Quantifying RhoA Facilitated Trafficking of the Epithelial Na+ Channel toward the Plasma Membrane with Total Internal Reflection Fluorescence-Fluorescence Recovery after Photobleaching

James D Stockand,Oleh Pochynyuk,Vladislav Bugaj,Alexander Staruschenko,Lila Lagrange

doi:10.1074/jbc.m701348200

Abstract

The epithelial Na(+) channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. Similar to other ion channels, ENaC activity is set, in part, by its membrane levels. 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. Few experimental methods are available to directly visualize trafficking of ion channels to the plasma membrane. Here we combine electrophysiology with two complementary imaging methods, total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching, to study the mechanistic basis of RhoA actions on ENaC. Patch clamp results demonstrate that RhoA increases ENaC activity in an additive manner with dominant-negative dynamin. This is consistent with a mechanism of increased ENaC trafficking to the membrane. Direct visualization of ENaC movement near the plasma membrane with total internal reflection fluorescence-fluorescence recovery after photobleaching revealed that RhoA accelerates ENaC trafficking toward the membrane. RhoA-facilitated movement of the channel was sensitive to disrupting the endomembrane system. Moreover, facilitating retrieval decreased ENaC activity but not trafficking toward the membrane. ENaC at the plasma membrane clustered and was laterally immobile suggesting that the cytoskeleton tethers or corrals membrane resident channels or membrane-directed vesicles containing ENaC. Disrupting microtubules but not microfilaments led to reorganization of ENaC clusters and slowed trafficking toward the membrane. The cytoskeleton is an established target for RhoA signaling. We conclude that RhoA, likely through effects on the cytoskeleton, promotes ENaC trafficking to the plasma membrane to increase channel membrane levels and activity.

Highlights

RhoA Promotes epithelial Na؉ channel (ENaC) Trafficking to the Plasma Membrane— RhoA increases ENaC activity by increasing the membrane levels of this channel [7, 8]
Co-expressing RhoAG14V plus DNDyn together with ENaC had additive effects significantly increasing channel activity above that for either alone. This finding is consistent with RhoA increasing ENaC activity through a mechanism independent of dynamin possibly involving increased channel trafficking to the membrane
We used TIRF-FRAP in conjunction with electrophysiology to define the mechanism by which RhoA increases ENaC activity

Summary

Introduction

Regulated trafficking of these proteins to and from the plasma membrane in part controls their activity This is true for ENaC, which is a nonvoltage-gated, Naϩ-selective ion channel. Regulation of ion channels, including several types of Kϩ channels (e.g. KCNA, KCNH, and KCNJ), by RhoA and other small G proteins is becoming widely appreciated (9 –11) Often, as they do for ENaC [7, 8] and TRPC5 [12], small G proteins in the RhoA family modulate channel activity by influencing their membrane levels. Rho family members localize to the plasma membrane, as well as to intracellular membrane compartments, including endosomes and the Golgi complex These proteins regulate cytoskeleton organization influencing both microfilaments and microtubules. Microtubules but not microfilaments played an important role in ENaC clustering and movement toward the membrane

Methods

Results

Conclusion