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WATER AND ELECTROLYTE HOMEOSTASIS

Role of lipid rafts in membrane delivery of renal epithelial Na⁺-K⁺-ATPase, thick ascending limb

¹Department of Anatomy and ²Biomedical Research Centre, Charité Universitätsmedizin Berlin, Berlin; and ³Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany

Submitted 10 March 2006 ; accepted in final form 20 October 2006

Lipid rafts are cholesterol- and shingolipid-enriched membrane microdomains implicated in membrane signaling and trafficking. To assess renal epithelial raft functions through the characterization of their associated membrane proteins, we have isolated lipid rafts from rat kidney by sucrose gradient fractionation after detergent treatment. The low-density fraction was enriched in cholesterol, sphingolipid, and flotillin-1 known as lipid raft markers. Based on proteomic analysis of the low-density fraction, the protein with the highest significance score was the -subunit of Na⁺-K⁺-ATPase (NKA), whose raft association was validated by simultaneous immunoblotting. The -subunit of NKA was copurified from the low-density fraction. To test the role of lipid rafts in sorting and membrane delivery of renal-transporting epithelia, we have chosen to study thick ascending limb (TAL) epithelium for its high NKA activity and the property to be stimulated by antidiuretic hormone (ADH). Cultured rabbit TAL cells were studied. Cholesterol depletion and detergent extraction at warmth caused a shift of NKA to the higher-density fractions. Comparative preparations from blood monocytes revealed the absence of NKA from rafts in these nonpolarized cells. Short-term exposure of rabbit TAL cells to ADH (1 h) caused translocation and enhanced raft association of NKA via cAMP activation. Preceding cholesterol depletion prevented this effect. TAL-specific, glycosylphosphatidylinositol-anchored Tamm Horsfall protein was copurified with NKA in the same raft fraction, suggesting functional interference between these products. These results may have functional implications regarding the turnover, trafficking, and regulated surface expression of NKA as the major basolateral ion transporter of TAL.

protein sorting; antidiuretic hormone; detergent resistant microdomains; sodium pump

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