Functional analysis of missense mutations in Kv8.2 causing cone dystrophy with supernormal rod electroretinogram.

Mutations in KCNV2 have been proposed as the molecular basis for cone dystrophy with supernormal rod electroretinogram. KCNV2 codes for the modulatory voltage-gated potassium channel α-subunit, Kv8.2, which is incapable of forming functional channels on its own. Functional heteromeric channels are however formed with Kv2.1 in heterologous expression systems, with both α-subunit genes expressed in rod and cone photoreceptors. Of the 30 mutations identified in the KCNV2 gene, we have selected three missense mutations localized in the potassium channel pore and two missense mutations localized in the tetramerization domain for analysis. We characterized the differences between homomeric Kv2.1 and heteromeric Kv2.1/Kv8.2 channels and investigated the influence of the selected mutations on the function of heteromeric channels. We found that two pore mutations (W467G and G478R) led to the formation of nonconducting heteromeric Kv2.1/Kv8.2 channels, whereas the mutations localized in the tetramerization domain prevented heteromer generation and resulted in the formation of homomeric Kv2.1 channels only. Consequently, our study suggests the existence of two distinct molecular mechanisms involved in the disease pathology.

Anion sensitivity and spectral tuning of middle- and long-wavelength-sensitive (MWS/LWS) visual pigments.

The long-wavelength-sensitive (LWS) opsins form one of four classes of vertebrate cone visual pigment and exhibit peak spectral sensitivities (λ(max)) that generally range from 525 to 560 nm for rhodopsin/vitamin-A(1) photopigments. Unique amongst the opsin classes, many LWS pigments show anion sensitivity through the interaction of chloride ions with a histidine residue at site 197 (H197) to give a long-wavelength spectral shift in peak sensitivity. Although it has been shown that amino acid substitutions at five sites (180, 197, 277, 285 and 308) are useful in predicting the λ(max) values of the LWS pigment class, some species, such as the elephant shark and most marine mammals, express LWS opsins that possess λ(max) values that are not consistent with this 'five-site' rule, indicating that other interactions may be involved. This study has taken advantage of the natural mutation at the chloride-binding site in the mouse LWS pigment. Through the use of a number of mutant pigments generated by site-directed mutagenesis, a new model has been formulated that takes into account the role of charge and steric properties of the side chains of residues at sites 197 and 308 in the function of the chloride-binding site in determining the peak sensitivity of LWS photopigments.

Disease mechanism for retinitis pigmentosa (RP11) caused by missense mutations in the splicing factor gene PRPF31.

PURPOSE: Missense mutations in the splicing factor gene PRPF31 cause a dominant form of retinitis pigmentosa (RP11) with reduced penetrance. Missense mutations in PRPF31 have previously been shown to cause reduced protein solubility, suggesting insufficiency of functional protein as the disease mechanism. Here we examine in further detail the effect of the A216P mutation on splicing function. METHODS: Splicing activity was assayed using an in vivo assay in transfected mammalian cells with rhodopsin (RHO) and transducin (GNAT1) splicing templates. Pull-down assays were used to study the interaction between PRPF31 and one of its cognate partners in the spliceosome, PRPF6. RESULTS: Splicing of RHO intron 3 and GNAT1 introns 3-5 mini-gene templates was inefficient with both spliced and unspliced products clearly detected. Assays using the RHO minigene template revealed a direct negative effect on splicing efficiency of the mutant. However, no effect of the mutation on splicing efficiency could be detected using the longer GNAT1 minigene template or using a full-length RHO transcript, splicing of which had an efficiency of 100%. No unspliced RHO transcripts could be detected in RNA from human retina. Pull-down assays between PRPF31 and PRPF6 proteins showed a stronger interaction for the mutant than wild type, suggesting a mechanism for the negative effect. CONCLUSIONS: Splicing of full-length RHO is more efficient than splicing of the minigene, and assays using a full-length template more accurately mimic splicing in photoreceptors. The RP11 missense mutations exert their pathology mainly via a mechanism based on protein insufficiency due to protein insolubility, but there is also a minor direct negative effect on function.

Genetic enhancement of cognition in a kindred with cone-rod dystrophy due to RIMS1 mutation.

