Retinoschisin and Cardiac Glycoside Crosstalk at the Retinal Na/K-ATPase

Abstract

PURPOSE. Mutations in the RS1 gene, which encodes retinoschisin, cause X-linked juvenile retinoschisis, a retinal dystrophy in males. Retinoschisin specifically interacts with the retinal sodium-potassium adenosine triphosphatase (Na/K-ATPase), a transmembrane ion pump. Na/K-ATPases also bind cardiac glycosides, which control the activity of the pump and have been linked to disturbances in retinal homeostasis. In this study, we investigated the crosstalk between retinoschisin and cardiac glycosides at the retinal Na/K-ATPase and the consequences of this interplay on retinal integrity. METHODS. The effect of cardiac glycosides (ouabain and digoxin) on the binding of retinoschisin to the retinal Na/K-ATPase was investigated via western blot and immunocytochemistry. Also, the influence of retinoschisin on the binding of cardiac glycosides was analyzed via enzymatic assays, which quantified cardiac glycoside-sensitive Na/K-ATPase pump activity. Moreover, retinoschisin-dependent binding of tritium-labeled ouabain to the Na/K-ATPase was determined. Finally, a reciprocal effect of retinoschisin and cardiac glycosides on Na/K-ATPase localization and photoreceptor degeneration was addressed using immunohistochemistry in retinoschisin-deficient murine retinal explants. RESULTS. Cardiac glycosides displaced retinoschisin from the retinal Na/K-ATPase; however, retinoschisin did not affect cardiac glycoside binding. Notably, cardiac glycosides reduced the capacity of retinoschisin to regulate Na/K-ATPase localization and to protect against photoreceptor degeneration. CONCLUSIONS. Our findings reveal opposing effects of retinoschisin and cardiac glycosides on retinal Na/K-ATPase binding and on retinal integrity, suggesting that a fine-tuned interplay between both components is required to maintain retinal homeostasis. This observation provides new insight into the mechanisms underlying the pathological effects of cardiac glycoside treatment on retinal integrity.

Altered Protein Function Caused by AMD-associated Variant rs704 Links Vitronectin to Disease Pathology

“…After 24 hours, the medium was changed to FCS-free medium containing 100 U/mL penicillin/streptomycin, 200 µg/mL dextran sulfate (Carl Roth GmbH, Karlsruhe, Germany), and 30 µg/mL ascorbic acid (Cayman Chemical, Ann Arbor, MI, USA), as described in McLenachan et al 38 Confluent ARPE-19 monolayers were cultured in this medium for 4 weeks with media changes three times per week. Transwell filter inserts were then decellularized by incubating them with 0.5% Triton X-100 and 20-mM NH 4 OH in DPBS for 5 minutes at 37°C, as described in Fernandez-Godino et al 39 Immunolabeling was performed as described in Friedrich et al 30 and Schmid et al 40 using a quarter of a 12-well transwell filter insert for each staining. Pictures were taken with a confocal laser scanning microscope (FV3000 Fluoview; Olympus Life Sciences, Hamburg, Germany).…”

Retinoschisin and novel Na/K-ATPase interaction partners Kv2.1 and Kv8.2 define a growing protein complex at the inner segments of mammalian photoreceptors

“…Retinoschisin binding to Hek293 cells heterologously expressing the retinal Na/K-ATPase, Kv2.1, Kv8.2, or Kv2.1 plus Kv8.2 was performed as described [ 6 , 16 , 17 , 19 ], but with a prolonged incubation time of 1 h. The supernatant of Hek293 cells stably secreting recombinant retinoschisin was used as input.…”

Retinoschisin and novel Na/K-ATPase interaction partners Kv2.1 and Kv8.2 define a growing protein complex at the inner segments of mammalian photoreceptors

“…Puri cation of recombinant retinoschisin from supernatant of Hek293 cells heterologously expressing Myc-tagged retinoschisin was performed as described before [21,50,52]. Supernatant of Hek293 cells transfected with the empty pCDNA3.1™ expression vector (Thermo Fisher Scienti c) was subjected to the identical puri cation procedure and served as control.…”