Abstract
Purpose: :
Na+/K+-ATPase in RPE apical microvilli plays a critical role in maintaining the photoreceptor microenvironment. In other cell types, Na+/K+-ATPase can be found in caveolae, where it serves both as an ion transporter and ligand-inducible signal transducer. The α1 subunit of Na+/K+-ATPase contains putative binding motifs for the "scaffolding domain" of the caveolar coat protein, caveolin-1 (Cav-1). We tested the hypothesis that Na+/K+-ATPase interacts with and is regulated by Cav-1 in RPE.
Methods: :
Caveolar membranes were isolated from bovine RPE and cofractionation of the Na/K-ATPase with Cav-1 was assayed by quantitative Western analysis. Bovine RPE microsomes were incubated with a peptide corresponding to the Cav-1 scaffolding domain ("Cav-SCD"), with a scrambled control peptide ("Cav-X"), or with no peptide, and total (ouabain-sensitive) Na+/K+-ATPase activity was determined. Separately, microsomes from Cav-1 null (KO) and control mouse eyecups were assayed for Na/K-ATPase activity. Correlative confocal immunohistochemistry of eyes from KO and control mice was performed to localize Na+/K+-ATPase and Cav-1 in RPE. The presence of caveolae in RPE was also assessed by TEM. Gene expression analysis with qRT-PCR validation was performed on KO and control mouse eyecups.
Results: :
Western analysis indicated ~60% of the total Na+/K+-ATPase α1 subunit cofractionates with Cav-1-enriched membranes. Na+/K+-ATPase and Cav-1 colocalized to the base of the apical RPE microvilli in both KO and control mice, coincident with ultrastructurally identified caveolae. Total Na+/K+-ATPase activity was increased 1.7-fold in KO RPE microsomes relative to controls; this corresponded to a decrease in affinity for K+, but not for Na+. Incubation of bovine RPE microsomes with Cav-SCD significantly inhibited Na+/K+-ATPase activity (3-fold, compared to Cav-X). In KO eyecups, microarray, qRT-PCR, and Western analyses revealed dramatic up-regulation of FXYD3, a regulator of Na+/K+-ATPase.
Conclusions: :
Our results suggest that Cav-1 associates with and is an endogenous regulator of Na+/K+-ATPase activity in RPE. The mechanism of this regulation does not involve changes in Na+/K+-ATPase localization or overall protein expression. Rather, regulation appears to be via interaction of Cav-1 with Na+/K+-ATPase.
Keywords: retinal pigment epithelium • NaK ATPase • ion transporters