BACKGROUND: The genetic basis of variation in human cognitive abilities is poorly understood. RIMS1 encodes a synapse active-zone protein with important roles in the maintenance of normal synaptic function: mice lacking this protein have greatly reduced learning ability and memory function. OBJECTIVE: An established paradigm examining the structural and functional effects of mutations in genes expressed in the eye and the brain was used to study a kindred with an inherited retinal dystrophy due to RIMS1 mutation. MATERIALS AND METHODS: Neuropsychological tests and high-resolution MRI brain scanning were undertaken in the kindred. In a population cohort, neuropsychological scores were associated with common variation in RIMS1. Additionally, RIMS1 was sequenced in top-scoring individuals. Evolution of RIMS1 was assessed, and its expression in developing human brain was studied. RESULTS: Affected individuals showed significantly enhanced cognitive abilities across a range of domains. Analysis suggests that factors other than RIMS1 mutation were unlikely to explain enhanced cognition. No association with common variation and verbal IQ was found in the population cohort, and no other mutations in RIMS1 were detected in the highest scoring individuals from this cohort. RIMS1 protein is expressed in developing human brain, but RIMS1 does not seem to have been subjected to accelerated evolution in man. CONCLUSIONS: A possible role for RIMS1 in the enhancement of cognitive function at least in this kindred is suggested. Although further work is clearly required to explore these findings before a role for RIMS1 in human cognition can be formally accepted, the findings suggest that genetic mutation may enhance human cognition in some cases.

A study of the nuclear trafficking of the splicing factor protein PRPF31 linked to autosomal dominant retinitis pigmentosa (ADRP).

In this study the mechanism of nuclear importation of the splicing factor PRPF31 is examined and the impact of two disease-linked mutations, A194E and A216P, assessed. Using pull-down assays with GST-tagged importin proteins, we demonstrate that His-tagged PRPF31 interacts with importin beta1 for translocation to the nucleus, with no requirement for importin alpha1. The A194E and A216P mutations have no affect on this interaction. Fluorescence recovery after photobleaching (FRAP) was used to estimate the rate of movement of EGFP-tagged PRPF31 into the nuclei of live cells. The kinetics indicated a two-component recovery process; a fast component with tau approximately 6 s and a slow component with tau approximately 80 s. The mutations affected neither component. We conclude that the two mutations have no negative effect on interaction with the nuclear importation machinery. Reduced mutant protein solubility resulting in an insufficiency of splicing activity in cells with a very high metabolic demand remains the most likely explanation for the disease pathology in ADRP patients.

Spectral tuning of shortwave-sensitive visual pigments in vertebrates.

Of the four classes of vertebrate cone visual pigments, the shortwave-sensitive SWS1 class shows some of the largest shifts in lambda(max), with values ranging in different species from 390-435 nm in the violet region of the spectrum to < 360 nm in the ultraviolet. Phylogenetic evidence indicates that the ancestral pigment most probably had a lambda(max) in the UV and that shifts between violet and UV have occurred many times during evolution. In violet-sensitive (VS) pigments, the Schiff base is protonated whereas in UV-sensitive (UVS) pigments, it is almost certainly unprotonated. The generation of VS pigments in amphibia, birds and mammals from ancestral UVS pigments must involve therefore the stabilization of protonation. Similarly, stabilization must be lost in the evolution of avian UVS pigments from a VS ancestral pigment. The key residues in the opsin protein for these shifts are at sites 86 and 90, both adjacent to the Schiff base and the counterion at Glu113. In this review, the various molecular mechanisms for the UV and violet shifts in the different vertebrate groups are presented and the changes in the opsin protein that are responsible for the spectral shifts are discussed in the context of the structural model of bovine rhodopsin.

Purification, characterisation and intracellular localisation of aryl hydrocarbon interacting protein-like 1 (AIPL1) and effects of mutations associated with inherited retinal dystrophies.

