Abstract
Regulation of renal sodium (Na+) excretion is crucial for the maintenance of extracellular salt and volume homeostasis and thus for blood pressure control. The epithelial sodium channel (ENaC) is composed of three subunits; α, β and γ; each subunit contains two transmembrane domains where both C- and N-terminal domains are cytoplasmic and allow interaction with regulatory proteins. For its sodium regulating properties, ENaC is principally present in the kidney collecting duct, reabsorbing ions from the urine to prevent unnecessary loss of salt and hence, water. This makes the channel vital for maintaining ECF homeostasis and consequently blood pressure. Channel activity is highly dependent on the density at the apical membrane, where sodium current is proportional to channel number. Dysregulation of ENaC or its associated trafficking proteins can lead to an array of problems which disrupt sodium homeostasis, leading to hypo/hypertension.
Although extensive research has gone into unravelling the downregulation of ENaC by endocytosis, there has been significantly less research into its exocytosis. The p11 protein is known to promote exocytosis of a number of other membrane channels. We hypothesized that addition of p11 would cause an increase ENaC trafficking, and subsequently increase the amiloride sensitive current in Xenopus laevis oocytes.
Previous research at the University of Otago confirmed the presence of an interaction between p11 and ENaC, and also identified that p11 is expressed endogenously within epithelial cells. To confirm a functional consequence of this interaction electrophysiological experiments were conducted. First, Xenopus laevis oocytes were injected with mRNA coding for α, β and γ-ENaC alone or together with mRNA coding for p11. Two electrode voltage clamp was carried out to measure ENaC current. Results from my experiments showed an increase in the amiloride sensitive current in the presence of p11 at both 0.75ng (12%) and 1.50ng (17%) p11 concentrations, however the results were insignificant for both 0.75ng (p=0.46) and 1.5ng (p=0.24). Preliminary results from the Condliffe lab show increased amiloride sensitive current for oocytes co-expressing ENaC + p11 as compared to oocytes expressing ENaC alone, indicating, that the presence of p11 promotes ENaC membrane insertion. Proteins from the oocytes were also used for western blotting to identify p11 within the oocytes, however, inconclusive results were obtained. Second, we wanted to determine if the amiloride sensitive current would reverse upon silencing of p11. Fisher rat thyroid epithelia were transfected with plasmids encoding ENaC subunits + si-p11 RNA, and their resistance and amiloride sensitive currents recorded using an Ussing chamber apparatus. Results show a significant decrease (average of 75%) (p=0.04) in the amiloride sensitive current for ENaC + si-p11, when compared to control (ENaC + si-control).
Overall, it is confirmed that p11 interacts with ENaC. Furthermore, it is highly likely that p11 plays a role in aiding the exocytosis of ENaC, as concluded from both the overexpression and knockdown experiments. A significant lack of any previous research on the interaction between ENaC and p11 contributed to the difficulty of this project, however, the resultant information significantly aids our understanding of the exocytic process of ENaC and the individual proteins involved, such as p11. The real-world applications of this information span across a wide spectrum including therapeutic approaches for both hyper and hypotension which are large contributors to mortality around the world.