Insights into congenital stationary night blindness based on the structure of G90D rhodopsin

We present active‐state structures of the G protein‐coupled receptor (GPCRs) rhodopsin carrying the disease‐causing mutation G90D. Mutations of G90 cause either retinitis pigmentosa (RP) or congenital stationary night blindness (CSNB), a milder, non‐progressive form of RP. Our analysis shows that the CSNB‐causing G90D mutation introduces a salt bridge with K296. The mutant thus interferes with the E113Q‐K296 activation switch and the covalent binding of the inverse agonist 11‐cis‐retinal, two interactions that are crucial for the deactivation of rhodopsin. Other mutations, including G90V causing RP, cannot promote similar interactions. We discuss our findings in context of a model in which CSNB is caused by constitutive activation of the visual signalling cascade.     

GUCY2D mutations in retinal guanylyl cyclase 1 provide biochemical reasons for dominant cone–rod dystrophy but not for stationary night blindness

Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber''s congenital amaurosis (LCA1), and dominant cone–rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro. The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca²⁺ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca²⁺ concentrations to decelerate GCAP-activated RetGC1 heterodimer—6-fold higher than WT and 2-fold higher than the Ser⁸³⁸-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.     

Characterization of rhodopsin congenital night blindness mutant T94I

The Thr94 --> Ile mutation in the second transmembrane segment of rhodopsin has been reported to be associated with a congenital night blindness phenotype in a large Irish pedigree. Previously, two other known rhodopsin mutants that cause congenital night blindness, A292E and G90D, have been shown in vitro to constitutively activate the G protein transducin in the absence of a chromophore. The proposed mechanism of constitutive activation of these two mutants is an electrostatic disruption of the active site salt bridge between Glu113 and Lys296 that contributes to stabilization of the protein in the inactive state. Here, the T94I rhodopsin mutant is characterized and compared to the two other known rhodopsin night blindness mutants. The T94I mutant opsin is shown also to constitutively activate transducin. The T94I mutant pigment (with a bound 11-cis-retinal chromophore), like the other known rhodopsin night blindness mutants, is not active in the dark and has wild-type activity upon exposure to light. Similar to the Gly90 --> Asp substitution, position 94 is close enough to the Schiff base nitrogen that an Asp at this position can functionally substitute for the Glu113 counterion. However, in contrast to the other night blindness mutants, the T94I MII intermediate decays with a half-life that is approximately 8-fold slower than in the wild-type MII intermediate. Thus, the one phenotype shared by all congenital night blindness mutants that is different from the wild-type protein is constitutive activation of the apoprotein.     

Structural, energetic, and mechanical perturbations in rhodopsin mutant that causes congenital stationary night blindness

Several point mutations in rhodopsin cause retinal diseases including congenital stationary night blindness and retinitis pigmentosa. The mechanism by which a single amino acid residue substitution leads to dysfunction is poorly understood at the molecular level. A G90D point mutation in rhodopsin causes constitutive activity and leads to congenital stationary night blindness. It is unclear which perturbations the mutation introduces and how they can cause the receptor to be constitutively active. To reveal insight into these mechanisms, we characterized the perturbations introduced into dark state G90D rhodopsin from a transgenic mouse model expressing exclusively the mutant rhodopsin in rod photoreceptor cells. UV-visible absorbance spectroscopy revealed hydroxylamine accessibility to the chromophore-binding pocket of dark state G90D rhodopsin, which is not detected in dark state wild-type rhodopsin but is detected in light-activated wild-type rhodopsin. Single-molecule force spectroscopy suggested that the structural changes introduced by the mutation are small. Dynamic single-molecule force spectroscopy revealed that, compared with dark state wild-type rhodopsin, the G90D mutation decreased energetic stability and increased mechanical rigidity of most structural regions in the dark state mutant receptor. The observed structural, energetic, and mechanical changes in dark state G90D rhodopsin provide insights into the nature of perturbations caused by a pathological point mutation. Moreover, these changed properties observed for dark state G90D rhodopsin are consistent with properties expected for an active state.     

