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Quantifying RhoA facilitated trafficking of ENaC to the plasma membrane with TIRF‐FRAP
Author(s) -
Pochynyuk Oleh,
Staruschenko Alexander,
Bugaj Vladislav,
LaGrange Lila,
Stockand James D
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a547-c
Subject(s) - rhoa , epithelial sodium channel , total internal reflection fluorescence microscope , microbiology and biotechnology , apical membrane , cytoskeleton , chemistry , ion channel , biophysics , membrane , biology , biochemistry , signal transduction , cell , sodium , receptor , organic chemistry
ENaC plays a central role in control of epithelial surface hydration and vascular volume. The small G protein RhoA increases ENaC activity by increasing the membrane levels of this channel. We hypothesize that RhoA increases ENaC activity by promoting channel trafficking to the plasma membrane. Here, we combine electrophysiology with total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) to study RhoA actions on ENaC. Patch clamp results demonstrate that RhoA increases ENaC activity in an additive manner with decreases in channel retrieval. Direct visualization of ENaC movement near the plasma membrane with TIRF‐FRAP revealed that RhoA accelerates ENaC trafficking towards the membrane. RhoA facilitated movement of ENaC was sensitive to disrupting the endomembrane system. Besides, facilitating retrieval decreased ENaC activity but not trafficking towards the membrane. ENaC at the plasma membrane clustered and was laterally immobile suggesting that the cytoskeleton tethers membrane resident channels or vesicles containing ENaC. Disrupting microtubules but not microfilaments led to reorganization of ENaC clusters and slowed trafficking towards the membrane. We conclude that RhoA, likely through effects on the cytoskeleton, promotes ENaC trafficking to the plasma membrane to increase channel levels and activity.