Function and Regulation of Polycystin-2 and Epithelial Sodium Channel by Qian Wang A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Physiology University of Alberta © Qian Wang, 2016 ABSTRACT 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 ii 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 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 iii 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. iv PREFACE This thesis is an original work of Qian Wang. Chapter 2 of this thesis has been published in the journal of PLoS One in 2012 as ‘Structural interaction and functional regulation of polycystin-2 by filamin’ by Qian Wang, Xiao-Qing Dai, Qiang Li, Zuocheng Wang, Maria del Rocio Cantero, Shu Li, Ji Shen, Jian-Cheng Tu, Horacio Cantiello and Xing-Zhen Chen (Conceived and designed the experiments: QW XQD QL JCT HC XZC; Performed the experiments: QW XQD QL ZW MRC SL JS HC; Analyzed the data: QW XQD QL ZW HC XZC; Contributed reagents/materials/analysis tools: JCT HC XZC; Wrote the paper: QW QL ZW HC XZC). Chapter 3 of this thesis has been published in the journal of PLoS One in 2015 as ‘Filamin-A increases the stability and plasma membrane expression of polycystin-2’ by Qian Wang, Wang Zheng, Zuocheng Wang, JungWoo Yang, Shaimaa Hussein, Jingfeng Tang and Xing-Zhen Chen (Conceived and designed the experiments: QW JFT XZC; Performed the experiments: QW WZ ZCW JWY; Analyzed the data: QW SH JFT; Wrote the paper: QW ZCW JFT XZC). Chapter 4 of this thesis forms part of an international research collaboration project with Dr. Richard Zimmermman from University of Saarland in Germany. The Ca2+ imaging experiments were conducted and analyzed by QW under the help of Dr. Zimmermman and his fellows. Dr. Horacio Cantiello from Universidad de Buenos Aires, Argentina, conducted the lipid bilayer experiments. Chapter 5 of this thesis has been published in Journal of Biological Chemistry in 2013 as ‘Filamin interacts with ENaC and inhibits its channel function’ by Qian Wang, Xiao-Qing Dai, v Qiang Li, Jagdeep Tuli, Genqing Liang, Shayla S.Li, and Xing-Zhen Chen (Conceived and designed the experiments: QW XZC; Performed the experiments: QW XQD QL JT GL SL; Analyzed the data: QW XQD; Wrote the paper: QW QL XZC). Chapter 1 and Chapter 6 are originally written by QW. vi ACKNOWLEDGEMENTS My 5 years’ PhD study is very rich and plentiful. I sincerely appreciated the attentive training and generous support from my supervisor Dr. Xing-Zhen Chen. Thank you for your careful and patient supervision on conducting research on all aspects and providing me a lot of opportunities to learn. Also, I want to express my deepest gratitude to my supervisory committees Dr. Yves Sauve and Dr. Ted Alllison, who have provided pertinent and valuable suggestions toward my PhD project and dissertation completion. Dr. Yves Sauve also taught me electroretinography to conduct research on retina and guide me in scientific writing. I would like to thank our Germany partner Dr. Richard Zimmerman for allowing me to study in his lab for 5 month and learn the Ca2+ imaging technique. I would also like to thank Dr. Klaus Ballanyi for spending time on discussion of a variety of questions. I want to say thank you to the previous lab members Jungwoo Yang, Zuocheng Wang, and current lab members Wang Zheng, Shaimaa Hussein for happy collaboration and knowledge sharing. My family has offered me strong support during my PhD study. I want to express my deep love to my husband, my mother and father. In particular, I would like to thank my uncle, who aroused my passion in biology, inspired me to learn, built my philosophy, and was always there with me facing all kinds of difficulties. Finally I want give a big kiss to my son who will be born in Feburary 2016 and has accompanied me to complete this thesis. vii Table of content CHAPTER 1 ........................................................................................................................... 1 INTRODUCTION .................................................................................................................. 1 1.1 Kidney physiology ........................................................................................................... 2 1.2 ADPKD ............................................................................................................................ 3 1.2.1 Clinical features ......................................................................................................... 3 1.2.2 Molecular mechanism ................................................................................................ 4 1.2.3 PKD1 (PC1) ............................................................................................................... 5 1.2.4 PKD2 (PC2) ............................................................................................................... 6 1.2.4.1 Molecular composition and structure ................................................................. 6 1.2.4.2 Oligomerization .................................................................................................. 9 1.2.4.3 Channel property and subcellular localization