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

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 membranedirected 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.Ion channels are integral membrane proteins. Regulated trafficking of these proteins to and from the plasma membrane in part controls their activity. This is true for ENaC, 3 which is a nonvoltage-gated, Na ϩ -selective ion channel. ENaC is localized to the apical plasma membrane of epithelia particularly that lining the distal renal nephron and colon and alveolar spaces (1-3). ENaC activity in this epithelia is limiting for Na ϩ (re)absorption. Thus, ENaC plays an important physiological role in negative feedback control of blood pressure, as well as epithelial surface hydration in the lungs. Indeed, improper regulation and dysfunction of ENaC are causative for some pulmonary and blood pressure disorders (4 -6).Corticosteroids are the primary endocrine modulators of ENaC activity. One mechanism by which these steroids increase ENaC activity is by increasing the levels of this channel in the apical plasma membrane (1-3). We recently reported that the small G protein RhoA similarly increases ENaC activity by elevating membrane levels of the channel (7, 8). Regulation of ion channels, including several types of K ϩ channels (e.g. KCNA, K...

Regulation of the epithelial sodium channel by membrane trafficking

“…Progress in this direction has been achieved with the combined application of total internal reflection microscopy (TIRF) and fluorescence recovery after photobleaching (FRAP) to the process of ENaC movement toward the plasma membrane (139). In their studies of Rho-dependent ENaC trafficking and involvement of the cytoskeleton, Pochynyuk and colleagues (139) combined electrophysiological measurements with these advanced imaging approaches.…”

Mechanisms Underlying Rapid Aldosterone Effects in the Kidney

“…Dynein motor-dependent vesicle trafficking relies on actin polymerization and bundling, and cytoskeletal structures also tether cellsurface ENaC within discrete domains at the apical membrane (53). The activity of phosphatidylinositol-4-phosphate 5-kinase, a type II phosphatidylinositol phosphate (PIP) kinase, is also coupled to RhoA activation and regulates Golgi fission to release ENaCcontaining transport vesicles (54).…”

Cholinergic regulation of epithelial sodium channels in rat alveolar type 2 epithelial cells

“…Others have shown that muscarinic agonists can activate the small G protein, RhoA (15,25), and that RhoA activation can increase ENaC activity (20,21,26). To determine whether muscarinic cholinergic agonists activated RhoA in AT2 cells, we used a commercially available ELISA assay for RhoA activation.…”