cyclic-gmp and Color-Vision-Defects

Humans with mutations in the phototransduction pathway develop forms of retinal degeneration, such as retinitis pigmentosa, cone dystrophy, or Leber congenital amaurosis. Similarly, numerous phototransduction mutant animal models resemble retinal degeneration. In our lab, using a zebrafish model, we study cone-specific phototransduction mutants. cGMP is the second messenger in the phototransduction pathway, and abnormal cGMP levels are associated with photoreceptor death. Rd1, a rod-specific phosphodiesterase 6 (Pde6) subunit mutant in mice, is one of the most widely used animal models for retinal degeneration. Rd1 mutant mice accumulate cGMP, causing rapid photoreceptor degeneration. However, much less is known about photoreceptor mutants producing abnormally low levels of cGMP. Here, focusing on Pde6 mutants in zebrafish and mice, we propose a correlation between cGMP levels and speed of photoreceptor degeneration.

Topics: Animals; Color Vision Defects; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Eye Proteins; Forecasting; Humans; Light Signal Transduction; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Zebrafish; Zebrafish Proteins

Topics: Animals; Color Vision Defects; Cyclic GMP; Dependovirus; Genetic Therapy; Genetic Vectors; HeLa Cells; Humans; Retina

Mutations in the

Topics: Animals; Biomarkers; Color Vision Defects; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type II; Cyclic Nucleotide-Gated Cation Channels; Disease Models, Animal; Disease Susceptibility; Endoplasmic Reticulum Stress; Fluorescent Antibody Technique; Gene Expression; Mice; Mice, Knockout; Microscopy, Confocal; Models, Biological; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Unfolded Protein Response

Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3',5'-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h(-/-)) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h(-/-) retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h(-/-) mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Color Vision Defects; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Electroretinography; Gene Deletion; Gene Expression; Humans; Light Signal Transduction; Mice; Mice, Knockout; Protein Isoforms; Protein Subunits; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Signal Transduction; Species Specificity

To determine if achromatopsia associated F525N and T383fsX mutations in the CNGB3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels increases susceptibility to cell death in photoreceptor-derived cells.. Photoreceptor-derived 661W cells were transfected with cDNA encoding wild-type (WT) CNGA3 subunits plus WT or mutant CNGB3 subunits, and incubated with the membrane-permeable CNG channel activators 8-(4-chlorophenylthio) guanosine 3',5'-cyclic monophosphate (CPT-cGMP) or CPT-adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Cell viability under these conditions was determined by measuring lactate dehydrogenase release. Channel ligand sensitivity was calibrated by patch-clamp recording after expression of WT or mutant channels in Xenopus oocytes.. Coexpression of CNGA3 with CNGB3 subunits containing F525N or T383fsX mutations produced channels exhibiting increased apparent affinity for CPT-cGMP compared to WT channels. Consistent with these effects, cytotoxicity in the presence of 0.1 μM CPT-cGMP was enhanced relative to WT channels, and the increase in cell death was more pronounced for the mutation with the largest gain-of-function effect on channel gating, F525N. Increased susceptibility to cell death was prevented by application of the CNG channel blocker L-cis-diltiazem. Increased cytotoxicity was also found to be dependent on the presence of extracellular calcium.. These results indicate a connection between disease-associated mutations in cone CNG channel subunits, altered CNG channel-activation properties, and photoreceptor cytotoxicity. The rescue of cell viability via CNG channel block or removal of extracellular calcium suggests that cytotoxicity in this model depends on calcium entry through hyperactive CNG channels.

Topics: Animals; Annexin A5; Calcium; Cell Death; Cell Survival; Color Vision Defects; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Diltiazem; Extracellular Space; Ion Channel Gating; Ligands; Mutation; Photoreceptor Cells, Vertebrate; Protective Agents; Xenopus

The CNGA3 gene encodes the A3 subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel, an essential component of the phototransduction cascade. Certain mutations in CNGA3 cause autosomal recessive achromatopsia, a retinal disorder characterized by severely reduced visual acuity, lack of color discrimination, photophobia, and nystagmus. We identified three novel mutations in the pore-forming region of CNGA3 (L363P, G367V, and E376K) in patients diagnosed with achromatopsia. We assessed the expression and function of channels with these three new and two previously described mutations (S341P and P372S) in a heterologous HEK293 cell expression system using Western blot, subcellular localization on the basis of immunocytochemistry, calcium imaging, and patch clamp recordings. In this first comparative functional analysis of disease-associated mutations in the pore of a CNG channel, we found impaired surface expression of S341P, L363P, and P372S mutants and reduced macroscopic currents for channels with the mutations S341P, G367V, and E376K. Calcium imaging and patch clamp experiments after incubation at 37 degrees C revealed nonfunctional homo- and heteromeric channels in all five mutants, but incubation at 27 degrees C combined with coexpression of the B3 subunit restored residual function of channels with the mutations S341P, G367V, and E376K.