Mutations in AIPL1 are associated with Leber Congenital Amaurosis (LCA), a major cause of childhood blindness, yet the cellular function of the encoded protein has yet to be fully elucidated. In order to investigate the biochemistry of AIPL1, we have developed a system for the expression of the recombinant protein in bacteria and its subsequent purification. The secondary structure and thermostability of wild-type and mutant proteins have been examined by circular dichroism (CD) spectroscopy. Some of the variants, notably W278X and P376S, had markedly different secondary structure compositions, indicating that the proteins had not folded properly, whilst W278X and T114I were particularly thermolabile. When eukaryotic cells were transfected with the AIPL1 expression constructs, we show by immunofluorescence microscopy that wild-type protein is distributed throughout the nucleus and cytoplasm. Several of the mutants give similar results. With two of the disease-associated variants (W278X and A336Delta2), however, the protein remains in the cytoplasm in aggresome-like particles. These particles were shown to be ubiquitinated, indicating that the mutant protein had been tagged for proteosomal degradation. On this basis, we can conclude that wild-type protein is expressed in a soluble and folded manner, and that some of the disease-associated mutant proteins are nonfunctional because they are insoluble and are degraded by the cell. Other mutations appear to have a more localised effect on secondary structure, which does not result in insolubility or affect protein targeting, but reduces the stability of the protein at human body temperature.

Characterisation of two genes for guanylate cyclase activator protein (GCAP1 and GCAP2) in the Japanese pufferfish, Fugu rubripes.

cDNA and genomic clones encoding guanylate cyclase activating proteins (GCAP1 and GCAP2) in the Japanese puffer fish (Fugu rubripes) were identified by probing, respectively, a retinal cDNA library and a whole genomic cosmid library with human GCAP1 and GCAP2 cDNA probes. Clones were identified as GCAP1 and GCAP2 on the basis of amino acid identity with the equivalent frog sequences and their placement into GCAP1 and GCAP2 clades within a GCAP phylogenetic tree. The Fugu genes have an identical four exon/three intron structure to GCAP1 and GCAP2 genes from other vertebrates but the introns are smaller, with the result that the four exons spread over approximately 1 kb of DNA in each case. The two genes are separated on to separate cosmids. However, the results of Southern analysis of the cosmids and of genomic DNA are consistent with a tail-to-tail gene arrangement, as in other species, but with a surprisingly large intergenic separation of around 18.7 kb. Recombinant Fugu GCAP1 failed to activate human retinal guanylate cyclase (retGC) in vitro although CD spectroscopy shows that the protein is folded with a similar secondary structure to that of human GCAP1. The failure to activate may be due therefore to a lack of molecular compatibility in this heterologous assay system.

Mutation in the gene GUCA1A, encoding guanylate cyclase-activating protein 1, causes cone, cone-rod, and macular dystrophy.

PURPOSE: To determine the underlying molecular genetic basis of a retinal dystrophy identified in a 4-generation family and to examine the phenotype and the degree of intrafamilial variability. DESIGN: Prospective case series. PARTICIPANTS: Six affected individuals from a nonconsanguineous British family. METHODS: Detailed ophthalmologic examination, color fundus photography, autofluorescence imaging, and electrophysiologic assessment were performed. Blood samples were taken for DNA extraction, and mutation screening of GUCA1A, the gene encoding guanylate cyclase-activating protein 1 (GCAP1), was undertaken. RESULTS: All affected subjects complained of mild photophobia and reduced central and color vision. Onset was between the third and fifth decade, with subsequent gradual deterioration of visual acuity and color vision. Visual acuity ranged between 6/9 and counting fingers. Color vision was either absent or markedly reduced along all 3 color axes. A range of macular appearances was seen, varying from mild retinal pigment epithelial disturbance to extensive atrophy. Electrophysiologic testing revealed a range of electrophysiologic abnormalities: isolated cone electroretinography abnormalities, reduced cone and rod responses (with cone loss greater than rod), and isolated macular dysfunction. The 4 coding exons of GUCA1A were screened for mutations in affected and unaffected family members. A single transition, A319G, causing a nonconservative missense substitution, Tyr99Cys, segregated uniquely in all affected subjects. CONCLUSIONS: The Tyr99Cys GUCA1A mutation has been previously shown to cause autosomal dominant progressive cone dystrophy. This is the first report of this mutation also causing both cone-rod dystrophy and isolated macular dysfunction. The phenotypic variation described here exemplifies the intrafamilial heterogeneity of retinal dysfunction that can be observed in persons harboring the same mutation and chromosomal segment.

Identification of novel mutations in the carbohydrate sulfotransferase gene (CHST6) causing macular corneal dystrophy.

