Small Molecule Activator of the Human Epithelial Sodium Channel

Lena Staszewski,Rachel Kimmich,Nancy Hwang,Andrew Patron,Erika Patron,David S Dahan,Dalia Kalabat,Hong Xu,Fernando Echeverri,Jing Ling,Raymond D Smith,Min Lu,Jeff Yamamoto,Bianca Laita,Peter Li,Bryan D Moyer,Walter Keung

doi:10.1074/jbc.m708001200

Abstract

The epithelial sodium channel (ENaC), a heterotrimeric complex composed of alpha, beta, and gamma subunits, belongs to the ENaC/degenerin family of ion channels and forms the principal route for apical Na(+) entry in many reabsorbing epithelia. Although high affinity ENaC blockers, including amiloride and derivatives, have been described, potent and specific small molecule ENaC activators have not been reported. Here we describe compound S3969 that fully and reversibly activates human ENaC (hENaC) in an amiloride-sensitive and dose-dependent manner in heterologous cells. Mechanistically, S3969 increases hENaC open probability through interactions requiring the extracellular domain of the beta subunit. hENaC activation by S3969 did not require cleavage by the furin protease, indicating that nonproteolyzed channels can be opened. Function of alphabetaG37Sgamma hENaC, a channel defective in gating that leads to the salt-wasting disease pseudohypoaldosteronism type I, was rescued by S3969. Small molecule activation of hENaC may find application in alleviating human disease, including pseudohypoaldosteronism type I, hypotension, and neonatal respiratory distress syndrome, when improved Na(+) flux across epithelial membranes is clinically desirable.

Highlights

This study describes the first compound, S3969, that fully and reversibly activates hENaC in heterologous cells
S3969 requires the extracellular domain of the ␤ subunit and increases hENaC open probability, thereby augmenting flux of Naϩ ions into cells
Identification of a Small Molecule Opener of ␣␤␥ hENaC—A high throughput screen was conducted using a fluorescent membrane potential dye to identify novel compounds that open hENaC channels expressed in heterologous cells [26]

Summary

Introduction

This study describes the first compound, S3969, that fully and reversibly activates hENaC in heterologous cells. S3969 requires the extracellular domain of the ␤ subunit and increases hENaC open probability, thereby augmenting flux of Naϩ ions into cells. S3969 activated ␣␤G37S␥ hENaC, a channel variant that exhibits reduced open probability and leads to the salt-wasting disease PHA1. Promoting hENaC-dependent Naϩ and fluid transport across epithelia may find utility in modulating extracellular volume and electrolyte homeostasis in both normal and diseased states

Methods

Results

Conclusion