Whole-Exome Sequencing Identifies LRIT3 Mutations as a Cause of Autosomal-Recessive Complete Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disorder. Two forms can be distinguished clinically: complete CSNB (cCSNB) and incomplete CSNB. Individuals with cCSNB have visual impairment under low-light conditions and show a characteristic electroretinogram (ERG). The b-wave amplitude is severely reduced in the dark-adapted state of the ERG, representing abnormal function of ON bipolar cells. Furthermore, individuals with cCSNB can show other ocular features such as nystagmus, myopia, and strabismus and can have reduced visual acuity and abnormalities of the cone ERG waveform. The mode of inheritance of this form can be X-linked or autosomal recessive, and the dysfunction of four genes (NYX, GRM6, TRPM1, and GPR179) has been described so far. Whole-exome sequencing in one simplex cCSNB case lacking mutations in the known genes led to the identification of a missense mutation (c.983G>A [p.Cys328Tyr]) and a nonsense mutation (c.1318C>T [p.Arg440^∗]) in LRIT3, encoding leucine-rich-repeat (LRR), immunoglobulin-like, and transmembrane-domain 3 (LRIT3). Subsequent Sanger sequencing of 89 individuals with CSNB identified another cCSNB case harboring a nonsense mutation (c.1151C>G [p.Ser384^∗]) and a deletion predicted to lead to a premature stop codon (c.1538_1539del [p.Ser513Cysfs^∗59]) in the same gene. Human LRIT3 antibody staining revealed in the outer plexiform layer of the human retina a punctate-labeling pattern resembling the dendritic tips of bipolar cells; similar patterns have been observed for other proteins implicated in cCSNB. The exact role of this LRR protein in cCSNB remains to be elucidated.     

Unusual thermal and conformational properties of the rhodopsin congenital night blindness mutant Thr-94 --> Ile

Naturally occurring point mutations in the opsin gene cause the retinal diseases retinitis pigmentosa and congenital night blindness. Although these diseases involve similar mutations in very close locations in rhodopsin, their progression is very different, with retinitis pigmentosa being severe and causing retinal degeneration. We report on the expression and characterization of the recently found T94I mutation associated with congenital night blindness, in the second transmembrane helix or rhodopsin, and mutations at the same site. T94I mutant rhodopsin folded properly and was able to bind 11-cis-retinal to form chromophore, but it showed a blue-shifted visible band at 478 nm and reduced molar extinction coefficient. Furthermore, T94I showed dramatically reduced thermal stability, extremely long lived metarhodopsin II intermediate, and highly increased reactivity toward hydroxylamine in the dark, when compared with wild type rhodopsin. The results are consistent with the location of Thr-94 in close proximity to Glu-113 counterion in the vicinity of the Schiff base linkage and suggest a role for this residue in maintaining the correct dark inactive conformation of the receptor. The reported results, together with previously published data on the other two known congenital night blindness mutants, suggest that the molecular mechanism underlying this disease may not be structural misfolding, as proposed for retinitis pigmentosa mutants, but abnormal functioning of the receptor by decreased thermal stability and/or constitutive activity.     

Role of the retinal hydrogen bond network in rhodopsin Schiff base stability and hydrolysis

Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin. We report here our investigation into this process focusing on the role of amino acids involved in a hydrogen bond network around the retinal Schiff base. We find conservative mutations in this network (T94I, E113Q, S186A, E181Q, Y192F, and Y268F) increase the activation energy (E(a)) and abolish the concave Arrhenius plot normally seen for Schiff base hydrolysis in dark state rhodopsin. Interestingly, two mutants (T94I and E113Q) show dramatically faster rates of Schiff base hydrolysis in dark state rhodopsin, yet slower hydrolysis rates in the active MII form. We find deuterium affects the hydrolysis process in wild-type rhodopsin, exhibiting a specific isotope effect of approximately 2.5, and proton inventory studies indicate that multiple proton transfer events occur during the process of Schiff base hydrolysis for both dark state and MII forms. Taken together, our study demonstrates the importance of the retinal hydrogen bond network both in maintaining Schiff base integrity in dark state rhodopsin, as well as in catalyzing the hydrolysis and release of retinal from the MII form. Finally, we note that the dramatic alteration of Schiff base stability caused by mutation T94I may play a causative role in congenital night blindness as has been suggested by the Oprian and Garriga laboratories.     

Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy

Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCRs) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state ²H NMR constitutes a powerful approach to study atomic-level dynamics of membrane proteins. In the present application, we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific ²H labels have been introduced into the methyl groups of retinal and solid-state ²H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryotrapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent ²H NMR line shapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the β-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangements of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all-trans retinal with the β4 strand of the E2 loop and the side chains of Glu¹²² and Trp²⁶⁵ within the binding pocket. The solid-state ²H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism.     

Mechanism of activation and inactivation of opsin: role of Glu113 and Lys296.

