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ARTICLE |
ENaC Mediates Elastase Activation of Na+ Transport
Correspondence to Robert J. Bridges: bob.bridges{at}rosalindfranklin.edu
The epithelial Na+ channel (ENaC) that mediates regulated Na+ reabsorption by epithelial cells in the kidney and lungs can be activated by endogenous proteases such as channel activating protease 1 and exogenous proteases such as trypsin and neutrophil elastase (NE). The mechanism by which exogenous proteases activate the channel is unknown. To test the hypothesis that residues on ENaC mediate protease-dependent channel activation wild-type and mutant ENaC were stably expressed in the FRT epithelial cell line using a tripromoter human ENaC construct, and protease-induced short-circuit current activation was measured in aprotinin-treated cells. The amiloride-sensitive short circuit current (INa) was stimulated by aldosterone (1.5-fold) and dexamethasone (8-fold). Dexamethasone-treated cells were used for all subsequent studies. The serum protease inhibitor aprotinin decreased baseline INa by approximately 50% and INa could be restored to baseline control values by the exogenous addition of trypsin, NE, and porcine pancreatic elastase (PE) but not by thrombin. All protease experiments were thus performed after exposure to aprotinin. Because NE recognition of substrates occurs with a preference for binding valines at the active site, several valines in the extracellular loops of 
and 
ENaC were sequentially substituted with glycines. This scan yielded two valine residues in ENaC at positions 182 and 193 that resulted in inhibited responses to NE when simultaneously changed to other amino acids. The mutations resulted in decreased rates of activation and decreased activated steady-state current levels. There was an 
20-fold difference in activation efficiency of NE against wild-type ENaC compared to a mutant with glycine substitutions at positions 182 and 193. However, the mutants remain susceptible to activation by trypsin and the related elastase, PE. Alanine is the preferred P1 position residue for PE and substitution of alanine 190 in the subunit eliminated INa activation by PE. Further, substitution with a novel thrombin consensus sequence (LVPRG) beginning at residue 186 in the subunit (Th) allowed for INa activation by thrombin, whereas wild-type ENaC was unresponsive. MALDI-TOF mass spectrometric evaluation of proteolytic digests of a 23-mer peptide encompassing the identified residues (T176-S198) showed that hydrolysis occurred between residues V193 and M194 for NE and between A190 and S191 for PE. In vitro translation studies demonstrated thrombin cleaved the Th but not the wild-type subunit. These results demonstrate that subunit valines 182 and 193 are critical for channel activation by NE, alanine 190 is critical for channel activation by PE, and that channel activation can be achieved by inserting a novel thrombin consensus sequence. These results support the conclusion that protease binding and perhaps cleavage of the subunit results in ENaC activation.
Abbreviations used in this paper: CAP, channel activating protease; ENaC, epithelial sodium channel; FRT, fisher rat thyroid; NE, human neutrophil elastase; PE, porcine pancreatic elastase; TFA, trifluoroacetic acid; TH, human alpha thrombin.
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