title: Proteolytic regulation of epithelial sodium channels by urokinase plasminogen activator: cutting edge and cleavage sites.
abstract: Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at ((177)GR↓KR(180)) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically "silent") channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions.
authors.affiliation: From the Department of Cellular and Molecular Biology and the Texas Lung Injury Institute, University of Texas Health Science Center, Tyler, Texas 75708, james.ji@uthct.edu.
authors.forename: Hong-Long
authors.initials: HL
authors.lastname: Ji
authors.affiliation: From the Department of Cellular and Molecular Biology and.
authors.forename: Runzhen
authors.initials: R
authors.lastname: Zhao
authors.affiliation: From the Department of Cellular and Molecular Biology and.
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Komissarov
authors.affiliation: the Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, Arizona 85013.
authors.forename: Yongchang
authors.initials: Y
authors.lastname: Chang
authors.affiliation: the College of Public Health, Xinxiang Medical University, Xinxiang, Henan 453100, China, and.
authors.forename: Yongfeng
authors.initials: Y
authors.lastname: Liu
authors.affiliation: the Departments of Medicine and Anesthesia, Cardiovascular Research Institute, University of California, San Francisco, California 94143.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Matthay
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D020134:Catalytic Domain; D053503:Epithelial Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D012964:Sodium / Q000378:metabolism; D015854:Up-Regulation; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D053503:Epithelial Sodium Channels; D017395:Plasminogen Activator Inhibitor 1; D012964:Sodium; D014568:Urokinase-Type Plasminogen Activator
keywords: Electrophysiology; Enzyme Catalysis; Epithelial Sodium Channel (ENaC); Gel Electrophoresis; Plasma Membrane; Proteolysis