In previous studies, mutation of either Lys296 or Glu113 in bovine rhodopsin has been shown to result in constitutive activation of the apoprotein form, opsin [Robinson et al. (1992) Neuron 9, 719-725]. In this report, pH-rate profiles for the rhodopsin-catalyzed exchange of GTPgS for GDP on transducin are established for the constitutively active opsin mutants. All of the mutants, including the double-mutant E113Q,K296G, show a bell-shaped pH-rate profile. Therefore, it is evident that at least two ionizable groups in addition to Lys296 and Glu113 control the formation of the active opsin state. The sole effect of mutation at position 113 or 296 is to alter the ionization constant of the group with the higher pKa, called pka2. pKa2 decreases in the following order: rhodopsin/light (9.0) > K296E = K296G = E113Q,K296G (8.0) > E113Q (6.8) > K296H (6.6) > wild-type opsin (< 5.0). These results are consistent with a model where activation of opsin involves (i) breaking of the salt bridge between Lys296 and Glu113, (ii) deprotonation of Lys296, and (iii) the net uptake of a proton from the solvent. Furthermore, exogenous addition of the chromophore all-trans-retinal shifts the wild-type and E113Q opsin equilibrium to favor the active state. In all these respects, the light-independent activation of the opsin mutants appears to proceed by a mechanism similar to that of light-activated rhodopsin.     

Solid-State H NMR spectroscopy of retinal proteins in aligned membranes

Solid-state ²H NMR spectroscopy gives a powerful avenue to investigating the structures of ligands and cofactors bound to integral membrane proteins. For bacteriorhodopsin (bR) and rhodopsin, retinal was site-specifically labeled by deuteration of the methyl groups followed by regeneration of the apoprotein. ²H NMR studies of aligned membrane samples were conducted under conditions where rotational and translational diffusion of the protein were absent on the NMR time scale. The theoretical lineshape treatment involved a static axial distribution of rotating C-C²H₃ groups about the local membrane frame, together with the static axial distribution of the local normal relative to the average normal. Simulation of solid-state ²H NMR lineshapes gave both the methyl group orientations and the alignment disorder (mosaic spread) of the membrane stack. The methyl bond orientations provided the angular restraints for structural analysis. In the case of bR the retinal chromophore is nearly planar in the dark- and all-trans light-adapted states, as well upon isomerization to 13-cis in the M state. The C13-methyl group at the “business end” of the chromophore changes its orientation to the membrane upon photon absorption, moving towards W182 and thus driving the proton pump in energy conservation. Moreover, rhodopsin was studied as a prototype for G protein-coupled receptors (GPCRs) implicated in many biological responses in humans. In contrast to bR, the retinal chromophore of rhodopsin has an 11-cis conformation and is highly twisted in the dark state. Three sites of interaction affect the torsional deformation of retinal, viz. the protonated Schiff base with its carboxylate counterion; the C9-methyl group of the polyene; and the β-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by ²H NMR are implicated in substituent control of activation. Retinal is locked in a conformation that is twisted in the direction of the photoisomerization, which explains the dark stability of rhodopsin and allows for ultra-fast isomerization upon absorption of a photon. Torsional strain is relaxed in the meta I state that precedes subsequent receptor activation. Comparison of the two retinal proteins using solid-state ²H NMR is thus illuminating in terms of their different biological functions.     

Cone dystrophy and ectopic synaptogenesis in a Cacna1f loss of function model of congenital stationary night blindness (CSNB2A)

Congenital stationary night blindness 2A (CSNB2A) is an X-linked retinal disorder, characterized by phenotypically variable signs and symptoms of impaired vision. CSNB2A is due to mutations in CACNA1F, which codes for the pore-forming α_1F subunit of a L-type voltage-gated calcium channel, Ca_v1.4. Mouse models of CSNB2A, used for characterizing the effects of various Cacna1f mutations, have revealed greater severity of defects than in human CSNB2A. Specifically, Cacna1f-knockout mice show an apparent lack of visual function, gradual retinal degeneration, and disruption of photoreceptor synaptic terminals. Several reports have also noted cone-specific disruptions, including axonal abnormalities, dystrophy, and cell death. We have explored further the involvement of cones in our ‘G305X’ mouse model of CSNB2A, which has a premature truncation, loss-of-function mutation in Cacna1f. We show that the expression of genes for several phototransduction-related cone markers is down-regulated, while that of several cellular stress- and damage-related markers is up-regulated; and that cone photoreceptor structure and photopic visual function – measured by immunohistochemistry, optokinetic response and electroretinography – deteriorate progressively with age. We also find that dystrophic cone axons establish synapse-like contacts with rod bipolar cell dendrites, which they normally do not contact in wild-type retinas – ectopically, among rod cell bodies in the outer nuclear layer. These data support a role for Ca_v1.4 in cone synaptic development, cell viability, and synaptic transmission of cone-dependent visual signals. Although our novel finding of cone-to-rod-bipolar cell contacts in this mouse model of a retinal channelopathy may challenge current views of the role of Ca_v1.4 in photopic vision, it also suggests a potential new target for restorative therapy.     