PURPOSE: Macular corneal dystrophy (MCD) is a rare corneal dystrophy that is characterized by abnormal deposits in the corneal stroma, keratocytes, Descemet's membrane, and endothelium, accompanied by progressive clouding. It has been classified into three immunophenotypes--MCD types I, IA, and II--according to the serum level of sulfated keratan sulfate (KS) and immunoreactivity of the corneal tissue. Recently, mutations in a new carbohydrate sulfotransferase gene (CHST6) encoding corneal glucosamine N-acetyl-6-sulfotransferase (C-GlcNac-6-ST) have been identified as the cause of MCD. Mutation screening of the CHST6 gene has been undertaken to identify the underlying mutations in five unrelated British families with MCD. METHODS: DNA was extracted from venous blood obtained from all participants, and the coding region of CHST6 was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by direct sequencing and restriction enzyme digestion. Enzyme-linked immunosorbent assay (ELISA) was performed to assess the presence of KS in serum from the probands of MCD-affected families participating in the study. RESULTS: Six novel missense mutations--four homozygous and two compound heterozygous--were identified in the CHST6 gene. The ELISA showed that the disease in all patients participating in the study was of MCD type I, including the subtype IA. CONCLUSIONS: These novel mutations are thought to result in loss of corneal sulfotransferase function, which would account for the MCD phenotype.

Dominant cone and cone-rod dystrophies: functional analysis of mutations in retGC1 and GCAP1.

The regulation of cGMP levels is central to the normal process of phototransduction in both cone and rod photoreceptor cells. Two of the proteins involved in this process are the enzyme, retinal guanylate cyclase (retGC), and its activating protein (GCAP) through which activity is regulated via changes in cellular Ca2+ levels. Dominant cone-rod dystrophies arising from changes in retGC1 are essentially restricted to mutations in codon 838 and result in the replacement of a conserved arginine residue with either cysteine, histidine or serine. In all three cases, the effect of the substitution on the in vitro cyclase activity is a loss of Ca2+ sensitivity arising from an increased stability of the coiled-coil domain of the protein dimer and retention of cyclase activity. In contrast, mutations in the Ca2+-coordinating EF hands of GCAP1 result in dominant cone dystrophy; the consequences of these mutations is a reduced ability of the mutant protein to regulate retGC activity in response to changes in Ca2+ levels. Functionally therefore, the retGC2 and GCAP2 mutations are similar in reducing the feedback inhibition of Ca2+ on cyclase activity and thereby on cGMP levels in the photoreceptors.

Guanylate cyclase activating proteins, guanylate cyclase and disease.

A range of cone and cone-rod dystrophies (CORD) have been observed in man, caused by mutations in retinal guanylate cyclase 1 (RetGC1) and guanylate cyclase activating protein 1 (GCAP 1). The CORD causing mutations in RetGC1 are located at a mutation "hot spot" within the dimerisation domain, where R838 is the key residue. Three disease causing mutations have been found in human GCAP1, resulting in cone or cone-rod degeneration. All three mutations are dominant in their effect although the mechanism by which the P50L mutation exerts its influence remains unclear although it might act due to a haplo-insufficiency, arising from increased susceptibility to protease activity and increased thermal instability. In contrast, loss of Ca2+ sensitivity appears to be the main cause of the diseased state for the Y99C and E155G mutations. The cone and cone-rod dystrophies that are caused by mutations in RetGC1 or GCAP1 arise from a perturbation of the delicate balance of Ca2+ and cGMP within the photoreceptor cells and it is this disruption that is believed to cause cell death. The diseases caused by mutations in RetGC1 and GCAP1 prominently affect cones, consistent with the higher concentrations of these proteins in cone cells.

Dominant cone-rod dystrophy: a mouse model generated by gene targeting of the GCAP1/Guca1a gene.

