Function and Regulation of Polycystin-2 and Epithelial Sodium Channel

  • Author / Creator
    Wang, Qian
  • Polycystin-2, encoded by the PKD2 gene, is mutated in ~15% of autosomal dominant polycystic kidney disease, and functions as a Ca2+ permeable non-selective cation channel. It is mainly localized on the endoplasmic reticulum membrane, and is also present on the plasma membrane and primary cilium. Polycystin-2 is critical for cellular homeostasis and thus a tight regulation of its expression and function is needed. In Chapter 2, filamin-A, a large cytoskeletal actin-binding protein, was identified as a novel polycystin-2 binding partner. Their physical interaction was confirmed by different molecular biology techniques, e.g., yeast two-hybrid, GST pull-down, and co-immunoprecipitation. Filamin-A C terminal fragment (FLNAC) mediates the interaction with both N- and C- termini of polycystin-2. Functional study in lipid bilayer reconstitution system showed that filamin substantially inhibits polycystin-2 channel activity. This study indicates that filamin is an important regulator of polycystin-2 channel function, and further links actin cytoskeletal dynamics to the regulation of this channel. In Chapter 3, further effect of filamin on polycystin-2 stability was studied using filamin-deficient and filamin-A replete human melanoma cells, as well other human cell lines together with filamin-A siRNA/shRNA knockdown. Filamin-A was found to repress polycystin-2 degradation and enhance its total expression and plasma membrane targeting. FLNAC overexpression reduced the physical binding between full length filamin-A and polycystin-2, as well as the expression level of polycystin-2, presumably by competing with filamin-A for binding polycystin-2. Further, filamin-A mediated polycystin-2 binding with actin by forming complex polycystin-2–filamin-A–actin. Finally, the physical interaction of polycystin-2 and filamin-A was found to be Ca2+-dependent, i.e., Ca2+ depletion weakened their binding strength. Taken together, this study indicates that filamin anchors polycystin-2 to the actin cytoskeleton through the polycystin-2–filamin-A–actin complex to reduce degradation and increase stability, and possibly regulates polycystin-2 function in a Ca2+-dependent manner. The dynamic regulation and the net effect of filamin on polycystin-2 were explored in Chapter 4. First, we found that the Ca2+-dependent binding of filamin-A with polycystin-2 N- differs from that with C- terminus. In addition, lipid bilayer experiment showed that filamin does not exhibit an inhibitory effect on polycystin-2 channel activity in the absence of Ca2+. These data indicate that filamin regulates/inhibits polycystin-2 activity in a Ca2+-dependent manner, which is probably through adjusting their physical interaction. The net effect and physiological relevance of polycystin-2-filamin binding were tested by live cell Ca2+ imaging. We found that filamin-A has a net inhibitory effect on polycystin-2 channel function through a combination of expression and functional regulations that are both important in maintaining intracellular Ca2+ homeostasis. The physiological role of filamin-A on regulating polycystin-2 channel function will be further investigated in animal models such as zebrafish and mice in the future study. Epithelial sodium channel (ENaC) in the kidneys mediates Na reabsorption across the epithelium, which is critical for Na+ balance, extracellular volume, and blood pressure. Abnormal ENaC function is associated with pseudohypoaldosteronism type 1, and Liddle syndrome. The channel function of ENaC is known to be regulated by many factors, such as hormones, chemicals and binding partners. Chapter 5 is about the structural interaction and functional regulation of ENaC by filamin. In this study, ENaC-filamin binding was detected by different in vitro and in vivo methods. Biotinylation and co-immunoprecipitation combined assays together revealed the presence of the ENaC-filamin complex on the cell surface. Functional study using Xenopus oocyte expression system and the two-electrode voltage clamp electrophysiology showed that co-expression of an ENaC-binding domain of filamin FLNAC dramatically reduces ENaC channel function. Lipid bilayer electrophysiology further confirmed the inhibition by showing that FLNAC reduces ENaC single channel open probability. This study demonstrated that filamin reduces ENaC channel function through direct interaction on the cell surface. In summary, the studies described in this thesis demonstrated that several properties of channel proteins polycystin-2 and ENaC are regulated by cytoskeleton protein filamin.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Doctor of Philosophy
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  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Physiology
  • Supervisor / co-supervisor and their department(s)
    • Xing-Zhen Chen (Physiology)
  • Examining committee members and their departments
    • Emmanuelle Cordat (Physiology)
    • Leonidas Tsiokas (Department of Cell Biology,University of Oklahoma Health Sciences Center)
    • Larry Fliegel (Biochemistry)
    • Yves Sauve (Physiology)