Covalent Bond between Ligand and Receptor Required for Efficient Activation in Rhodopsin

Rhodopsin is an extensively studied member of the G protein-coupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation.     

Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness

Congenital stationary night blindness (CSNB) is a heterogeneous retinal disorder characterized by visual impairment under low light conditions. This disorder is due to a signal transmission defect from rod photoreceptors to adjacent bipolar cells in the retina. Two forms can be distinguished clinically, complete CSNB (cCSNB) or incomplete CSNB; the two forms are distinguished on the basis of the affected signaling pathway. Mutations in NYX, GRM6, and TRPM1, expressed in the outer plexiform layer (OPL) lead to disruption of the ON-bipolar cell response and have been seen in patients with cCSNB. Whole-exome sequencing in cCSNB patients lacking mutations in the known genes led to the identification of a homozygous missense mutation (c.1807C>T [p.His603Tyr]) in one consanguineous autosomal-recessive cCSNB family and a homozygous frameshift mutation in GPR179 (c.278delC [p.Pro93Glnfs^∗57]) in a simplex male cCSNB patient. Additional screening with Sanger sequencing of 40 patients identified three other cCSNB patients harboring additional allelic mutations in GPR179. Although, immunhistological studies revealed Gpr179 in the OPL in wild-type mouse retina, Gpr179 did not colocalize with specific ON-bipolar markers. Interestingly, Gpr179 was highly concentrated in horizontal cells and Müller cell endfeet. The involvement of these cells in cCSNB and the specific function of GPR179 remain to be elucidated.     

Physical mapping and exclusion of<Emphasis Type="Italic"> GPR34</Emphasis> as the causative gene for congenital stationary night blindness type 1

X-linked congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder characterized by impaired night vision, variably involving high myopia, nystagmus, decreased visual acuity, and strabismus. Linkage studies have identified two distinct loci for X-linked CSNB1 and CSNB2 on the short arm of chromosome X. The gene mutated in families displaying the "incomplete phenotype" of CSNB (i.e., CSNB2) has recently been identified. To identify novel candidate genes for the "complete form" of CSNB (i.e., CSNB1) we screened the physically vast region Xp11.3-Xp11.4 for cDNA sequences. This led us to identify and map the G protein coupled receptor (GPCR) gene GPR34 to Xp11.4 within 650 kb of the marker DXS993. Deletion screening via Southern blotting and direct sequencing of GPR34 revealed no mutations in 19 unrelated men with CSNB1, excluding a causal role in the disease. However, because of its expression in retinal and neural tissue and the involvement of GPCRs in transmembrane signal transduction, GPR34 remains a putative candidate gene for a number of ocular diseases which also map to the Xp11.4 region.     

Mutations in TRPM1 Are a Common Cause of Complete Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impaired night vision and variable decreased visual acuity. We report here that six out of eight female probands with autosomal-recessive complete CSNB (cCSNB) had mutations in TRPM1, a retinal transient receptor potential (TRP) cation channel gene. These data suggest that TRMP1 mutations are a major cause of autosomal-recessive CSNB in individuals of European ancestry. We localized TRPM1 in human retina to the ON bipolar cell dendrites in the outer plexifom layer. Our results suggest that in humans, TRPM1 is the channel gated by the mGluR6 (GRM6) signaling cascade, which results in the light-evoked response of ON bipolar cells. Finally, we showed that detailed electroretinography is an effective way to discriminate among patients with mutations in either TRPM1 or GRM6, another autosomal-recessive cCSNB disease gene. These results add to the growing importance of the diverse group of TRP channels in human disease and also provide new insights into retinal circuitry.     