Topics: Amino Acid Sequence; Biological Transport; Blotting, Western; Calcium; Cell Line; Color Vision Defects; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Dose-Response Relationship, Drug; Humans; Immunohistochemistry; Membrane Potentials; Models, Molecular; Molecular Sequence Data; Mutant Proteins; Mutation; Protein Conformation; Protein Multimerization; Retinal Cone Photoreceptor Cells; Sequence Homology, Amino Acid; Transfection

CNGA3 encodes the A-subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel, which is a crucial component of the phototransduction cascade in cone outer segments. Mutations in the CNGA3 gene have been associated with complete and incomplete forms of achromatopsia (ACHR), a congenital, autosomal recessively inherited retinal disorder characterized by lack of color discrimination, reduced visual acuity, nystagmus, and photophobia. Here we report the identification of three novel CNGA3 missense mutations in ACHR patients: c.682G>A (p.E228 K), c.1315C>T (p.R439W), and c.1405G>A (p.A469 T), and the detailed functional analyses of these new as well as five previously reported mutations (R283Q, T291R, F547L, G557R, and E590 K), in conjunction with clinical data of patients carrying these mutations, to establish genotype-phenotype correlations. The functional characterization of mutant CNGA3 channels was performed with calcium imaging and patch clamp recordings in a heterologous HEK293 cell expression system. Results were corroborated by immunostaining and colocalization experiments of the channel protein with the plasma membrane. Several mutations evoked pronounced alterations of the apparent cGMP sensitivity of mutant channels. These functional defects were fully or partially compensated by coexpressing the mutant CNGA3 subunit with the wild-type CNGB3 subunit for channels with the mutations R439W, A469 T, F547L, and E590 K. We could show that several mutant channels with agonist dose-response relationships similar to the wild-type exhibited severely impaired membrane targeting. In addition, this study presents the positive effect of reduced cell culture temperature on surface expression and functional performance of mutant CNG channels with protein folding or trafficking defects.

Topics: Color Vision Defects; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Humans; Immunohistochemistry; Models, Biological; Mutation; Patch-Clamp Techniques; Protein Transport

Thirty-nine missense mutations, which had been identified in rod monochromacy or related disorders, in the CNGA3 subunit of cone photoreceptor cGMP-gated channels were analyzed. HEK293 cells were transfected with cDNA of the human CNGA3 subunit harboring each of these mutations in an expression vector. Patch-clamp recordings demonstrated that 32 of the 39 mutants did not show cGMP-activated current, suggesting that these 32 mutations cause a loss of function of the channels. From the remaining 7 mutants that showed cGMP-activated current, two mutations in the cyclic nucleotide-binding domain, T565M or E593K, were further studied. The half-maximal activating concentration (K(1/2)) for cGMP in the homomeric CNGA3-T565M channels (160microM) was 17.8-fold higher than that of the homomeric wild-type CNGA3 channels (9.0microM). Conversely, the K(1/2) for cGMP in the homomeric CNGA3-E593K channels (3.0microM) was 3-fold lower than that of the homomeric wild-type CNGA3 channels. These results suggest that the T565M and E593K mutations alter the apparent affinity for cGMP of the channels to cause cone dysfunction, resulting in rod monochromacy.

Topics: Cell Line; Cloning, Molecular; Color Vision Defects; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Dimerization; Dose-Response Relationship, Drug; Humans; Ion Channels; Kinetics; Mutation; Mutation, Missense; Patch-Clamp Techniques; Photoreceptor Cells; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells

Achromatopsia 2, an inherited retinal disorder resulting in attenuation or loss of cone function, is caused by mutations in the alpha subunit of the cone cyclic nucleotide-gated (CNG) channel gene CNGA3. Examination of mutations that cluster in the first transmembrane segment of the protein may provide insight into its role in CNG channel structure, function, biogenesis, and pathophysiology.. The human CNGA3 gene was tagged at the C terminus with green fluorescent protein. Four mutations, Y181C, N182Y, L186F, and C191Y, were expressed in human embryonic kidney cells. Protein expression was evaluated with immunoblot analysis and cellular localization was determined by immunocytochemistry. Channel function was evaluated by patch-clamp electrophysiology.. All the mutations result in loss of channel function, as determined by the failure of cGMP to activate wild-type currents in excised patches. Full-length mutant proteins were synthesized but retained in the endoplasmic reticulum. Glycerol treatment did not rescue channel function nor did coexpression with CNGB3, a subunit of native hetero-tetrameric cone channels. A control mutant, C191S, exhibited cGMP current activation with significantly reduced cooperativity, suggesting that mutations in the first transmembrane domain alter in inter- or intrasubunit communication.. The results implicate the first transmembrane segment in both maturation and function of CNG channels. The defects are not reversed with glycerol, a chemical chaperone that rescues channel function in some channelopathies. Molecular analysis of achromatopsia 2 mutations may be useful in evaluating potential therapeutic approaches for treatment of this channelopathy.

