Na+ transport through epithelial cells mediated by the epithelial Na+ channel (ENaC) is important for maintaining body fluid Na+ homeostasis, alveolar fluid clearance and normal airway mucocilliary function. A large body of evidence shows significant correlation between serine protease activity, channel fragmentation and transepithelial movement of Na+. The extracellular protease dependent regulation may play an important role in epithelial cells where the channel activity is intended more for the control of the extracellular environment, such as in airway cells, than in the control of the internal fluid status of the organism. Presented here is evidence supporting the hypothesis that Na+ channels are inserted into the apical membrane as inactive precursors whereupon they are acted upon by membrane resident serine proteases resulting in active channels. The effects of the serine protease inhibitor, aprotinin, on ENaC single channel properties were studied using transepithelial fluctuation analysis in the A6 amphibian kidney epithelium. Aprotinin causes a potent specific time-dependent inhibition of Na+ transport. Analysis of blocker induced fluctuations in Na+ current (INa) showed linear rate-concentration plots with the same blocker on- and off-rates in control and aprotinin inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. It was shown that protease regulation of INa is mediated by increasing the number of active channels in the apical membrane. To test the hypothesis that residues on ENaC mediate protease dependent channel activation ENaC was subjected to site-directed mutagenesis and heterologously expressed in Fisher rat thyroid (FRT) model epithelial cells. Activation by exogenous proteases depended on the presence of substrate specific residues in ENaC which dictated the rates of activation and the steady-state current levels.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07272006-132858 |
| Date | 04 August 2006 |
| Creators | Adebamiro, Adedotun |
| Contributors | Robert J. Bridges, Peter F. Drain, John P. Horn, John P. Johnson, Daniel C. Devor |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-07272006-132858/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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