice. Comparison of the two PDE5/6cd structures shows an overlap between the sildenafil and Pc 70À87 -binding sites, thereby providing critical insights into the side effects of PDE5 inhibitors on vision.

Phosphodiesterase 6 (PDE6) is the effector enzyme in the phototransduction cascade and is critical for the health of both rod and cone photoreceptors. Its dysfunction, caused by mutations in either the enzyme itself or AIPL1 (aryl hydrocarbon receptor-interacting protein-like 1), leads to retinal diseases culminating in blindness. Progress in research on PDE6 and AIPL1 has been severely hampered by failure to express functional PDE6 in a heterologous expression system. Here, we demonstrated that AIPL1 is an obligate chaperone of PDE6 and that it enables low yield functional folding of cone PDE6C in cultured cells. We further show that the AIPL1-mediated production of folded PDE6C is markedly elevated in the presence of the inhibitory P␥-subunit of PDE6. As illustrated in this study, a simple and sensitive system in which AIPL1 and P␥ are co-expressed with PDE6 represents an effective tool for probing structure-function relationships of AIPL1 and reliably establishing the pathogenicity of its variants.Cyclic nucleotide phosphodiesterases of the sixth family (PDE6) 2 are the key effectors in the visual transduction cascade in rod and cone photoreceptors. In the dark, activity of the PDE6 catalytic dimers is restrained by two tightly bound inhibitory ␥-subunits (P␥). This allows cGMP to maintain depolarizing "dark" current through a cGMP-gated channel in the photoreceptor plasma membrane. Photoexcitation leads to G-protein-mediated activation of PDE6 followed by a drop in cytoplasmic cGMP, channel closure, and propagation of an electrical signal to downstream retinal neurons (1, 2). In addition to being essential to photoreceptor physiology, PDE6 is critical to the health and survival of rods and cones. Malfunctions caused by mutations in genes that encode either PDE6 or its putative chaperone, aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), lead to severe blinding retinal diseases.Mutations in the PDE6A, PDE6B, and PDE6G genes, which encode the catalytic PDE6AB subunits and P␥ of rod PDE6, respectively, are responsible for a significant proportion of cases of recessive retinitis pigmentosa (3-5) and can also lead to autosomal dominant congenital stationary night blindness (6). Mutations in their cone counterparts, PDE6C and PDE6H, cause autosomal recessive achromatopsia (7-10).PDE6 deficiency also appears to underlie one of the most severe forms of Leber congenital amaurosis (LCA type 4), a condition caused by mutations in the AIPL1 gene (11,12). LCA is an early onset inherited retinopathy and one of the main causes of blindness in children (13). The link between PDE6 and AIPL1 was discovered in studies of AIPL1 knock-out and knockdown mouse models, which revealed rapid severe retinal degeneration and a marked reduction in PDE6 protein levels and activity prior to the loss of photoreceptor cells (14, 15). Thus, the animal models recapitulated the hallmarks of LCA and suggested that AIPL1 is a potential chaperone of PDE6. This notion is consistent with the fact that AIPL1 contains an FK506-binding pro...

Inherited retinal degenerations are a common cause of untreatable blindness worldwide, with retinitis pigmentosa and cone dystrophy affecting approximately 1 in 3500 and 1 in 10,000 individuals, respectively. A major limitation to the development of effective therapies is the lack of availability of animal models that fully replicate the human condition. Particularly for cone disorders, rodent, canine, and feline models with no true macula have substantive limitations. By contrast, the cone-rich macula of a nonhuman primate (NHP) closely mirrors that of the human retina. Consequently, well-defined NHP models of heritable retinal diseases, particularly cone disorders that are predictive of human conditions, are necessary to more efficiently advance new therapies for patients. We have identified 4 related NHPs at the California National Primate Research Center with visual impairment and findings from clinical ophthalmic examination, advanced retinal imaging, and electrophysiology consistent with achromatopsia. Genetic sequencing confirmed a homozygous R565Q missense mutation in the catalytic domain of PDE6C, a cone-specific phototransduction enzyme associated with achromatopsia in humans. Biochemical studies demonstrate that the mutant mRNA is translated into a stable protein that displays normal cellular localization but is unable to hydrolyze cyclic GMP (cGMP). This NHP model of a cone disorder will not only serve as a therapeutic testing ground for achromatopsia gene replacement, but also for optimization of gene editing in the macula and of cone cell replacement in general.

Phosphodiesterase-6 (PDE6) is the key effector enzyme of the phototransduction cascade in rods and cones. The catalytic core of rod PDE6 is a unique heterodimer of PDE6A and PDE6B catalytic subunits. The functional significance of rod PDE6 heterodimerization and conserved differences between PDE6AB and cone PDE6C and the individual properties of PDE6A and PDE6B are unknown. To address these outstanding questions, we expressed chimeric homodimeric enzymes, enhanced GFP (EGFP)-PDE6C-A and EGFP-PDE6C-B, containing the PDE6A and PDE6B catalytic domains, respectively, in transgenic Xenopus laevis. Similar to EGFP-PDE6C, EGFP-PDE6C-A and EGFP-PDE6C-B were targeted to the rod outer segments and concentrated at the disc rims. PDE6C, PDE6C-A, and PDE6C-B were isolated following selective immunoprecipitation of the EGFP fusion proteins. All three enzymes, PDE6C, PDE6C-A, and PDE6C-B, hydrolyzed cGMP with similar K m (20 -23 M) and k cat (4200 -5100 s ؊1 ) values. Likewise, the K i values for PDE6C, PDE6C-A, and PDE6C-B inhibition by the cone-and rod-specific PDE6 ␥-subunits (P␥) were comparable. Recombinant cone transducin-␣ (G␣ t2 ) and native rod G␣ t1 fully and potently activated PDE6C, PDE6C-A, and PDE6C-B. In contrast, the half-maximal activation of bovine rod PDE6 required markedly higher concentrations of G␣ t2 or G␣ t1 . Our results suggest that PDE6A and PDE6B are enzymatically equivalent. Furthermore, PDE6A and PDE6B are similar to PDE6C with respect to catalytic properties and the interaction with P␥ but differ in the interaction with transducin. This study significantly limits the range of mechanisms by which conserved differences between PDE6A, PDE6B, and PDE6C may contribute to remarkable differences in rod and cone physiology.Vertebrates rely on two types of photoreceptor cells, rods and cones, for vision. The phototransduction cascades in rods and cones are principally similar. The central components of the rod and cone signaling pathways, visual pigments, transducins (G t ), and retinal cGMP-phosphodiesterases (PDE6) 2 are distinct but highly homologous proteins (1-3). In contrast, the physiology of rods and cones is strikingly different. Rods are exceptionally sensitive to light and provide for nighttime (scotopic) vision, whereas cones are markedly less sensitive and signal during daytime (photopic receptors). Cone electrical responses to light are smaller in amplitude and much faster than rod responses. Furthermore, cones adapt to a much broader range of illumination conditions than rods and can function in intensely bright light (1-3). The molecular origin(s) of the differences in physiology of rods and cones is one of the key unresolved questions of vertebrate phototransduction (3). The physiological differences may be due to sequence and concentration differences between signaling proteins in rods and cones, as well as to characteristic photoreceptor morphologies of rods and cones (3, 4).Sequence differences in rod and cone transduction components are limited, but well conserved, among vertebrate sp...

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