Expression and purification of the alpha subunit of the epithelial sodium channel, ENaC.

Academic Article

Abstract

  • The epithelial sodium channel (ENaC) plays a critical role in maintaining Na(+) homeostasis in various tissues throughout the body. An understanding of the structure of the ENaC subunits has been developed from homology modeling based on the related acid sensing ion channel 1 (ASIC1) protein structure, as well as electrophysiological approaches. However, ENaC has several notable functional differences compared to ASIC1, thereby providing justification for determination of its three-dimensional structure. Unfortunately, this goal remains elusive due to several experimental challenges. Of the subunits that comprise a physiological hetero-trimeric αβγENaC, the α-subunit is unique in that it is capable of forming a homo-trimeric structure that conducts Na(+) ions. Despite functional and structural interest in αENaC, a key factor complicating structural studies has been its interaction with multiple other proteins, disrupting its homogeneity. In order to address this issue, a novel protocol was used to reduce the number of proteins that associate and co-purify with αENaC. In this study, we describe a novel expression system coupled with a two-step affinity purification approach using NiNTA, followed by a GFP antibody column as a rapid procedure to improve the purity and yield of rat αENaC.
  • Published In

    Keywords

  • ENaC, Epithelial sodium channel, GFP, GFP Affinity Column, Membrane Protein Expression, Membrane Protein Purification, Animals, Epithelial Sodium Channels, Gene Expression, HEK293 Cells, Humans, Mice, Mice, Inbred BALB C, Rats, Recombinant Proteins
  • Digital Object Identifier (doi)

    Author List

  • Reddy BG; Dai Q; McNicholas CM; Fuller CM; Kappes JC; DeLucas LJ
  • Start Page

  • 67
  • End Page

  • 75
  • Volume

  • 117