Protein tyrosine kinase is expressed and regulates ROMK1 location in the cortical collecting duct

Abstract

We previously demonstrated that dietary K intake regulates the expression of Src family PTK, which plays an important role in controlling the expression of ROMK1 in plasma membrane (Wei Y, Bloom P, Lin D-H, Gu RM, and Wang WH. Am J Physiol Renal Physiol 281: F206–F212, 2001). In the present study, we used the immunofluorescence staining technique to demonstrate the presence of c-Src, a member of Src family PTK, in the thick ascending limb (TAL) and collecting duct. Confocal microscopy shows that c-Src is highly expressed in the renal cortex and outer medulla. Moreover, c-Src and ROMK are coexpressed in the same nephron segment. Also, the positive staining of c-Src is visible in tubules stained with Tamm-Horsfall glycoprotein or aquaporin-2. This suggests that c-Src is present in the TAL, cortical collecting duct (CCD), and outer medullary collecting duct (OMCD). To study the role of PTK in the regulation of ROMK membrane expression in the TAL and CCD, we carried out immunocytochemical staining with ROMK antibody in the CCD or TAL from rats on either a high-K (HK) or K-deficient (KD) diet. A sharp membrane staining of ROMK can be observed in the TAL from rats on both HK and KD diets. However, a clear plasma membrane staining can be observed only in the CCD from rats on a HK diet but not from those on a KD diet. Treatment of the CCD from rats on a HK diet with phenylarsine oxide (PAO) decreases the positive staining in the plasma/subapical membrane and increases the ROMK staining in the intracellular compartment. However, PAO treatment did not significantly alter the staining pattern of ROMK in the TAL. Moreover, the biotinylation technique has also confirmed that neither herbimycin A nor PAO has significantly changed the biotin-labeled ROMK2 in HEK293 cells transfected with ROMK2 and c-Src. We conclude that c-Src is expressed in the TAL, CCD, and OMCD and that stimulation of PTK increases the ROMK channels in the intracellular compartment but decreases them in the apical/subapical membrane in the CCD.