Abstract
Purpose :
Mutations in the RS1 gene cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy in juvenile or adolescent males. Recently, we and others showed that the retinoschisin protein encoded by RS1 binds to the retinal Na/K-ATPase and modulates its localization in the plasma membrane of photoreceptors. In this study, we aimed to identify interaction partners of the Na/K-ATPase-retinoschisin complex. Specifically, we were interested to examine an influence of retinoschisin on the localization of selected interaction partners, namely the voltage-gated potassium ion channels (Kv). This should provide new insights into the role of retinoschisin as a putative regulator of photoreceptor membrane compartmentalization.
Methods :
Porcine retinal lysates were subjected to co-immunoprecipitation targeting the α3-subunit of the Na/K-ATPase (ATP1A3). Bound proteins were eluted and separated by SDS-PAGE for mass spectrometric analysis. Recognized proteins were verified in co-immunoprecipitation experiments with murine retinal lysates. Localization of Kv was investigated via immunohistochemistry in eyes from wildtype (wt) and retinoschisin-deficient (Rs1h knockout, Rs1tm1Web) mice. Effect of retinoschisin on thermal stability and total protein level was investigated in Y79 cells or retinoschisin-deficient murine retinal lysates, respectively.
Results :
Mass spectrometry from co-immunoprecipitates of porcine retinal lysates targeting ATP1A3 identified the Kv subunits Kv2.1 and Kv8.2. Binding to the retinal Na/K-ATPase was verified in further co-immunoprecipitations with murine retinal lysates. Immunohistochemical analyses in murine retinal cryosections revealed Kv localization to the inner segments and plexiform layers, like observed for the Na/K-ATPase. In retinae from retinoschisin-deficient mice, Kv2.1 and Kv8.2 distribution and total protein amount was altered compared to wt retinae. In contrast, no effect of retinoschisin was noted on thermal stability for Kv2.1 and Kv8.2.
Conclusions :
Our data suggest that Kv subunits Kv2.1. and Kv8.2 are part of a macromolecular complex together with the Na/K-ATPase and retinoschisin. Defective compartmentalization of the retinal Na/K-ATPase and its complexing partners may be an initial step in XLRS pathogenesis.
This is a 2021 ARVO Annual Meeting abstract.