title: Filamin interacts with epithelial sodium channel and inhibits its channel function.
abstract: Epithelial sodium channel (ENaC) in the kidneys is critical for Na(+) balance, extracellular volume, and blood pressure. Altered ENaC function is associated with respiratory disorders, pseudohypoaldosteronism type 1, and Liddle syndrome. ENaC is known to interact with components of the cytoskeleton, but the functional roles remain largely unclear. Here, we examined the interaction between ENaC and filamins, important actin filament components. We first discovered by yeast two-hybrid screening that the C termini of ENaC α and β subunits bind filamin A, B, and C, and we then confirmed the binding by in vitro biochemical assays. We demonstrated by co-immunoprecipitation that ENaC, either overexpressed in HEK, HeLa, and melanoma A7 cells or natively expressed in LLC-PK1 and IMCD cells, is in the same complex with native filamin. Furthermore, the biotinylation and co-immunoprecipitation combined assays showed the ENaC-filamin interaction on the cell surface. Using Xenopus oocyte expression and two-electrode voltage clamp electrophysiology, we found that co-expression of an ENaC-binding domain of filamin substantially reduces ENaC channel function. Western blot and immunohistochemistry experiments revealed that the filamin A C terminus (FLNAC) modestly reduces the expression of the ENaC α subunit in oocytes and A7 cells. After normalizing the current by plasma membrane expression, we found that FLNAC results in ~50% reduction in the ENaC channel activity. The inhibitory effect of FLNAC was confirmed by lipid bilayer electrophysiology experiments using purified ENaC and FLNAC proteins, which showed that FLNAC substantially reduces ENaC single channel open probability. Taken together, our study demonstrated that filamin reduces ENaC channel function through direct interaction on the cell surface.
authors.affiliation: Membrane Protein Disease Research Group, Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.forename: Xiao-Qing
authors.initials: XQ
authors.lastname: Dai
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.forename: Jagdeep
authors.initials: J
authors.lastname: Tuli
authors.forename: Gengqing
authors.initials: G
authors.lastname: Liang
authors.forename: Shayla S
authors.initials: SS
authors.lastname: Li
authors.forename: Xing-Zhen
authors.initials: XZ
authors.lastname: Chen
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D003285:Contractile Proteins / Q000737:chemistry*; D003599:Cytoskeleton / Q000378:metabolism; D004285:Dogs; D053503:Epithelial Sodium Channels / Q000737:chemistry*; D064448:Filamins; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006706:Homeostasis; D006801:Humans; D007668:Kidney / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry*; D009865:Oocytes / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism; D013552:Swine; D020798:Two-Hybrid System Techniques; D014981:Xenopus
chemical_list: D003285:Contractile Proteins; D053503:Epithelial Sodium Channels; D064448:Filamins; D008840:Microfilament Proteins; D015222:Sodium Channels; D012964:Sodium; D005982:Glutathione Transferase