Topics: Amino Acid Sequence; Color Vision Defects; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Eye Proteins; Fluorescent Antibody Technique, Indirect; Green Fluorescent Proteins; Humans; Immunoblotting; Ion Channels; Kidney; Molecular Sequence Data; Mutation; Patch-Clamp Techniques; Recombinant Fusion Proteins; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Transfection

To characterize the functional consequences of disease-associated mutations in the CNGB3 (B3) subunit of human cone photoreceptor cyclic nucleotide-gated channels in order to gain insight into disease mechanisms.. Three separate disease-associated mutations were generated in CNGB3: F525N, R403Q, and T383fsX. These mutant subunits were then heterologously expressed in Xenopus oocytes in combination with wild type CNGA3 (A3) subunits, and characterized by patch-clamp recording in the inside-out configuration.. Co-expression of A3 and B3F525N, A3 and B3R403Q, or A3 and B3R403Q and B3T383fsX subunits resulted in channels that exhibited an increase in ligand sensitivity without a reduction in current density compared to wild-type heteromeric channels. Each simulated disease state produced channels that exhibited greater sensitivity to block by L-cis-diltiazem than homomeric CNGA3 channels, confirming that the mutant CNGB3 subunits were competent to form functional heteromeric channels. Each combination of subunits displayed an increase in apparent affinity for cGMP relative to wild-type heteromeric channels. However, F525N enhanced cGMP apparent affinity to a significantly greater extent than the other two modeled disease states.. We have examined the gating effects of two previously uncharacterized disease-associated mutations in the CNGB3 subunit and found that in each case, the mutations resulted in a gain of function molecular phenotype. Furthermore, the magnitude of the effect on channel function correlated with the severity of the associated disease. The complete achromatopsia-associated F525N mutation resulted in more pronounced alterations in channel function than the mutation combinations linked to macular degeneration or progressive cone dystrophy.

Topics: Animals; Color Vision Defects; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Eye Proteins; Female; Gene Expression; Humans; Ion Channels; Macular Degeneration; Oocytes; Patch-Clamp Techniques; Point Mutation; Polymerase Chain Reaction; Retinal Cone Photoreceptor Cells; RNA, Messenger; Xenopus laevis

Cone photoreceptor cyclic nucleotide-gated (CNG) channels are thought to form by assembly of two different subunit types, CNGA3 and CNGB3. Recently, mutations in the gene encoding the CNGB3 subunit have been linked to achromatopsia in humans. Here we describe the functional consequences of two achromatopsia-associated mutations in human CNGB3 (hCNGB3). Co-expression in Xenopus oocytes of human CNGA3 (hCNGA3) subunits with hCNGB3 subunits containing an achromatopsia-associated mutation in the S6 transmembrane domain (S435F) generated functional heteromeric channels that exhibited an increase in apparent affinity for both cAMP and cGMP compared with wild type heteromeric channels. In contrast, co-expression of a presumptive null mutation of hCNGB3 (T383f.s.Delta C) with hCNGA3 produced channels with properties indistinguishable from homomeric hCNGA3 channels. The effect of hCNGB3 S435F subunits on cell-surface expression of green fluorescent protein-tagged hCNGA3 subunits and of non-tagged hCNGA3 subunits on surface expression of green fluorescent protein-hCNGB3 S435F subunits were similar to those observed for wild type hCNGB3 subunits, suggesting that the mutation does not grossly disturb subunit assembly or plasma membrane targeting. The S435F mutation was also found to produce changes in the pore properties of the channel, including decreased single channel conductance and decreased sensitivity to block by l-cis-diltiazem. Overall, these results suggest that the functional properties of cone CNG channels may be altered in patients with the S435F mutation, providing evidence supporting the pathogenicity of this mutation in humans. Thus, achromatopsia may arise from a disturbance of cone CNG channel gating and permeation or from the absence of functional CNGB3 subunits.

