Dissociation of spectrin-ankyrin complex as a basis for loss of Na-K-ATPase polarity after ischemia.
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 spectrin-ankyrin cytoskeleton. In this study, we have explored the mechanism underlying the loss of Na-K-ATPase polarity after ischemic injury in Madin-Darby canine kidney (MDCK) cells, utilizing a novel antibody raised against a recently described kidney-specific isoform of ankyrin. In control MDCK cells, ankyrin was colocalized 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. Colocalization studies showed a partial overlap between the distribution of ankyrin and Na-K-ATPase at all periods after ATP depletion. By immunoprecipitation with anti-ankyrin antibody, the mislocated Na-K-ATPase remained bound to ankyrin at all time points after 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 glutathione S-transferase with the ankyrin binding domain of Na-K-ATPase, a complex with ankyrin was detected at all time points after 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 after ischemic injury and reaffirm a critical adapter role for ankyrin in the normal maintenance of Na-K-ATPase polarity.
Woroniecki
Robert
R
Division of Pediatric Nephrology, Albert Einstein College of Medicine, New York, New York 10467, USA.
Ferdinand
Jean R
JR
Morrow
Jon S
JS
Devarajan
Prasad
P
D000255:Adenosine Triphosphate / Q000172:deficiency; D000818:Animals; D017487:Ankyrins / Q000378:metabolism*; D002460:Cell Line; D004285:Dogs; D007511:Ischemia / Q000378:metabolism*; D007684:Kidney Tubules / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D012079:Renal Circulation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013049:Spectrin / Q000378:metabolism*; D013997:Time Factors; D014018:Tissue Distribution
D017487:Ankyrins; D013049:Spectrin; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase