Dissociation of the Spectrin-Ankyrin Complex as a Basis for the Loss of Na,K- ATPase Polarity Following Ischemic Injury in MDCK Cells

The polarized distribution of Na,K-ATPase at the basolateral membranes of renal tubule epithelial cells is maintained via a tethering interaction with the underlying spectrinankyrin cytoskeleton. In this study, we have explored the mechanism underlying the loss of Na,K-ATPase polarity following ischemic injury in MDCK cells, utilizing a novel antibody raised against a recently-described kidney-specific isoform of ankyrin. In control MDCK cells, ankyrin was co-localized with Na,K-ATPase at the basolateral membrane. ATP depletion resulted in a duration-dependent mislocation of Na,K-ATPase and ankyrin throughout the cytoplasm. Co-localization studies showed partial overlap between the distribution of ankyrin and Na,K-ATPase at all periods following ATP depletion. By immunoprecipitation with anti-ankyrin antibody, the mislocated Na,KATPase remained bound to ankyrin at all time points following ATP depletion. However, the interaction between ankyrin and spectrin was markedly diminished within 3 h of ATP depletion, and was completely lost after 6 h. In solution binding assays using a fusion peptide of GST with the ankyrin binding domain of Na,K-ATPase, a complex with ankyrin was detected at all time points following ATP depletion, but spectrin was lost from the complex in a duration-dependent manner. The loss of spectrin binding was not attributable to spectrin degradation, but was associated with hyperphosphorylation of ankyrin. The results suggest that a dissociation of the membrane-cytoskeleton complex at the spectrin-ankyrin interface may contribute to the loss of Na,K-ATPase polarity following ischemic injury, and reaffirm a critical adapter role for ankyrin in the normal maintenance of Na,K-ATPase polarity.