Slow binding of retinal to rhodopsin mutants G90D and T94D

In an examination of the effect of three rhodopsin night blindness mutations on the rate of association of 11-cis-retinal with opsin, one of the mutations (G90D) was found to slow the rate of reaction by more than 80-fold. This effect does not appear to be general to night blindness mutations as the two other mutants (A292E and T94I) were not found to bind retinal with slowed kinetics. However, T94D was similar to G90D in that the rate of retinal binding was dramatically slowed. Gly90 and Thr94 are both located in the active site of the protein close to the Schiff base counterion Glu113. Thus, the slow kinetics of Schiff base formation appear to correlate with the introduction of a negative charge close to the Schiff base counterion, suggesting a possible role for Glu113 as a catalytic base in this reaction. Consistent with this model, the E113Q mutant was also found to bind retinal more slowly than the wild type.     

Chromcrphore-Environment Interaction of Visual Pigments in Model System

SEVERAL laboratories^1–6 have recently been concerned with the mechanism of the bathochromic shift of about 120 nm which results when 11-cis retinal (λ _max 380 nm) combines with the protein opsin to form rhodopsin (λ_max 498 nm). A red shift of up to 186 nm is involved in the formation of iodopsin from 11-cis retinal and cone opsin^7,8. The active site of bovine rhodopsin consists of the 11-cis retinylidene chromophore attached to a primary amine group of the protein forming a Schiff-base linkage of the type shown in Fig. 1, Ia. On the basis of the chemical reactions of rhodopsin and its derivatives it has been suggested that an interaction between a protonated form of the chromophore (structure of the type Ib) and a lipophilic environment contributes¹¹ to the red shift.     

TRPM1 Is Mutated in Patients with Autosomal-Recessive Complete Congenital Stationary Night Blindness

Night vision requires signaling from rod photoreceptors to adjacent bipolar cells in the retina. Mutations in the genes NYX and GRM6, expressed in ON bipolar cells, lead to a disruption of the ON bipolar cell response. This dysfunction is present in patients with complete X-linked and autosomal-recessive congenital stationary night blindness (CSNB) and can be assessed by standard full-field electroretinography (ERG), showing severely reduced rod b-wave amplitude and slightly altered cone responses. Although many cases of complete CSNB (cCSNB) are caused by mutations in NYX and GRM6, in ∼60% of the patients the gene defect remains unknown. Animal models of human diseases are a good source for candidate genes, and we noted that a cCSNB phenotype present in homozygous Appaloosa horses is associated with downregulation of TRPM1. TRPM1, belonging to the family of transient receptor potential channels, is expressed in ON bipolar cells and therefore qualifies as an excellent candidate. Indeed, mutation analysis of 38 patients with CSNB identified ten unrelated cCSNB patients with 14 different mutations in this gene. The mutation spectrum comprises missense, splice-site, deletion, and nonsense mutations. We propose that the cCSNB phenotype in these patients is due to the absence of functional TRPM1 in retinal ON bipolar cells.     

Identification of novel rhodopsin mutations responsible for retinitis pigmentosa: Implications for the structure and function of rhodopsin

Ten rhodopsin mutations have been found in a screen of 282 subjects with retinitis pigmentosa (RP), 76 subjects with Leber congenital amaurosis, and 3 subjects with congenital stationary night blindness. Eight of these mutations (gly⁵¹-to-ala, val¹⁰⁴-to-ile, gly¹⁰⁶-to-arg, arg¹³⁵-to-gly, cys¹⁴⁰-to-ser, gly¹⁸⁸-to-glu, val²⁰⁹-to-met, and his²¹¹-to-arg) produce amino acid substitutions, one (gln⁶⁴-to-ter) introduces a stop codon, and one changes a guanosine in the intron 4 consensus splice donor sequence to thymidine. Cosegregation of RP with gln⁶⁴-to-ter, gly¹⁰⁶-to-arg, arg¹³⁵-to-gly, cys¹⁴⁰-to-ser, gly¹⁸⁸-to-glu, his²¹¹-to-arg, and the splice site guanosine-to-thymidine indicates that these mutations are likely to cause retinal disease. Val¹⁰⁴-to-ile does not cosegregate and is therefore unlikely to be related to retinal disease. The relevance of gly⁵¹-to-ala and val²⁰⁹-to-met remains to be determined. The finding of gln⁶⁴-to-ter in a family with autosomal dominant RP is in contrast to a recent report of a recessive disease phenotype associated with the rhodopsin mutation glu²⁴⁹-to-ter. In the present screen, all of the mutations that cosegregate with retinal disease were found among patients with RP. The mutations described here bring to 35 the total number of amino acid substitutions identified thus far in rhodopsin that are associated with RP. The distribution of the substitutions along the polypeptide chain is significantly nonrandom: 63% of the substitutions involve those 19% of amino acids that are identical among vertebrate visual pigments sequenced to date.