In tight epithelia, electrogenic sodium reabsorption is mediated by a sodium-selective amiloride-sensitive channel (ENaC), located in the apical membrane. Regulatory mechanisms include kinetic effects at the single channel level, protein synthesis and recruitment of proteins to the cell surface from intracellular pools. Syntaxins mediate vesicle trafficking and are members of the NSF-SNAP-SNAREs fusion complex. Recent data suggest that the syntaxins interact with and functionally regulate a number of ion channels and pumps. Syntaxin isoforms increase the net externalization of the ENaC channel complex, but functional regulation is isoform-specific (syntaxin 1A inhibits while syntaxin 3 stimulates the amiloride-sensitive currents when expressed in Xenopus oocytes). It is hypothesized that syntaxins affect ENaC expression and activity depending on the functional domains of the syntaxins and the ENaC subunits. The mpkCCDC14 cell line is a physiologically relevant system derived from mouse principal cells which has ENaC expression, and responds to aldosterone with an increase in amiloride-sensitive currents. Syntaxin 1a, 3 and 4 and SNARE proteins (SNAP25 and Munc18) are readily detectable; confocal images show the syntaxins to be expressed in the apical membrane. Data obtained with anti-sense oligonucleotides suggest an important role for syntaxins in regulating ENaC function.
The specific aims i nclude: 1): To define the physical expression of syntaxins, their accessory proteins and other SNARE in a Novel Immortalized Mouse Collecting Duct Principal Cell Line (mpkCCDC14). 2). To define the functional interactions between specific Syntaxins and other SNARE proteins with the ENaC Complex in mpkCCDC14cells. 3). To understand the differences between Syntaxin 1A and Syntaxin 3 on ENaC activity and expression in the oocyte expression system and in mpkCCDC14 cells. 4). To define the interacting domains of mouse ENaC subunits and syntaxin isoforms and the SNARES, using the Xenopus oocyte expression system and mpkCCDC14 cells. 5). To study the regulation of ENaC by aldosterone in the context of Syntaxins and SNARES expressed in mpkCCDC14 cells. A variety of methods will be used in this proposal, including voltage clamp measurements and single channel measurements, immunoprecipitation, confocal microscopy, and metabolic labeling will be utilized. Epitope mapping will be used to optimize the specificity of polyclonal antibodies, and anti-sense oligonucleotides will be used to modulate the level of endogenous proteins in the mpkCCDC14 line.
