Dark-light: model for nightblindness from the human rhodopsin Gly-90-->Asp mutation.

Abstract

MATERIALS AND METHODSElectroretinography. Corneal electroretinograms (ERGs) were elicited by full-field 10-gs xenon photostrobe flashes and recorded at 0.1-1000 Hz by using methods and control values presented previously (6). Molecular Biology. Genomic DNA was isolated from peripheral blood samples. The Gly9OAsp mutation was found by sequencing the entire rhodopsin coding region, with target DNA generated by PCR amplification (7) of genomic DNA during 40 rounds of thermal cycling [94°C for 90 s, 54°C for 60 s, and 72°C for 180 s] followed by 72°C for 10 min. Exon 1 primers were 5'-AGCTCAGGCCTTCGCAGCAT-3' and 5'-GAGGGCTTTGGATAACATTG-3'. The codon 90 region was sequenced by the Sanger dideoxynucleotide chaintermination method (8) with primer 5'-ACGCAGCCCCTTC-GAGTAC-3'. The presence of the Gly9OAsp mutation was assayed in a population of normal subjects by allele-specific oligonucleotide hybridization with primers 5'-TGGT-GAAGCCACCTAGGAC-3' for Gly-90 and 5'-TGGTGA-AGTCACCTAGGAC-3' for Asp-90.Densitometry. The 530-nm test and 880-nm reference beams of a modified Florida retinal densitometer (9) were reflected from the human retina in vivo onto a photomultiplier tube. The 70 test field was centered on the 9.10 bleaching field. Measurements were made at 17.80 in the temporal retina of the left eye of the 38-year-old proband. A bleaching beam was either "white" or "monochromatic" from 10-nm interference filters (Baird-Atomic type B-1). Dark-corrected photon counts of test and reference beams were counted separately in 2-s intervals, and the ratio was averaged to give T, with To at the end of a long full bleach and Td after full dark recovery.(To -Td)/To = (1 -s)[1 -e-23(Am) [1] in which s is the fraction of dark-corrected test counts evoked by photons that have not passed twice through rhodopsin (i.e., stray light) and ,B is rhodopsin density (loge) expressed as a(Am)cl where a(Am) is the molecular extinction coefficient at the test wavelength Am, c is the concentration, and 1 is the optical path length through rhodopsin in the rod outer segment. Since s is not easily measured, it is common to assume that s = 0 and then to solve Eq. 1 for 2,. Converting to loglo gives the "two-way density" A, which is the frequently used parameter of retinal densitometry. At equilibrium, the kinetic equation of rhodopsin bleaching (10) is:Abbreviations: adRP, autosomal dominant retinitis pigmentosa; ERG, electroretinogram; sc-td, scotopic trolands; tvi, threshold versus retinal illuminance curve; Gly9OAsp, Gly-90 -> Asp. tTo whom reprint requests should be addressed at: W. K.

11-cis- and All-trans-Retinols Can Activate Rod Opsin: Rational Design of the Visual Cycle

“…Such a mechanism is likely the cause for some forms of congenital night blindness. For example, the G90D rhodopsin mutant (26) opsin is constitutively active (27). A rod containing G90D rhodopsin seems to have a significant amount of apoprotein present in the dark (28), and its phenotype is that they appear as if they were light-adapted in the dark (26,29).…”

Unusual Thermal and Conformational Properties of the Rhodopsin Congenital Night Blindness Mutant Thr-94 → Ile

“…In contrast, it has been shown that constitutive activity is not the cause of photoreceptor degeneration in transgenic mice carrying RP mutations (20). An alternative mechanism, involving thermal instability of the mutant rhodopsins, has also been proposed for CNB mutations, like G90D (12,13). Thus, different molecular mechanisms underlying RP and CNB have been outlined previously (34).…”

“…Another possible explanation for the observed G90D mutant phenotype has been proposed, i.e. enhanced rate of thermal isomerization due to lowered activation energy caused by the mutation (12,13). More recently, a third mutation associated with CNB, T94I, in the second transmembrane helix of rhodopsin, has been described (14).…”

“…Although no misfolding is detected for this mutant, the high hydroxylamine reactivity indicates that its dark structure is significantly altered. Also, the low thermal stability in the dark may be relevant to the molecular mechanism of CNB (12,13). The results obtained for the Thr-94 mutants highlight the tight coupling between the structural domains of rhodopsin in in vivo folding of this receptor.…”

Hereditary Degenerative Retinopathies: Optimism for Somatic Gene Therapy

“…Whereas autosomal dominant (Ad) and autosomal recessive (Ar) RP are progressive disorders, congenital stationary night blindness is a nonprogressive disorder (9). Moreover, affected members of a speci c kindred having lifelong night blindness and carrying the rhodopsin mutation (G90D) show preservation of rod functions (10). In addition, how any of the gene defects (mutation) leads to photoreceptor death rather than functional impairment of the cell, as is observed in the case of color blindness, is not known.…”