The epithelial sodium channel (ENaC) is the rate-determining step in Na entry at the apical membranes of distal nephron epithelial cells where the fine regulation of Na balance occurs. The importance of ENaC in this process is illustrated by genetic diseases, in which channel mutations produce defects in salt and water homeostasis. The cloning of ENaC and delineation of its subunit structure has enabled experiments designed to define the mechanisms of channel function and regulation. The control of apical membrane ENaC density is an important, if not central, mechanism governing Na entry rate;yet, we do not understand the pathways and protein interactions that regulate ENaC insertion and recycling at the apical membrane. Our work has indicated that physical interactions (binding) of ENaC with acknowledged traffic regulatory (SNARE) proteins (syntaxins, munc 18, VAMPs and cysteine string protein, Csp) provide for regulated insertion and recycling of ENaC. In this project, we will selectively manipulate the physical interactions between ENaC and SNAREs (and other traffic proteins) in a polarized cortical collecting duct cell line (mCCD) and in MDCK cells to examine the physiological mechanisms by which these interactions control apical membrane ENaC density. Botulinum or Tetanus toxins will provide for acute manipulation of the activity of several proteins;effects will be rescued by toxin-insensitive SNAREs. FRET will be used to determine SNARE pairs that interact in regulated apical ENaC insertion. Binding sites will be mapped on both ENaC and SNARE proteins and binding incompetent ENaC subunits and SNAREs will be expressed to selectively assess the role of these interactions in ENaC traffic. We will examine the hypotheses that constitutive and regulated traffic pathways are mediated by different SNARE pairs, that ENaC and SNARE proteins are clustered in different microdomains to regulate apical channel insertion, and that phosphorylation of munc 18 in response to aldosterone or vasopressin controls syntaxin availability for apical ENaC trafficking. We will evaluate the possibility that an ENaC-Csp-alphaGDI complex controls an apical Rab3 cycle that mediates ENaC insertion. With the help of a new assay for the recycling of functional ENaC channels, we will express dominant negative regulators of specific steps in protein trafficking and degradation to determine the control points for regulated apical ENaC insertion and recycling. Current fluctuation analysis, patch clamp and cell surface biotinylation will be used to assess the effects of manipulating traffic protein expression on apical membrane channel number (N) and open probability (P0) in polarized mCCD and MDCK epithelia under basal conditions and in response to the ENaC modulators, vasopressin and aldosterone. These studies will determine how ENaC's interactions with traffic regulatory proteins controls apical membrane channel density under normal conditions and in response to physiological modulators of Na transport.