Molecular Proximity of Cystic Fibrosis Transmembrane Conductance Regulator and Epithelial Sodium Channel Assessed by Fluorescence Resonance Energy Transfer

Henderika M.J. Oosterveld-Hut,Estelle Cormet-Boyaka,Gergely L. Lukacs,Patricia A. Gonzales,Cathy M. Fuller,Jeong S. Hong,Bakhrom K. Berdiev,Albert Tousson,Eric J. Sorscher,Yawar J. Qadri,Dale J. Benos

doi:10.1074/jbc.m708089200

Abstract

We present the evidence for a direct physical association of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC), two major ion channels implicated in the pathophysiology of cystic fibrosis, a devastating inherited disease. We employed fluorescence resonance energy transfer, a distance-dependent imaging technique with capability to detect molecular complexes with near angstrom resolution, to estimate the proximity of CFTR and ENaC, an essential variable for possible physical interaction to occur. Fluorescence resonance energy transfer studies were complemented with a classic biochemical approach: coimmunoprecipitation. Our results place CFTR and ENaC within reach of each other, suggestive of a direct interaction between these two proteins.

Highlights

We present the evidence for a direct physical association of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC), two major ion channels implicated in the pathophysiology of cystic fibrosis, a devastating inherited disease
CF2 is the most common lethal autosomal recessive disorder in Caucasians. It is caused by mutations in the CFTR protein [1,2,3], a ClϪ channel that is widely expressed in epithelia [4]
CFTR and ENaC both contribute to the pathology of CF, only CFTR is affected by the genetic mutations

Summary

Molecular Proximity of Cystic Fibrosis Transmembrane

Conductance Regulator and Epithelial Sodium Channel Assessed by Fluorescence Resonance Energy Transfer*□S. Our results place CFTR and ENaC within reach of each other, suggestive of a direct interaction between these two proteins. Some of the earliest electrophysiological studies of CF patient airways demonstrated that an amiloride-sensitive Naϩ transport pathway contributed to the raised transepithelial potential difference across CF nasal epithelia [12]. This sensitivity to the diuretic amiloride suggested an involvement of the ENaC family of proteins. These results place CFTR and ENaC proteins in very close proximity to each other, consistent with a direct interaction of these two proteins This renders plausible a possibility that these two proteins interact directly in vivo to contribute to CF airway pathology

MATERIALS AND METHODS

ENaC Subunits Interact with

Findings

Soon after the molecular cloning of