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.
Background: Mutations in AIPL1, a chaperone of the lipidated visual effector phosphodiesterase-6, cause severe childhood blindness. Results: AIPL1 binds the farnesyl lipid moiety. The unique insert region of AIPL1 is critical for this interaction. Conclusion: The AIPL1-farnesyl interaction suggests its role in the interaction with phosphodiesterase-6 and normal function of AIPL1. Significance: This study describes a novel mechanism of AIPL1 in retina disease.
The retinal degeneration model rd10 contains a missense mutation of the catalytic PDE6 β subunit, which hydrolyzes cGMP in response to light. This model produces cell death more slowly than others caused by PDE6 loss of function, making it of particular interest for studying potential therapeutics. We used morphology, biochemistry, and single-cell physiology to examine the mechanism of rd10 degeneration. Our results show that the mutation produces no alteration of RNA but does dramatically decrease maximal and basal PDE6 activity, apparently caused by a decrease in protein stability and transport. The enzymatic properties of the remaining mutant PDE6 appear to be nearly normal. We demonstrate that an increase in free cGMP, which would result from decreased PDE6 activity and serve to increase opening of the cGMP-gated channels and calcium influx, is an underlying cause of cell death: degeneration of rd10/ double mutants is slower than the parent rd10 line. Paradoxically, degeneration in rd10/ is also slower than in This rescue is correlated with a lowering of cGMP content in retinas and suggests that it may be caused by mislocalization of active PDE6. Single-cell recordings from rd10 rods show that the rates of rise and decay of the response are significantly slower; simulations indicate that these changes are primarily the result of the decrease in PDE6 concentration and rod collecting area. Together, these results provide insights into the complex mechanisms that underlie rd10-mediated retinal degeneration and a cautionary note for analysis of therapeutic interventions.
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
Mutations in the primate-specific proline-rich domain (PRD) of aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) are thought to cause Leber congenital amaurosis or dominant conerod dystrophy. The role of PRD and the mechanisms of PRD mutations are poorly understood. Here, we have examined properties of hAIPL1 and effects of the PRD mutations on protein structure and function. Solution structures of hAIPL1, hAIPL1 1-316 with PRD truncation, and the P351D12 and P376S mutants were examined by small angle X-ray scattering. Our analysis suggests that PRD assumes an extended conformation and does not interact with the FK506-binding and tetratricopeptide domains. The PRD truncation, but not PRD mutations, reduced the molecule's radius of gyration and maximum dimension. We demonstrate that hAIPL1 is a monomeric protein, and its secondary structure and stability are not affected by the PRD mutations. PRD itself is an extended monomeric random coil. The PRD mutations caused little or no changes in hAIPL1 binding to known partners, phosphodiesterase-6A and HSP90. We also identified the c-subunit of phosphodiesterase-6 as a novel partner of hAIPL1 and hypothesize that this interaction is altered by P351D12. Our results highlight the complexity of mechanisms of PRD mutations in disease and the possibility that certain mutations are benign variants. Keywords: AIPL1, HSP90, phosphodiesterase-6, photoreceptor, retina, SAXS. Uniquely, AIPL1 proteins in primates contain a third proline-rich region (PRD) located C-terminally to the TPR domain (Fig. 1a). Mutations associated with LCA are found in all three domains of AIPL1 (Sohocki et al. 2000b;Dharmaraj et al. 2004;Stone 2007;Tan et al. 2012). The key role of AIPL1 in rods and cones is its function as a specialized chaperone for cGMP-specific phosphodiesterase-6 (PDE6), the effector enzyme in the phototransduction cascade (Liu et al. 2004;Ramamurthy et al. 2004). AIPL1-knockout in mice revealed markedly reduced stability and activity of PDE6, which was followed by rapid retina Address correspondence and reprint requests to Nikolai O. Artemyev, Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA. E-mail: nikolai-artemyev@uiowa.eduAbbreviations used: AIPL1, aryl hydrocarbon receptor-interacting protein-like 1; AMCA, 6-((7-amino-4-methylcoumarin-3-acetyl)amino) hexanoic acid, succinimidyl ester; BC, 3-(bromo acetyl)-7-diethyl aminocoumarine; CD, circular dichroism; DLS, dynamic light scattering; FKBP, FK506-binding proteins; FRET, fluorescence resonance energy transfer; LCA, leber congenital amaurosis; PDE6AB, catalytic subunits of rod PDE6; PDE6, photoreceptor phosphodiesterase-6; PRD, prolinerich domain of human AIPL1; Pc, the inhibitory c-subunit of PDE6; SAXS, small angle X-ray scattering; SLS, static light scattering; TPR, tetratricopeptide repeat.
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