Cone dystrophy 3 (COD3) is a severe dominantly inherited retinal degeneration caused by missense mutations in GUCA1A, the gene encoding Guanylate Cyclase Activating Protein 1 (GCAP1). The role of GCAP1 in controlling cyclic nucleotide levels in photoreceptors has largely been elucidated using knock-out mice, but the disease pathology in these mice cannot be extrapolated directly to COD3 as this involves altered, rather than loss of, GCAP1 function. Therefore, in order to evaluate the pathology of this dominant disorder, we have introduced a point mutation into the murine Guca1a gene that causes an E155G amino acid substitution; this is one of the disease-causing mutations found in COD3 patients. Disease progression in this novel mouse model of cone dystrophy was determined by a variety of techniques including electroretinography (ERG), retinal histology, immunohistochemistry and measurement of cGMP levels. It was established that although retinal development was normal up to 3 months of age, there was a subsequent progressive decline in retinal function, with a far greater alteration in cone than rod responses, associated with a corresponding loss of photoreceptors. In addition, we have demonstrated that accumulation of cyclic GMP precedes the observed retinal degeneration and is likely to contribute to the disease mechanism. Importantly, this knock-in mutant mouse has many features in common with the human disease, thereby making it an excellent model to further probe disease pathogenesis and investigate therapeutic interventions.

The molecular evolution of avian ultraviolet- and violet-sensitive visual pigments.

The shortwave-sensitive SWS1 class of vertebrate visual pigments range in lambda(max) from the violet (385-445 nm) to the ultraviolet (UV) (365-355 nm), with UV-sensitivity almost certainly ancestral. In birds, however, the UV-sensitive pigments present in a number of species have evolved secondarily from an avian violet-sensitive (VS) pigment. All avian VS pigments expressed in vitro to date encode Ser86 whereas Phe86 is present in all non-avian ultraviolet sensitive (UVS) pigments. In this paper, we show by site directed mutagenesis of avian VS pigments that Ser86 is required in an avian VS pigment to maintain violet-sensitivity and therefore underlies the evolution of avian VS pigments. The major mechanism for the evolution of avian UVS pigments from an ancestral avian VS pigment is undoubtedly a Ser90Cys substitution. However, Phe86, as found in the Blue-crowned trogon, will also short-wave shift the pigeon VS pigment into the UV whereas Ala86 and Cys86 which are also found in natural avian pigments do not generate short-wave shifts when substituted into the pigeon pigment. From available data on avian SWS1 pigments, it would appear that UVS pigments have evolved on at least 5 separate occasions and utilize 2 different mechanisms for the short-wave shift.

Vertebrate opsins belonging to different classes vary in constitutively active properties resulting from salt-bridge mutations.

Vertebrate opsins are classified into one of five classes on the basis of amino acid similarity. These classes are short wavelength sensitive 1 and 2 (SWS1, SWS2), medium/long wavelength sensitive (M/LWS), and rod opsin like 1 and 2 (RH1, RH2). In bovine rod opsin (RH1), two critical amino acids form a salt bridge in the apoprotein that maintains the opsin in an inactive state. These residues are K296, which functions as the chromophore binding site, and E113, which functions as the counterion to the protonated Schiff base. Corresponding residues in each of the other vertebrate opsin classes are believed to play similar roles. Previous reports have demonstrated that mutations in these critical residues result in constitutive activation of transducin by RH1 class opsins in the absence of chromophore. Additionally, recent reports have shown that an E113Q mutation in SWS1 opsin is constitutively active. Here we ask if the other classes of vertebrate opsins maintain activation characteristics similar to that of bovine RH1 opsin. We approach this question by making the corresponding substitutions which disrupt the K296/E113 salt bridge in opsins belonging to the other vertebrate opsin classes. The mutant opsins are tested for their ability to constitutively activate bovine transducin. We demonstrate that mutations disrupting this key salt bridge produce constitutive activation in all classes. However, the mutant opsins differ in their ability to be quenched in the dark state by the addition of chromophore as well as in their level of constitutive activation. The differences in constitutive activation profiles suggest that structural differences exist among the opsin classes that may translate into a difference in activation properties.

Mutations in the gene KCNV2 encoding a voltage-gated potassium channel subunit cause "cone dystrophy with supernormal rod electroretinogram" in humans.

"Cone dystrophy with supernormal rod electroretinogram (ERG)" is an autosomal recessive disorder that causes lifelong visual loss combined with a supernormal ERG response to a bright flash of light. We have linked the disorder to a 0.98-cM (1.5-Mb) region on chromosome 9p24, flanked by rs1112534 and rs1074449, using homozygosity mapping in one large consanguineous pedigree. Analysis of one gene within this region, KCNV2, showed a homozygous nonsense mutation. Mutations were also found in 17 alleles of 10 other unrelated families with the same disorder. In situ hybridization demonstrated KCNV2 expression in human rod and cone photoreceptors. The precise function of KCNV2 in human photoreceptors remains to be determined, although this work suggests that mutations might perturb or abrogate I(KX), the potassium current within vertebrate photoreceptor inner segments, which has been shown to set their resting potential and voltage response.

