cyclic-gmp and Macular-Degeneration

Macular dystrophy leads to progressive loss of central vision and shows symptoms similar to age-related macular degeneration. Genetic screening of patients diagnosed with macular dystrophy disclosed a novel mutation in the GUCA1A gene, namely a c.526C > T substitution leading to the amino acid substitution p.L176F in the guanylate cyclase-activating protein 1 (GCAP1). The same variant was found in three families showing an autosomal dominant mode of inheritance. For a full functional characterization of the L176F mutant we expressed and purified the mutant protein and measured key parameters of its activating properties, its Ca2+/Mg2+-binding, and its Ca2+-induced conformational changes in comparison to the wildtype protein. The mutant was less sensitive to changes in free Ca2+, resulting in a constitutively active form under physiological Ca2+-concentration, showed significantly higher activation rates than the wildtype (90-fold versus 20-fold) and interacted with an higher apparent affinity with its target guanylate cyclase. However, direct Ca2+-binding of the mutant was nearly similar to the wildtype; binding of Mg2+ occurred with higher affinity. We performed molecular dynamics simulations for comparing the Ca2+-saturated inhibiting state of GCAP1 with the Mg2+-bound activating states. The L176F mutant exhibited significantly lower flexibility, when three Ca2+ or two Mg2+ were bound forming probably the structural basis for the modified GCAP1 function.

Topics: Adolescent; Adult; Calcium; Cyclic GMP; Female; Guanylate Cyclase-Activating Proteins; Humans; Macular Degeneration; Male; Middle Aged; Molecular Dynamics Simulation; Mutation; Pedigree; Protein Conformation; Retinal Cone Photoreceptor Cells; Young Adult

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

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