Luminal proteolytic activation of collecting duct enac and atp-gated ppx receptors and its potential role in regulation of renal sodium reabsorption

The epithelial sodium channel (ENaC) is a transmembrane ion channel that is highly permeable to sodium (Na). ENaC is a key regulator of Na transport across the apical membrane of the distal nephron; it largely determines final urinary Na excretion, and thereby body Na balance and systemic blood pressure. By necessity, its function is tightly regulated through a variety of local and systemic factors. Recently, trypsin, acting from the luminal side (apical cell membrane) of collecting duct principal cells, has been shown to increase ENaC activity in vitro; however, there have been no in vivo studies to test its physiological significance for renal Na reabsorption; urine contains a variety of proteases that could potentially activate ENaC in vivo. The FIRST AIM of this studentship application is to assess directly the effect of luminal trypsin (as a prototypical protease) on ENaC function in vivo using the distal tubule micro-injection technique (with radioactive Na) in rats on a normal Na diet (when baseline ENaC activity is low), in the absence and presence of a trypsin inhibitor, or the ENaC blocker amiloride. Interestingly, a family of ATP-gated ion channels called P2X receptors, which are also expressed apically in the collecting duct principal cells, have a similar structure to ENaC, i.e. they have a large extracellular loop rich in basic amino acid residues that contain the consensus sequence of Arg-X-X-Arg, where X is any residue. As yet, there have been no studies on the effects of proteases on P2 receptor activity. Thus, the SECOND AIM of this studentship application is to assess the effect of extracellular proteases on P2X receptor activity in the presence and absence of ATP using the Xenopus oocyte heterologous expression system, and a twin-electrode voltage-clamp electrophysiological technique. We will examine the effects of proteases on those luminal P2X receptors found in the collecting duct principal cell (i.e. 4 homomeric and 4 heteromeric assemblies). To explore the potential to manipulate protease activation of ENaC (and its interaction with P2X receptors - see below), it is important to have a series of small molecule compounds that can mimic, inhibit, or potentiate, protease activity. The THIRD AIM of this application, to be undertaken in the laboratories of Discovery BioMed Inc (Birmingham, Alabama, USA), is to screen their compound libraries (which come from diverse synthetic and natural-product sources, and are based on the Company's expertise in renal physiology and biochemistry) using modern high-throughput bioassays on mammalian (primarily human)-cell-based platforms to develop suitable pharmacological agents. We have recently shown that renal ENaC activity is inhibited by activation of apical P2X(4 and/or 4/6) receptors when luminal concentrations of Na are high, and that these receptors switch to being potentiators of ENaC activity when concentrations of extracellular Na are lowered. We have proposed that these P2X receptors are Na sensors responsible for the local regulation of ENaC activity. Interestingly, in the original study of trypsin activation of ENaC, amiloride did not block trypsin's effect on ENaC, but in a more recent study it did. This suggests that proteases may act in more than one way to alter ENaC function, and raises the possibility that another local regulator (such as P2X) of ENaC might also be protease-sensitive. The effect of proteolytic factors on ENaC regulation by P2X receptors has not been investigated. The FOURTH AIM of this application is to assess the effect of luminal proteases on P2X-mediated regulation of ENaC activity in vitro using the rat isolated split-open collecting duct technique (with whole-cell patch clamp electrophysiology) in rats on a normal or low Na diet, and to test suitable compounds identified above.