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Am J Physiol Renal Physiol (June 21, 2005). doi:10.1152/ajprenal.00390.2003
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Submitted on November 3, 2003
Accepted on June 13, 2005

PKA-Dependent ENaC Trafficking Requires the SNARE-Binding Protein Complexin

M. B Butterworth1*, R. A Frizzell1, J. P. Johnson2, K. W. Peters1, and R. S. Edinger2

1 Department of Cell Biology and Physiology, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
2 Department of Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA

* To whom correspondence should be addressed. E-mail: michael7{at}pitt.edu.

Acute regulation of epithelial sodium channel (ENaC) function at the apical surface of polarized kidney cortical collecting duct (CCD) epithelial cells occurs in large part by changes in channel number, mediated by membrane vesicle trafficking. Several soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNARE) have been implicated in this process. A novel SNARE binding protein, complexin has been identified in nervous tissue which specifically binds to and stabilizes SNARE complexes at synaptic membranes to promote vesicle fusion. To test whether this protein is present in mouse CCD (mCCD) cells and its possible involvement in acute ENaC regulation, we cloned complexin (isoform II) from a mouse kidney cDNA library. Complexin II mRNA co-expressed with {alpha}, {beta} and {gamma} ENaC subunits in Xenopus laevis oocytes reduced sodium currents to 16 ± 3% (n=19) of control values. Short-circuit current (ISC) measurements on mCCD cell lines stably over- or under-expressing complexin produced similar results. Basal ISC was reduced from 12.0 ±1.0 (n=15) to 2.0 ±0.4 (n=15) and 1.8 ±0.3 (n=17) µA/cm2 respectively. Similarly forskolin-stimulated ISC was reduced from control values of 20.0 ±2 to 2.7 ± 0.5 and 2.3 ± 0.4 µA/cm2 by either increasing or decreasing complexin expression. Surface biotinylation demonstrated that the complexin-induced reduction in basal ISC was due to a reduction in apical membrane-resident ENaC and the inhibition in forskolin stimulation was due to the lack of ENaC insertion into the apical membrane to increase surface channel number. Immunofluorescent localization of SNARE proteins in polarized mCCD epithelia detected the presence of syntaxins 1 and 3 and synaptosomal-associated protein of 23 kDa (SNAP-23) at the apical membrane, vesicle-associated membrane protein (VAMP2) was localized to intracellular compartments. These findings identify SNAREs that may mediate ENaC-containing vesicle insertion in mCCD epithelia and suggest that stabilization of SNARE interactions by complexin is an essential aspect of the regulated trafficking events that increase apical membrane ENaC density either by constitutive or regulated trafficking pathways.




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