The actin cytoskeleton and small G protein RhoA are not involved in flow-dependent activation of ENaC

Alexey V Karpushev,Alexander Staruschenko,Daria V Ilatovskaya

doi:10.1186/1756-0500-3-210

Alexey V Karpushev, Alexander Staruschenko + Show 1 more

Open Access

https://doi.org/10.1186/1756-0500-3-210

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Abstract

BackgroundEpithelial cells are exposed to a variety of mechanical stimuli. Epithelial Na+ channels (ENaC) mediate sodium transport across apical membranes of epithelial cells that line the distal nephron, airway and alveoli, and distal colon. Early investigations into stretch sensitivity of ENaC were controversial. However, recent studies are supportive of ENaC's mechanosensitivity. This work studied whether flow-dependent activation of ENaC is modulated by changes in the state of the actin cytoskeleton and whether small GTPase RhoA is involved in flow-mediated increase of ENaC activity.FindingsPretreatment with Cytochalasin D and Latrunculin B for 20 min and 1-2 hrs to disassemble F-actin had no effect on flow-mediated increase of amiloride-sensitive current. Overexpression of ENaC with constitutively active (G14V) or dominant negative (T19N) RhoA similarly had no effect on flow-dependent activation of ENaC activity. In addition, we did not observe changes when we inhibited Rho-kinase with Y27632.ConclusionsOur results suggest that the flow-dependent activation of ENaC is not influenced by small GTPase RhoA and modifications in the actin cytoskeleton.

Highlights

The long term control of blood pressure involves Na+ homeostasis through the precise regulation of the Epithelial Na+ Channel (ENaC) in the aldosterone-sensitive distal nephron [1,2]
Our results suggest that the flow-dependent activation of Epithelial Na+ channels (ENaC) is not influenced by small GTPase RhoA and modifications in the actin cytoskeleton
To investigate flow-dependent activation of ENaC activity, we reconstituted the channel in Chinese hamster ovary (CHO) cells. mENaC was reconstituted by coexpressing a, b- and g-channel subunits

Summary

Introduction

The long term control of blood pressure involves Na+ homeostasis through the precise regulation of the Epithelial Na+ Channel (ENaC) in the aldosterone-sensitive distal nephron [1,2]. Palmer and Frindt studied whether mechanical perturbations could influence ENaC kinetics or gating mode in freshly isolated rat CCDs. In most cases negative pressure applied to the patch clamp pipette had no effect on channel behaviour. In most cases negative pressure applied to the patch clamp pipette had no effect on channel behaviour They observed a rapid and reversible increase in channel open probability (Po) in 6 out of 22 patches [4]. They proposed that the variability in the response could reflect differences in the mechanical deformations of the apical membrane within the tip of the pipette [4]. This work studied whether flow-dependent activation of ENaC is modulated by changes in the state of the actin cytoskeleton and whether small GTPase RhoA is involved in flowmediated increase of ENaC activity

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