Serine protease activation of near-silent epithelial Na+ channels.
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 alpha-, beta-, and gamma-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 microg/ml bath trypsin superfusion, NPo increased approximately 66-fold (n = 7). In patches observed to contain a single functional channel, trypsin increased Po from 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 Po by 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.
Caldwell
Ray A
RA
Cystic Fibrosis/Pulmonary Research and Treatment Center, CB#7248, University of North Carolina, Chapel Hill, NC 27599-7248, USA. ray_caldwell@med.unc.edu
Boucher
Richard C
RC
Stutts
M Jackson
MJ
D016475:3T3 Cells; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D053503:Epithelial Sodium Channels; D051379:Mice; D018408:Patch-Clamp Techniques; D051381:Rats; D012697:Serine Endopeptidases / Q000502:physiology*; D015222:Sodium Channels / Q000378:metabolism / Q000502:physiology*; D014357:Trypsin / Q000494:pharmacology
D053503:Epithelial Sodium Channels; D015222:Sodium Channels; D012697:Serine Endopeptidases; D014357:Trypsin