Cytosolic Internalization of the Na/K ATPase Ouabain Receptor Complex (NORC)

Abstract

In early (1960–80) classic work, digitalis (ouabain) binding to the Na/K ATPase (NKA) was considered mainly a disruption of the cellular ionic steady state and in terms of ligand dependency. During the past 2–3 decades, the role of NKA as signal transducer involving protein/protein interactions with subsequent protein phosphorylation and gene transcriptional upregulation was elucidated regarding cell survival and apoptosis. Recently re‐established, the significance of a cytosolic internalization of the NKA‐ouabain receptor‐complex (here called NORC) is not yet understood in terms of details of participating proteins and organelles, and its potential for gene transcriptional upregulation. The fate of NORC was followed by immunocytochemistry, and enhanced dark field hyperspectral microscopy (EDHM) in two different lens epithelial cells (LECs) (FHL124 and B3) with the aim to characterize some of the protein partners of the hypothetical membrane‐to‐cytosol escalator involved in this process. Partners of interest were the Bcl‐2 protein BclXL, proposed by us to interact with NKA's N‐terminal domain (Lauf et al., Cell Physiol Biochem 31:257–276, 2013) and detected by co‐immunoprecipitation (Lauf et al., Am J Physiol Cell Physiol. 308, C51–60, 2015), α‐tubulin (Zampar et al. Biochem J. 422:129,2009), caveolin (Cui & Zie, Molecules 22:990, 2017), and the endosomal and lysosomal proteins EEA1 and LAMP1 (Cheriavsky et al., JBC 289:1049, 2014). The presence of these protein partners was also studied in B3 LECs and verified by PAGEL and Western blots. Protein interactions were tested with Bodipy‐green fluorescent ouabain (BFO) and green‐ or red‐fluorescent labeled antibodies against all protein components, verified immuno‐cytochemically by overlapping yellow/orange fluorescence and further refined by MATLAB photo‐editing for background noise. Results: Based on immuno‐colocalization of BFO with Cy‐3 Red‐stained anti‐α1 NKA isoform antibody, NORC transgressed the plasmalemma into cytosol 45 min after exposure to μM BFO. This was further supported by finding of intracellular BFO labeling using EDHM in FHL124 cells. Higher ouabain concentrations affected cell size and NKA distribution. However, interaction of NKA with Bcl‐XS, a shorter variant of BclXL lacking the B1 motif, and caveolin‐1 in B3 LECs was independent of ouabain concentrations, and not seen in all cells. Increase in ouabain concentration enhanced the interaction of NKA with α‐tubulin, which was attenuated by preventing its deacetylation with Trichostatin A, a histone deacetylase inhibitor. Colocalization of NKA with EEA1 was not affected by ouabain, which appeared to increase NKA/Lamp1 immuno‐colocalization. A model is proposed by which NORC is translocated across the plasma membranes within minutes after its generation. In some B3 cells, by interaction with BclXS, caveolin and α‐tubulin, NORC may be guided to endosomes and, if not recycled back to the plasma membrane, subsequently to lysosomes. The future fate of NORC, especially of a putative nuclear receptor capture of lysosome‐processed ouabain to mediate gene transcriptional upregulation of various pro‐apoptotic and pro‐inflammatory proteins is under study and reported elsewhere at this meeting.Support or Funding InformationWright State University Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.