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Bernhard H. Weber, Ulrike Friedrich, Thomas Loenhardt, Thomas Langmann, Heidi Stöhr; The Na/K ATPase is Obligatory for Membrane Anchorage of Retinoschisin the Protein Involved in the Pathogenesis of X-linked Juvenile Retinoschisis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5379.
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Mutations in the retina-specific RS1 gene which encodes the discoidin-domain containing retinoschisin cause X-linked juvenile retinoschisis (XLRS), a prevalent macular degeneration in males. Retinoschisin is secreted as a disulfide-linked homo-oligomeric complex and associates with the surface of photoreceptor and bipolar cells. The aim of this study is to analyze the molecular mechanisms by which retinoschisin is linked to retinal membranes in vivo.
The binding of retinoschisin to phospholipids was determined by ELISA and Western blotting. Retinoschisin knock-out (Rs1h-/Y) and Na/K-ATPase subunit β2 deficient mice (Atp1b2-/-) were analyzed for gene expression and protein distribution of retinoschisin, ATP1B2, ATP1A3 and SARM1 by RT-PCR, Western blotting and immunohistochemistry. Hek293 cells expressing different Na/K-ATPase subunits and membranes isolated from retina and other tissues of Rs1h-/Y and Rs1h-/Y/Atp1b2-/- mice were used to analyze the interaction with retinoschisin.
We show that retinoschisin fails to bind to immobilized phospholipids or unilamellar lipid vesicles. Instead, binding assays with membranes from Rs1h-/Y and Rs1h-/Y/Atp1b2-/- mice indicate that retinoschisin membrane association is implicitly dependent on the presence of the Na/K-ATPase subunits. Also, co-expression of ATP1A3 and ATP1B2 is required for retinoschisin binding to intact Hek293 cells. In addition, we demonstrate a significant loss of retinoschisin in vivo in the Atp1b2-/- retina lacking ATP1A3 and ATP1B2 protein. Finally, we observed reduced ATP1A3 and ATP1B2 protein expression in the photoreceptor inner segments accompanied by an increase of ATP1A3 and ATP1B2 in the ONL at the time of eye opening in Rs1h-/Y mice.
Our data provide evidence for a predominant role of Na/K-ATPase in anchoring retinoschisin to retinal cell surfaces. Furthermore, altered localization of ATP1A3 and ATP1B2 is a notable consequence of retinoschisin deficiency and thus may be an important downstream aspect of cellular pathology in XLRS.
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