Cell-Surface Expression of the Channel Activating Protease xCAP-1 Is Required for Activation of ENaC in the (2002)

Sodium balance, extracellular fluid volume, and ul- timately BP are maintained by precise regulation of the activity of the epithelial sodium channel (ENaC). Using a functional complementation assay in the Xenopus laevis oocyte expres- sion system, a channel-activating protease (CAP-1) that in- creases ENaC activity two to threefold in the Xenopus oocyte expression system is here identified. External application of trypsin mimics the effect of Xenopus CAP-1 (xCAP-1) on ENaC activity, which can be blocked by aprotinin, a serine protease inhibitor, suggesting the existence of a novel extra- cellular pathway for controlling ENaC activity. Sequence anal- ysis predicts that CAP-1 is a secreted and/or glycosyl-phos- phatidyl-inositol (GPI)-anchored protein. The aim of the present study was to determine whether cell-surface expression of xCAP-1 is required for ENaC activation. By site-directed mutagenesis of xCAP-1, the importance of the catalytic site, N-glycosylation, and the GPI anchor of xCAP-1 on ENaC activity were analyzed. Glycosylation or catalytic activity is not required for cell-surface expression of xCAP-1, whereas the deletion of the GPI anchor consensus motif at the C- terminus of xCAP-1 (G305Stop) abolishes cell-surface expres- sion and ENaC activation. G305Stop-mutated xCAP-1 is re- covered as a secreted protein in the external medium. A catalytic mutant of xCAP-1 significantly decreased ENaC ac- tivation but did not fully abolish the effect of xCAP-1. The data indicate the critical role of the GPI anchor in ENaC activation and suggest that catalytic and noncatalytic mechanisms are involved. The epithelial amiloride-sensitive sodium channel (ENaC) is expressed at the apical membrane of the epithelial cells of aldosterone-sensitive organs, such as the kidney, the colon, and the sweat and salivary glands (1). In the kidney, ENaC medi- ates the aldosterone-dependent sodium reabsorption in the dis- tal part of the nephron, thereby playing a major role in the control of sodium balance and BP. We have recently identified a Xenopus serine protease (xCAP-1) that increases ENaC ac- tivity when coexpressed with ENaC , , and subunits in the Xenopus oocyte (2,3). The analysis of xCAP-1 primary se- quence indicates that this protein is a serine protease with its characteristic histidine-aspartate-serine catalytic triad (HDS). The sequence analysis predicts one asparagine-linked glyco- sylation site and a glycosyl-phosphatidyl-inositol (GPI) con- sensus motif, indicating that the protein may be anchored at the outer leaflet of the membrane bilayer (4). This observation suggests that xCAP-1 activates ENaC by its extracellular serine protease activity at the plasma membrane. In addition, patch clamp experiments have shown that extracellular application of trypsin inside the pipette can mimic xCAP-1 activation while trypsin outside the pipette is unable to activate ENaC (5). Overall, the data suggest that xCAP-1 is colocalized with ENaC channel protein and that the serine protease acts directly on ENaC by its catalytic activity and/or by protein-protein interaction with the extracellular domain of ENaC or an asso- ciated protein. The aim of this study was to elucidate structural features of xCAP-1 that are important for ENaC activation in the Xenopus oocyte system. Site-directed mutagenesis of the catalytic triad, the N-glycosylation site, and the GPI-anchored motif demon- strate the critical role of the GPI anchor to observe ENaC activation at the plasma membrane.