Distinguishing Roles of the Membrane-Cytoskeleton and Cadherin Mediated Cell-Cell Adhesion in Generating Different Na+,K+-ATPase Distributions in Polarized Epithelia

In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the epithelium. In the choroid plexus, Na+,K+-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion and production of cerebrospinal fluid; in contrast, Na+,K+-ATPase is localized to the basal-lateral membrane of cells in the kidney nephron where it regulates ion and solute reabsorption. The mechanisms involved in restricting Na+,K+-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies have indicated a role for E-cadherin mediated cell-cell adhesion and membrane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K+-ATPase distribution in absorptive kidney epithelial cells. Confocal immunofluorescence microscopy reveals that in chicken and rat choroid plexus epithelium, fodrin, and ankyrin colocalize with Na+,K+-ATPase at the apical plasma membrane, but fodrin, ankyrin, and adducin also localize at the lateral plasma membrane where Na+,K+-ATPase is absent. Biochemical analysis shows that fodrin, ankyrin, and Na+,K+-ATPase are relatively resistant to extraction from cells in buffers containing Triton X-100. The fractions of Na+,K+-ATPase, fodrin, and ankyrin that are extracted from cells cosediment in sucrose gradients at ∼10.5 S. Further separation of the 10.5 S peak of proteins by electrophoresis in nondenaturing polyacrylamide gels revealed that fodrin, ankyrin, and Na+,K+-ATPase comigrate, indicating that these proteins are in a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a marker protein of the basal-lateral plasma membrane in the choroid plexus, did not cosediment in sucrose gradients or comigrate in nondenaturing polyacrylamide gels with the complex of Na+,K+-ATPase, ankyrin, and fodrin. Ca++-dependent cell adhesion molecules (cadherins) were detected at lateral membranes of the choroid plexus epithelium and colocalized with a distinct fraction of ankyrin, fodrin, and adducin. Cadherins did not colocalize with Na+,K+-ATPase and were absent from the apical membrane. The fraction of cadherins that was extracted with buffers containing Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin an