Serine protease activation of near-silent epithelial Na+channels

Abstract

The regulation of epithelial Na+channel (ENaC) function is critical for normal salt and water balance. This regulation is achieved through cell surface insertion/retrieval of channels, by changes in channel open probability ( Po), or through a combination of these processes. Epithelium-derived serine proteases, including channel activating protease (CAP) and prostasin, regulate epithelial Na+transport, but the molecular mechanism is unknown. We tested the hypothesis that extracellular serine proteases activate a near-silent ENaC population resident in the plasma membrane. Single-channel events were recorded in outside-out patches from fibroblasts (NIH/3T3) stably expressing rat α-, β-, and γ-subunits (rENaC), before and during exposure to trypsin, a serine protease homologous to CAP and prostasin. Under baseline conditions, near-silent patches were defined as having rENaC activity ( NPo) < 0.03, where N is the number of channels. Within 1–5 min of 3 μg/ml bath trypsin superfusion, NPoincreased ∼66-fold ( n = 7). In patches observed to contain a single functional channel, trypsin increased Pofrom 0.02 ± 0.01 to 0.57 ± 0.03 ( n = 3, mean ± SE), resulting from the combination of an increased channel open time and decreased channel closed time. Catalytic activity was required for activation of near-silent ENaC. Channel conductance and the Na+/Li+current ratio with trypsin were similar to control values. Modulation of ENaC Poby endogenous epithelial serine proteases is a potentially important regulator of epithelial Na+transport, distinct from the regulation achieved by hormone-induced plasma membrane insertion of channels.