Spectral tuning and evolution of short wave-sensitive cone pigments in cottoid fish from Lake Baikal.

The cottoid fishes of Lake Baikal in eastern Siberia provide a unique opportunity to study the evolution of visual pigments in a group of closely related species exposed to different photic environments. Members of this species flock are adapted to different depth habitats down to >1000 m, and both the rod and cone visual pigments display short wave shifts as depth increases. The blue-sensitive cone pigments of the SWS2 class cluster into two species groups with lambda(max) values of 450 and 430 nm, with the pigment in Cottus gobio, a cottoid fish native to Britain, forming a third group with a lambda(max) of 467 nm. The sequences of the SWS2 opsin gene from C. gobio and from two representatives of the 450 and 430 nm Baikal groups are presented. Approximately 6 nm of the spectral difference between C. gobio and the 450 nm Baikal group can be ascribed to the presence of a porphyropsin/rhodopin mixture in C. gobio. Subsequent analysis of amino acid substitutions by site-directed mutagenesis demonstrates that the remainder of the shift from 461 to 450 nm arises from a Thr269Ala substitution and the shift from 450 to 430 nm at least partly from Thr118Ala and Thr118Gly substitutions. The underlying adaptive significance of these substitutions in terms of spectral tuning and signal-to-noise ratio is discussed.

Divergent mechanisms for the tuning of shortwave sensitive visual pigments in vertebrates.

Of the four classes of vertebrate cone visual pigments, the shortwave-sensitive SWS1 class shows the shortest lambda(max) values with peaks in different species in either the violet (390-435 nm) or ultraviolet (around 365 nm) regions of the spectrum. Phylogenetic evidence indicates that the ancestral pigment was probably UV-sensitive (UVS) and that the shifts between violet and UV have occurred many times during evolution. This is supported by the different mechanisms for these shifts in different species. All visual pigments possess a chromophore linked via a Schiff base to a Lys residue in opsin protein. In violet-sensitive (VS) pigments, the Schiff base is protonated whereas in UVS pigments, it is almost certainly unprotonated. The generation of VS from ancestral UVS pigments most likely involved amino acid substitutions in the opsin protein that serve to stabilise protonation. The key residues in the opsin protein for this are at sites 86 and 90 that are adjacent to the Schiff base and the counterion at Glu113. In this review, the different molecular mechanisms for the UV or violet shifts are presented and discussed in the context of the structural model of bovine rhodopsin.

The molecular mechanism for the spectral shifts between vertebrate ultraviolet- and violet-sensitive cone visual pigments.

The short-wave-sensitive (SWS) visual pigments of vertebrate cone photoreceptors are divided into two classes on the basis of molecular identity, SWS1 and SWS2. Only the SWS1 class are present in mammals. The SWS1 pigments can be further subdivided into violet-sensitive (VS), with lambda(max) (the peak of maximal absorbance) values generally between 400 and 430 nm, and ultraviolet-sensitive (UVS), with a lambda(max)<380 nm. Phylogenetic evidence indicates that the ancestral pigment was UVS and that VS pigments have evolved separately from UVS pigments in the different vertebrate lineages. In this study, we have examined the mechanism of evolution of VS pigments in the mammalian lineage leading to present day ungulates (cow and pig). Amino acid sequence comparisons of the UVS pigments of teleost fish, amphibia, reptiles and rodents show that site 86 is invariably occupied by Phe but is replaced in bovine and porcine VS pigments by Tyr. Using site-directed mutagenesis of goldfish UVS opsin, we have shown that a Phe-86-->Tyr substitution is sufficient by itself to shift the lambda(max) of the goldfish pigment from a wild-type value of 360 nm to around 420 nm, and the reverse substitution of Tyr-86-Phe into bovine VS opsin produces a similar shift in the opposite direction. The substitution of this single amino acid is sufficient to account therefore for the evolution of bovine and porcine VS pigments. The replacement of Phe with polar Tyr at site 86 is consistent with the stabilization of Schiff-base protonation in VS pigments and the absence of protonation in UVS pigments.