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Updated: Jul 19, 2026

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation
Published on: December 23, 2009
PKA-dependent ENaC trafficking requires the SNARE-binding protein complexin
M B Butterworth1, R A Frizzell, J P Johnson
1Dept. of Cell Biology and Physiology, University of Pittsburgh, PA 15261, USA. michael7@pitt.edu
Complexin protein regulates epithelial sodium channel (ENaC) trafficking in kidney cells. This protein stabilizes SNARE interactions, controlling ENaC insertion into the apical membrane and affecting sodium transport.
Area of Science:
- Nephrology
- Cell Biology
- Molecular Biology
Background:
- Epithelial sodium channel (ENaC) function in kidney collecting ducts is acutely regulated by trafficking of membrane vesicles.
- Soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs) are involved in this vesicle trafficking process.
Purpose of the Study:
- To investigate the presence and role of complexin, a novel SNARE-binding protein, in mouse kidney cortical collecting duct (mCCD) cells.
- To determine complexin's involvement in the acute regulation of ENaC function.
Main Methods:
- Cloning of complexin (isoform II) from a mouse kidney cDNA library.
- Coexpression of complexin II mRNA with ENaC subunits in Xenopus laevis oocytes.
- Short-circuit current (I(sc)) measurements in mCCD cell lines with altered complexin expression.
- Surface biotinylation assays to assess ENaC localization.
- Immunofluorescent localization of SNARE proteins in polarized mCCD epithelia.
Main Results:
- Complexin II expression reduced sodium currents in oocytes and basal/forskolin-stimulated short-circuit currents in mCCD cells.
- Reduced ENaC at the apical membrane and inhibited ENaC insertion were observed with altered complexin levels.
- Specific SNARE proteins (syntaxins 1 and 3, SNAP-23, VAMP2) were localized to the apical membrane and intracellular compartments, respectively.
Conclusions:
- Complexin is present in mCCD cells and plays a role in regulating ENaC trafficking.
- Complexin stabilizes SNARE interactions, essential for ENaC-containing vesicle insertion into the apical membrane.
- This mechanism is crucial for modulating apical ENaC density via constitutive and regulated trafficking pathways.
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