Quantifying RhoA facilitated trafficking of ENaC to the plasma membrane with TIRF‐FRAP

Abstract

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.

Dynamin 2 and human diseases

“…On the other hand, DNM2 mutations may lead to a prolonged 14. Durieux half-life of various proteins at the cell surface due to a defect in protein removal, similar to that as suggested for the EnaC sodium channel [109,110], KCNQ1 potassium channel subunits [111] or the GLUT4 glucose transporter [91,[112][113][114]. A deregulation of glucose transport in patients with DNM2 mutations could have a strong impact on muscle fibers given their high glucose consumption.…”

Molecular Determinants of PI(4,5)P2 and PI(3,4,5)P3 Regulation of the Epithelial Na+ Channel

“…We interpret this fi nding as meaning that either none of the regions probed here play a role in regulation by PI(4,5)P 2 or that a response to chronic elevations in PI(4,5)P 2 is complex, involving possible changes in either the number of channels in the membrane or channel P o or both. In consideration of earlier work (Staruschenko et al, 2004b;Pochynyuk et al, 2006a;Pochynyuk et al, 2007a), we suggest that chronic elevations in PI(4,5)P 2 increase the number of ENaC in the membrane and that the regions mutated in the current study do not have a role in this regulation. If this is correct, then possible effects on P o may have been masked during chronic elevation of PI(4,5)P 2 .…”