PKA-dependent ENaC trafficking requires the SNARE-binding protein complexin.
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 alpha-, beta-, and gamma-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) microA/cm(2), 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 microA/cm(2) by either increasing or decreasing complexin expression. Surface biotinylation demonstrated that the complexin-induced reduction in basal I(sc)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, 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.
Dept. of Cell Biology and Physiology, University of Pittsburgh, PA 15261, USA. michael7@pitt.edu
M B
MB
Butterworth
R A
RA
Frizzell
J P
JP
Johnson
K W
KW
Peters
R S
RS
Edinger
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D003001:Cloning, Molecular; D053503:Epithelial Sodium Channels; D015723:Gene Library; D007685:Kidney Tubules, Collecting / Q000502:physiology*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D009865:Oocytes; D012964:Sodium / Q000493:pharmacokinetics; D015222:Sodium Channels / Q000502:physiology*; D013572:Synaptic Vesicles / Q000502:physiology; D014982:Xenopus laevis
D033942:Adaptor Proteins, Vesicular Transport; D053503:Epithelial Sodium Channels; D009419:Nerve Tissue Proteins; D015222:Sodium Channels; C095993:complexin II; D012964:Sodium