Electrogenic transepithelial Na+ transport is mediated by an apical membrane, amiloride sensitive Na+ channel (ENaC) and plays an important role in the physiology of a number of organs including the kidney, colon, sa1ivary, and sweat glands as well as the lungs. Sodium transport in the lungs has an important impact on mucociliary clearance, which is impaired in a number respiratory diseases including cystic fibrosis, chronic obstructive pulmonary disease, asthma and acute and chronic bronchitis. Pharmacological modulation of Na+ transport is expected to improve mucociliary clearance in these diseases. In this application, we present results that demonstrate 70 percent of Na+ transport, in primary cultures of human bronchial epithelial cells (HBE), is regulated by a novel extracellular protease-mediated mechanism. We will show that HBE cells express an endogenous protease, prostasin, that we hypothesize is the ENaC channel activating protease (CAP). In addition, we will show that an endogenous protease inhibitor, bikunin, inhibits Na+ transport and this inhibitory effect can be reversed upon washout of the inhibitor or by the addition of exogenous protease. We are convinced this protease/protease inhibitor mediated mechanism represents a paradigm shift in how we view the regulation of ENaC with important basic science and therapeutic implications.
Our specific aims are designed to test several hypothesis predicated on a model we have proposed to explain the mechanism of protease/protease inhibitor mediated regulation of ENaC in the airways. The hypotheses we will test include: (i) prostasin is the HBE CAP, (ii) that prostasin directly binds to ENaC and proteolysis one or more of the subunits to cause ENaC activation, (iii) that inactive ENaC is inserted into the apical membrane where it is activated by prostasin and has a short half-life as an active channel before being retrieved and degraded, (iv) that bikunin inhibits prostasin via its Kunitz' domains and (v) that the binding site on ENaC for prostasin is at the Kunitz-like domain in the second cysteine rich domain of the channel. We will use short circuit current measurements, fluctuation analysis, site directed mutagenesis protein biochemical and high resolution field emission scanning electron microscopy methods to test our hypotheses. The successful completion of our proposed studies is certain to provide new an important insight about how ENaC is regulated by this novel extracellular mechanism.
| Adebamiro, Adedotun; Cheng, Yi; Rao, U Subrahmanyeswara et al. (2007) A segment of gamma ENaC mediates elastase activation of Na+ transport. J Gen Physiol 130:611-29 |
| Adebamiro, Adedotun; Cheng, Yi; Johnson, John P et al. (2005) Endogenous protease activation of ENaC: effect of serine protease inhibition on ENaC single channel properties. J Gen Physiol 126:339-52 |
