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Adebamiro, Adedotun (2006) SERINE PROTEASE REGULATION OF THE EPITHELIAL SODIUM CHANNEL. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Adebamiro, Adedotunadast15@pitt.eduADAST15
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairDevor, Daniel Cdd2@pitt.eduDD2
Committee MemberHorn, John Pjph@pitt.eduJPH
Committee MemberJohnson, John Pjohnson@dom.pitt.eduJOHNSONJ
Committee MemberDrain, Peter Fdrain@pitt.eduDRAIN
Committee MemberBridges, Robert
Date: 4 August 2006
Date Type: Completion
Defense Date: 13 July 2006
Approval Date: 4 August 2006
Submission Date: 27 July 2006
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Cell Biology and Molecular Physiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: aprotinin; epithelial sodium channel; noise analysis; serine protease; short-circuit current; sodium transport
Other ID:, etd-07272006-132858
Date Deposited: 10 Nov 2011 19:54
Last Modified: 15 Nov 2016 13:47


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