Topics: Amino Acid Sequence; Animals; Calcium Channel Blockers; Cell Membrane; Color Vision Defects; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Diltiazem; Dimerization; Dose-Response Relationship, Drug; Electrophysiology; Green Fluorescent Proteins; Humans; Ion Channels; Kinetics; Ligands; Luminescent Proteins; Microscopy, Confocal; Models, Biological; Molecular Sequence Data; Mutation; Oocytes; Photoreceptor Cells; Potassium; Protein Structure, Tertiary; Recombinant Fusion Proteins; Recombinant Proteins; Retinal Cone Photoreceptor Cells; RNA, Messenger; Sequence Homology, Amino Acid; Xenopus laevis

To describe the clinical phenotype, with emphasis on the electrophysiological findings, of patients with autosomal recessive rod monochromacy (RM) and defined mutations in the CNGA3/CNGB3 genes.. RM patients from eight different families were included in the study. Their genotypes were determined by DNA sequencing and/or RFLP analysis of PCR-amplified genomic segments of the CNGA3 and CNGB3 genes. For comparison, we investigated one patient with blue-cone monochromacy (BCM). The clinical examination included best-corrected visual acuity, fundus examination, and full-field ERG. In six patients, the examination was complemented by multifocal ERG (MERG).. Three patients had three different CNG3A genotypes. Five patients were homozygous and one patient compound heterozygous for a 1-bp deletion (1148delC) in the CNGB3 gene. All patients examined presented with a visual acuity of 0.1-0.15. Small residual cone responses were noted in four young RM patients. The oldest patient examined (age 47 years) presented with pigmentary changes in the mid-peripheral retina and concentric constrictions of the visual fields.. Patients with RM and mutations in the CNGA3/CNGB3 genes presented a similar clinical phenotype, confirming the essential function of both the alpha- and beta-subunits of the cGMP-gated cation channel in cone photoreceptor function. Small remaining cone responses in a few of the younger patients and mid-peripheral pigmentary degenerations in the oldest patient examined indicate that there could be some degree of progression in retinal dysfunction in at least some patients with RM.

Topics: Adolescent; Adult; Child; Child, Preschool; Color Vision Defects; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Electroretinography; Female; Fluorescein Angiography; Genotype; Humans; Ion Channels; Male; Middle Aged; Mutation; Pedigree; Phenotype; Photoreceptor Cells; Photoreceptor Cells, Vertebrate; Sweden

Total colourblindness (OMIM 216900), also referred to as rod monochromacy (RM) or complete achromatopsia, is a rare, autosomal recessive inherited and congenital disorder characterized by photophobia, reduced visual acuity, nystagmus and the complete inability to discriminate between colours. Electroretinographic recordings show that in RM, rod photoreceptor function is normal, whereas cone photoreceptor responses are absent. The locus for RM has been mapped to chromosome 2q11 (ref. 2), however the gene underlying RM has not yet been identified. Recently, a suitable candidate gene, CNGA3, encoding the alpha-subunit of the cone photoreceptor cGMP-gated cation channel, a key component of the phototransduction pathway, has been cloned and assigned to human chromosome 2q11 (refs 3,4). We report the identification of missense mutations in CNGA3 in five families with RM. Homozygous mutations are present in two families, whereas the remaining families show compound heterozygous mutations. In all cases, the segregation pattern of the mutations is consistent with the autosomal recessive inheritance of the disease and all mutations affect amino acids that are highly conserved among cyclic nucleotide gated channels (CNG) in various species. This is the first report of a colour vision disorder caused by defects other than mutations in the cone pigment genes, and implies at least in this instance a common genetic basis for phototransduction in the three different cone photoreceptors of the human retina.

Topics: Base Sequence; Color Vision Defects; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; DNA, Complementary; Female; Humans; Ion Channel Gating; Ion Channels; Male; Molecular Sequence Data; Mutation; Pedigree; Retinal Cone Photoreceptor Cells

GCAP1 stimulates photoreceptor guanylate cyclase (GC) in bleached vertebrate photoreceptors when [Ca2+]free decreases but is inactivated when cytoplasmic [Ca2+]free increase after dark adaptation. A Y99C mutation in GCAP1 has recently been found to be associated with autosomal dominant cone dystrophy. We show that the GCAP1(Y99C) mutant and native GCAP1 are highly effective in stimulation of photoreceptor GC1. The Ca2+ sensitivity of the mutant GCAP1, however, is markedly altered, causing reduced but persistent stimulation of GC1 under physiological dark conditions. These results are consistent with a model in which enhanced GC activity in dark-adapted cones leads to elevated levels of cytoplasmic cGMP. Alterations in physiological cGMP levels are also associated with other retinal degenerations, including Leber's congenital amaurosis.

Topics: Adaptation, Physiological; Animals; Calcium; Calcium-Binding Proteins; Cattle; Cell Line; Cloning, Molecular; Color Vision Defects; Cyclic GMP; Darkness; Enzyme Activation; Eye Diseases, Hereditary; Eye Proteins; Genes, Dominant; Guanylate Cyclase; Guanylate Cyclase-Activating Proteins; Humans; Macular Degeneration; Moths; Mutagenesis, Site-Directed; Optic Atrophies, Hereditary; Protein Conformation; Recombinant Fusion Proteins; Retinal Cone Photoreceptor Cells; Signal Transduction; Structure-Activity Relationship