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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.sub.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.sub.Na] 6-10-fold in the Xenopus oocyte expression system. [I.sub.Na] and the number (N) of channels expressed at the cell surface (measured by binding of a FLAG monoclonal [I.sup.125]-radioiodinated antibody) were measured in the same oocyte. Using this assay, we show that mCAP1 increases [I.sub.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 (wc[P.sub.o]). The serum- and glucocorticoid-regulated kinase (Sgk1) involved in the aldosterone-dependent signaling cascade enhances [I.sub.Na] by 2.5-fold (P < 0.001) and N by 1.6-fold (P < 0.001), indicating a dual effect on N and wc[P.sub.o]. Compared with Sgk1 alone, coexpression of Sgk1 with mCAP1 leads to a ninefold increase in [I.sub.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.sub.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. KEY WORDS: epithelial sodium channel * amiloride * serum glucocorticoid-regulated kinase 1 (Sgk1) * aldosterone * NEDD4

Aldosterone controls sodium balance by regulating an epithelial sodium channel (ENaC)-mediated sodium transport along the aldosterone-sensitive distal nephron, which expresses both mineralocorticoid (MR) and glucocorticoid receptors (GR). Mineralocorticoid specificity is ensured by 11beta-hydroxysteroid dehydrogenase type 2, which metabolizes cortisol or corticosterone into inactive metabolites that are unable to bind MR and/or GR. The fractional occupancy of MR and GR by aldosterone mediating the sodium transport response in the aldosterone-sensitive distal nephron cannot be studied in vivo. For answering this question, a novel mouse cortical collecting duct cell line (mCCD(cl1)), which expresses significant levels of MR and GR and a robust aldosterone sodium transport response, was used. Aldosterone elicited a biphasic response: Low doses (K(1/2) = approximately 0.5 nM) induced a transient and early increase of sodium transport (peaking at 3 h), whereas high doses (K(1/2) = approximately 90 nM) entailed an approximately threefold larger, long-lasting response. At 3 h, the corticosterone dose-response curve was shifted to the right compared with that of aldosterone by more than two log concentrations, an effect that was fully reverted in the presence of the 11beta-hydroxysteroid dehydrogenase type 2 inhibitor carbenoxolone. Low doses of dexamethasone (0.1 to 1 nM) failed to induce an early response, but high doses elicited a long-lasting response (K(1/2) = approximately 8 nM), similar to that observed for high aldosterone concentrations. Equilibrium binding assays showed that both aldosterone and corticosterone bind to a high-affinity, low-capacity site, whereas dexamethasone binds to one site. Within the physiologic range of aldosterone concentrations, sodium transport is predicted to be controlled by MR occupancy during circadian cycles and by MR and GR occupancy during salt restriction or acute stress.