COMMD10 in epithelial sodium channel (ENaC) trafficking
The epithelial sodium channel (ENaC), is assembled as a heterotrimer composed of three homologous subunits α or δ, β, and γ and is selectively permeable to Na+ ions. ENaC plays an essential role in the regulation of sodium transport in the renal connecting tubule and collecting duct. Mutations in ENaC subunits cause various disorders including the low and high blood pressure conditions pseudohypoaldosteronism-I and Liddle syndrome respectively. In renal principal cells, ENaC cell surface population is tightly regulated by hormones such as aldosterone and vasopressin, and through protein trafficking pathways that transport ENaC to and from the cell surface. ENaC is internalized from the plasma membrane mainly via clathrin-coated vesicles after ubiquitination of ENaC subunits. Like most endocytic cargos, ENaC can follow the degradative pathway promoted by ubiquitin fusion or a recycling pathway after deubiquitination and sorting on early endosomes (EE). One of the endosomal protein sorting machines is the retromer complex that concentrates membrane proteins into nascent tubules formed through the action of sorting nexin (SNX-BAR) proteins. Thus, on the early endosomes, retromer binds to its cargos through SNX3, then recruits Wiskott–Aldrich syndrome and SCAR homologue (WASH) complex to endosomes which mediates CCC (COMMD1-10/CCDC22/CCDC93) complex recruitment in turn. The WASH complex promotes the formation of endosomal branched F-actin networks and stabilizes recycling tubules for cargo entry. For the release of tubules, the CCC complex phosphorylates myotubularin-related protein 2 (MTMR2). MTMR2 then converts endosomal phosphatidylinositol-3-phosphate (PI(3)P) to phosphoinositide (PI) which causes disassembling of WASH complex that likely leads to the endosomal actin depletion and thereby release of cargo tubules. Previous studies in our lab showed that all COMMD (COpper Metabolism MURR1 or COMM domain containing) family proteins of the CCC complex interact with ENaC and further studies revealed that when COMMD1, -3 and -9 were overexpressed but interestingly when COMMD10 knocked down, ENaC surface activity is reduced. The aim of this study is to investigate the role of COMMDs in ENaC trafficking. For this study, Fischer Rat Thyroid (FRT) cells with stable COMMD10 knockdown (C10 KD), mouse cortical collecting duct (mCCDcl1), and U2OS cells were used. The C10 KD FRT epithelia were transiently transfected with αβγ-ENaC for ENaC study. Cell surface activity of ENaC was studied by Ussing assay measuring ENaC amiloride-sensitive current (Isc). Then cell surface population and internalization of ENaC was quantified by cell surface biotinylation and endocytosis assay, respectively and the results suggested that ENaC cell surface population is reduced by COMMD10 KD. Endogenous ENaC cell surface activity was also studied and the results suggested that transient COMMD10 KD also reduces ENaC cell surface activity in the mCCDcl1 cell line. Further, recycling assays suggested the reduction of ENaC cell surface population is linked with a recycling defect deriving from COMMD10 depletion. Immunocytochemistry was used to show the endosomal localization of COMMD10 on Rab5-, and Rab7-positive endosomes confirming a role for COMMD10 in endosomal sorting, while localization of COMMD10 on Rab11-positive endosomes proposes that COMMD10 may be involved in the translocation of recycling vesicles to the plasma membrane. To investigate regulation of COMMDs the effect of aldosterone on COMMD mRNA levels was tested and showed no significant changes, while aldosterone reduced protein levels of COMMD1 and 10. In contrast, calcium treatment induced protein levels of COMMD1 and 10. The findings of this study furthers our understanding of the role of COMMD10 in ENaC trafficking, endosomal sorting and recycling of ENaC. Understanding this pathway could lead to advancing knowledge in diseases like hypertension.
Advisor: McDonald, Fiona
Degree Name: Doctor of Philosophy
Degree Discipline: Physiology
Publisher: University of Otago
Keywords: New Zealand; epithelial sodium channel, ENaC, COMMD, COMMD10, endosomal recycling, protein trafficking, early endosome, recycling endosome, Rab, retromer
Research Type: Thesis