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Abstract: TH-PO489

Dissecting the Na/K-ATPase Signaling Complex in Renal Proximal Tubule: A Cross-Linking Approach

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Liu, Jiang, Marshall University School of Medicine, Huntington, West Virginia, United States
  • Bai, Fang, Marshall University School of Medicine, Huntington, West Virginia, United States
  • Pratt, Rebecca, Marshall University School of Medicine, Huntington, West Virginia, United States
  • Xie, Zi-jian, Marshall University, Huntington, West Virginia, United States
  • Shapiro, Joseph I., Marshall University School of Medicine, Huntington, West Virginia, United States
Background

The Na/K-ATPase α1 subunit and c-Src have been shown to form a signaling complex. Activation this signaling cascade regulates sodium handling in proximal tubule, systemic oxidative stress, and uremic cardiomyopathy. The present study is to investigate the formation of the Na/K-ATPase signaling complex, especially the binding of the α1 subunit and c-Src under native condition in live cells.

Methods

Crosslinking studies were performed in live LLC-PK1 cells with sulfhydryle-sulfhydryle crosslinkers BMH (non-cleavable) and DTME (cleavable). SDS-PAGE and WES system were used to determine the crosslinking efficiency and molecular weight shifts under denatured condition. Blue Native gel electrophoresis (BN-PAGE) was used to identify the molecular weight of the complex under native condition. A BN-PAGE/SDS-PAGE 2D system was used to separate and identify the components of the Na/K-ATPase complex by silver staining and Western blot.

Results

(1) Both BMH and DTMH effectively crosslink the α1 subunit and c-Src, demonstrated by Western blot analysis in both SDS-PAGE and WES system. (2) In BN-PAGE system, like in control, both BMH and DTME-treated samples showed protein bands closer to marker of 720 kDa (between 480 and 720 kDa markers). Western blot analysis showed co-existence of the α1 subunit, β1 subunit, c-Src, and caveolin-1. (3) In WES system, like seen in control, there is a clear crosslinking between the α1 subunit and c-Src, by comparing the data amongst BMH and DTME crosslinking treatments, combined with or without DTT/SDS cleavage. (4) In the BN-PAGE/SDS-PAGE 2D system, preliminary data showed that the complex contains the α1 subunit, c-Src, and caveolin-1. (5) Interestingly, pretreatment with pNaKtide (a peptide derived from the α1 subunit), an antagonist of the Na/K-ATPase signaling, increases ouabain-sensitive 86Rb+ uptake in LLC-PK1 cells. Moreover, pNaKtide also co-exists with the Na/K-ATPase complex.

Conclusion

Our data indicated that the Na/K-ATPase signaling complex contains the Na/K-ATPase α1 subunit, c-Src, and caveolin-1. Further studies with mass spectrometry need to be performed to confirm the findings, and might be able to find more component(s) of the complex.

Funding

  • Other NIH Support