Distribution of epithelial ankyrin (Ank3) spliceoforms in renal proximal and distal tubules.

ABSTRACT

In diverse cell types, ankyrin tethers a variety of ion transport and cell adhesion molecules to the spectrin-based membrane skeleton. In the whole kidney, epithelial ankyrin (Ank3) is the predominantly expressed ankyrin and is expressed as distinct spliceoforms. Antibodies against a portion of the Ank3 regulatory domain detected four major spliceoforms at 215, 200, 170, and 120 kDa. Immunoblotting of the renal cortex, which is 80% proximal tubule (PT), detected all four spliceoforms but showed significantly diminished Ank3(200/215). To determine the Ank3 spliceoforms present in the mouse PT cells, PT fragments were purified to 100% from the renal cortex. Isolation was performed by incubating cortical tubule segments with fluorescein and isolating the fluorescein-laden PT fragments or fluorescein-deplete non-PT (distal) fragments under fluorescence microscopy. Distal tubule (DT) fragments displayed abundance of the Ank3(200/215) but no Ank3(170) or Ank3(120). Isolated PT segments contained all four spliceoforms but dramatically diminished Ank3(200/215). These larger spliceoforms bind Na-K-ATPase in diverse cell types. Densitometric analysis of Ank3(200/215) and Na-K-ATPase abundance measured a lower Ank3(200/215)-to-Na-K-ATPase ratio in the PT vs. the renal cortex. These proximal vs. distal differences in Ank3 spliceoforms were displayed in LLC-PK1 cells, a proximal cell line, and MDCK cells, a distal cell line. The lower PT content of Ank3(200/215) suggests Na-K-ATPase in PT may be organized differently than in DT. Likely reflecting their cell-specific organization, regulation, and function, these studies indicate the different renal cell types express distinct Ank3 spliceoforms.