NYX(Nyctalopin on Chromosome X), the Gene Mutated in Congenital Stationary Night Blindness, Encodes a Cell Surface Protein

Abstract

These data provide evidence that human and mouse nyctalopin are membrane-bound extracellular proteins and are functionally conserved.

Night blindness and abnormal cone electroretinogram ON responses in patients with mutations in the GRM6 gene encoding mGluR6

“…Like the GRM6 patients, the NYX patients have residual rod responses that are possibly mediated through the gap junctions with cones (57). The similarities in the GRM6 and NYX phenotypes support the speculation that the NYX gene product, a membranebound extracellular protein whose exact function is currently obscure (58), is involved in ON bipolar cell signaling. A distinctive electrical defect is found in patients with the incomplete form of X-linked stationary night blindness due to mutations in the CACNA1F gene; their ERGs suggest abnormalities in both the ON and OFF bipolar pathways (59).…”

Congenital stationary night blindness: An analysis and update of genotype–phenotype correlations and pathogenic mechanisms

“…16D). We previously investigated three of these mutations, p.Glu244_Ala247del, p.Leu347Pro and p.Gly370Val, with respect to their impact on subcellular localization (Zeitz et al, 2003). These preliminary results suggest that the location of nyctalopin at the cell surface is necessary for correct function but is not influenced by the mutations.…”

Night blindness-associated mutations in the ligand-binding, cysteine-rich, and intracellular domains of the metabotropic glutamate receptor 6 abolish protein trafficking

“…Similarly, cells were always washed between different staining or fixation steps during the protocol. To observe proteins that localize exclusively extracellularly, cells were stained with preabsorbed [Zeitz et al, 2003] rabbit anti-V5 antibody (Sigma, Buchs, Switzerland), directed against the N-terminal V5-tag (Invitrogen) in 0.1% BSA, 1 Â PBS at a dilution 1:1,000 for 1 hr and then incubated with anti-rabbit FITC antibody at a dilution of 1:500 in 0.1% BSA, 1 Â PBS for 30 min. Thereafter, stained surface proteins were fixed in 4% paraformaldehyde.…”

“…Thereafter, stained surface proteins were fixed in 4% paraformaldehyde. To stain the intracellular part of the glutamate receptor, cells were permeabilized with 0.2% TritonX-100, 1 Â PBS, and incubated for 30 min with mouse anti-myc antibody (Roche), directed against the C-terminal preabsorbed myc-tag, at a dilution of 1:500 in PBGT [Zeitz et al, 2003]. Thereafter, cells were incubated with anti-mouse Cy3 antibody at a dilution of 1:500 for 30 min, shortly fixed with 4% paraformaldehyde, and embedded in antifade mounting media (Vectashield mounting media with DAPI; Reactolabs S.A., Servion, Switzerland).…”

“…A detectable amount of the expressed proteins (10 ml) was loaded on an 8% SDS polyacrylamide gel. Protein was transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech, Otelfingen, Switzerland) by electroblotting and detected in PBST with mouse anti-V5 (Invitrogen) and anti-mouse HRP (NEN Life Science Products, Perkin Elmer, Rodgau-Jügesheim, Germany) antibodies, as described previously [Zeitz et al, 2003]. For immunoprecipitation studies, confluent cells from two bottles (75 mm) of stably-expressing cell lines were harvested with a cell scraper, as described above, and homogenized in 1.5 ml of lysis buffer with a pestle.…”