Lateral mobility of Na,K-ATPase and membrane lipids in renal cells. Importance of cytoskeletal integrity

Because membrane fluidity is an important determinant of membrane function, the lateral diffusion rate (D_L) of the membrane protein Na,K-ATPase was determined in intact renal proximal tubule epithelial cells by the technique of fluorescence redistribution after photobleaching (FRAP). In normal cells the D_Lof Na,K-ATPase in the basal membrane was 3.31×10^-10 cm²/ sec. Treatment with cytochalasin D to promote actin filament depolymerization caused a sevenfold increase in D_L. Exposure of cells to a Ca²⁺-free medium or to hypoxia and reoxygenation, which have similar disruptive effects on the cytoskeleton, also caused increases in D_L. Disruption of actin microfilament structure also increased the mobile fraction of Na,K-ATPase. Using a confocal laser microscopic technique only 14.9% of total Na,K-ATPase was observed to reside in the apical membrane domain of normal cells. Microfilament depolymerization caused this fraction to increase to 47.7%. Thus, the translocation of Na,K-ATPase from the basolateral to the apical domain induced by cytoskeletal protein dysfunction was enabled by an increased rate of lateral diffusion of Na,K-ATPase. The behavior of a variety of membrane lipids following actin depolymerization was more heterogeneous. Some lipids showed a similar increase in D_Lwhereas others showed very little dependence upon the cytoskeleton for lateral restraint.