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PKA-dependent ENaC trafficking requires the SNARE-binding protein complexin

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

Submitted 3 November 2003 ; accepted in final form 13 June 2005

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 coexpressed with -, -, and -ENaC subunits in Xenopus laevis oocytes reduced sodium currents to 16 ± 3% (n = 19) of control values. Short-circuit current (I_sc) measurements on mCCD cell lines stably over- or underexpressing complexin produced similar results. Basal I_sc 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 I_sc 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 I_scwas 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, and 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.

kidney cortical collecting duct; transcellular sodium reabsorption

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