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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 
, 
and 
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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