Prostasin-dependent activation of epithelial Na+ channels by low plasmin concentrations.
Several pathophysiological conditions, including nephrotic syndrome, are characterized by increased renal activity of the epithelial Na(+) channel (ENaC). We recently identified plasmin in nephrotic urine as a stimulator of ENaC activity and undertook this study to investigate the mechanism by which plasmin stimulates ENaC activity. Cy3-labeled plasmin was found to bind to the surface of the mouse cortical collecting duct cell line, M-1. Binding depended on a glycosylphosphatidylinositol (GPI)-anchored protein. Biotin-label transfer showed that plasmin interacted with the GPI-anchored protein prostasin on M-1 cells and that plasmin cleaved prostasin. Prostasin activates ENaC by cleavage of the gamma-subunit, which releases an inhibitory peptide from the extracellular domain. Removal of GPI-anchored proteins from the M-1 cells with phosphatidylinositol-specific phospholipase C (PI-PLC) inhibited plasmin-stimulated ENaC current in monolayers of M-1 cells at low plasmin concentration (1-4 microg/ml). At a high plasmin concentration of 30 microg/ml, there was no difference between cell layers treated with or without PI-PLC. Knockdown of prostasin attenuated binding of plasmin to M1 cells and blocked plasmin-stimulated ENaC current in single M-1 cells, as measured by whole-cell patch clamp. In M-1 cells expressing heterologous FLAG-tagged prostasin, gammaENaC and prostasin were colocalized. A monoclonal antibody directed against the inhibitory peptide of gammaENaC produced specific immunofluorescence labeling of M-1 cells. Pretreatment with plasmin abolished labeling of M-1 cells in a prostasin-dependent way. We conclude that, at low concentrations, plasmin interacts with GPI-anchored prostasin, which leads to cleavage of the gamma-subunit and activation of ENaC, while at higher concentrations, plasmin directly activates ENaC.
Department of Physiology and Pharmacology, Institute of Medical Biology, Univeristy of Southern Denmark, DK-5000 Odense C., Denmark.
Per
P
Svenningsen
Torben R
TR
Uhrenholt
Yaseelan
Y
Palarasah
Karsten
K
Skjødt
Boye L
BL
Jensen
Ole
O
Skøtt
D000818:Animals; D019921:Biotinylation; D002232:Carbocyanines / Q000378:metabolism; D002460:Cell Line; D004195:Disease Models, Animal; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000378:metabolism; D015640:Ion Channel Gating; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D008564:Membrane Potentials; D051379:Mice; D009404:Nephrotic Syndrome / Q000652:urine; D009842:Oligopeptides; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C / Q000378:metabolism; D011485:Protein Binding; D034622:RNA Interference; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D000099271:Prostasin
D002232:Carbocyanines; D053503:Epithelial Sodium Channels; D005341:Fibrinolysin; D005456:Fluorescent Dyes; D009842:Oligopeptides; D010455:Peptides; D054801:Phosphoinositide Phospholipase C; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D000099271:Prostasin; C507053:Scnn1g protein, mouse; C089187:cyanine dye 3; C085706:FLAG peptide