Date of Degree
PhD (Doctor of Philosophy)
Molecular Physiology and Biophysics
Peter M. Snyder
The epithelial sodium channel, ENaC, forms the rate-limiting step for sodium reabsorption in the cortical collecting duct of the kidney. It is known that ENaC is important in maintaining fluid homeostasis and ultimately blood pressure as mutations in ENaC result in inherited forms of hyper- and hypotension (Liddle's syndrome and Pseudohypoaldosteronism (PHA type I), respectively). Clinically, ENaC activity can be blocked by treatment with the potassium sparing diuretic amiloride. However, due to difficulties in dosing and the transient nature of channel block, treatment goals are seldom achieved. It is, therefore, necessary to better understand the function and regulation of ENaC activity.
ENaC is a member of the DEG/ENaC family of ion channels. Each family member is composed of multiple subunits - each subunit contains two transmembrane domains, short cytoplasmic amino and carboxy termini, and a relatively large extracellular domain. ENaC is a heterotrimer of homologous subunits Α-,Β-, and ΓENaC. ENaC is a constitutively active ion channel. It is not ligand gated or voltage activated. However, channel activity can be modulated by a variety of stimuli. I hypothesize that the extracellular domain functions as a sensor, allowing the channel to detect and respond to changes in extracellular conditions.
To test this, we expressed human ΑΒΓENaC in Xenopus oocytes and used the two-electrode voltage clamp technique to measure changes in ENaC activity in response to changing extracellular conditions. Using this technique, I identified several novel means of regulating ENaC activity. I found that ENaC activity can be rapidly and reversibly stimulated or suppressed in response to extracellular acidification depending on the balance of extracellular sodium and chloride concentrations and have identified several key residues involved. I found that extracellular chloride inhibits ENaC activity through putative binding sites in the extracellular domain located between the Α- and Β- and Β- and ΓENaC subunits. This allowed us to determine that ENaC adopts an ΑΓΒ channel architecture. Additionally, I have made progress in understanding channel movement by identifying length dependent intersubunit interactions that alter channel gating. Based on our data we conclude that the extracellular domain is integral to modulation of channel activity. The work described herein has significantly advanced the field by improving our understanding of ENaC structure and function.
Copyright 2011 Daniel Mohr Collier