Synergistic Activation of ENaC by Three Membrane-bound Channel-activating Serine Proteases (mCAP1, mCAP2, and mCAP3) and Serum- and Glucocorticoid-regulated Kinase (Sgk1) in Xenopus Oocytes

Sodium balance is maintained by the precise regulation of the activity of the epithelial sodium channel (ENaC) in the kidney. We have recently reported an extracellular activation of ENaC-mediated sodium transport (I_Na) by a GPI-anchored serine protease (mouse channel–activating protein, mCAP1) that was isolated from a cortical collecting duct cell line derived from mouse kidney. In the present study, we have identified two additional membrane-bound serine proteases (mCAP2 and mCAP3) that are expressed in the same cell line. We show that each of these proteases is able to increase I_Na 6–10-fold in the Xenopus oocyte expression system. I_Na and the number (N) of channels expressed at the cell surface (measured by binding of a FLAG monoclonal I¹²⁵-radioiodinated antibody) were measured in the same oocyte. Using this assay, we show that mCAP1 increases I_Na 10-fold (P < 0.001) but N remained unchanged (P = 0.9), indicating that mCAP1 regulates ENaC activity by increasing its average open probability of the whole cell (wcP_o). The serum- and glucocorticoid-regulated kinase (Sgk1) involved in the aldosterone-dependent signaling cascade enhances I_Na by 2.5-fold (P < 0.001) and N by 1.6-fold (P < 0.001), indicating a dual effect on N and wcP_o. Compared with Sgk1 alone, coexpression of Sgk1 with mCAP1 leads to a ninefold increase in I_Na (P < 0.001) and 1.3-fold in N (P < 0.02). Similar results were observed for mCAP2 and mCAP3. The synergism between CAPs and Sgk1 on I_Na was always more than additive, indicating a true potentiation. The synergistic effect of the two activation pathways allows a large dynamic range for ENaC-mediated sodium regulation crucial for a tight control of sodium homeostasis.