Atomic force microscopy imaging reveals the formation of ASIC/ENaC cross-clade ion channels

Pia Jeggle; Ewan St J Smith; Andrew P Stewart; Silke Haerteis; Christoph Korbmacher; J Michael Edwardson

doi:10.1016/j.bbrc.2015.05.091

Atomic force microscopy imaging reveals the formation of ASIC/ENaC cross-clade ion channels

Biochem Biophys Res Commun. 2015 Aug 14;464(1):38-44. doi: 10.1016/j.bbrc.2015.05.091. Epub 2015 May 29.

Authors

Pia Jeggle¹, Ewan St J Smith¹, Andrew P Stewart¹, Silke Haerteis², Christoph Korbmacher², J Michael Edwardson³

Affiliations

¹ Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
² Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Waldstrasse 6, 91054 Erlangen, Germany.
³ Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom. Electronic address: [email protected].

PMID: 26032502
DOI: 10.1016/j.bbrc.2015.05.091

Abstract

ASIC and ENaC are co-expressed in various cell types, and there is evidence for a close association between them. Here, we used atomic force microscopy (AFM) to determine whether ASIC1a and ENaC subunits are able to form cross-clade hybrid ion channels. ASIC1a and ENaC could be co-isolated from detergent extracts of tsA 201 cells co-expressing the two subunits. Isolated proteins were incubated with antibodies against ENaC and Fab fragments against ASIC1a. AFM imaging revealed proteins that were decorated by both an antibody and a Fab fragment with an angle of ∼120° between them, indicating the formation of ASIC1a/ENaC heterotrimers.

Keywords: Acid-sensing ion channel (ASIC); Atomic force microscopy; Channel assembly; Epithelial Na(+) channel (ENaC).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Acid Sensing Ion Channels / chemistry*
Acid Sensing Ion Channels / genetics
Acid Sensing Ion Channels / metabolism
Animals
Antibodies / chemistry
CHO Cells
Cell Line, Transformed
Cricetulus
Epithelial Sodium Channels / chemistry*
Epithelial Sodium Channels / genetics
Epithelial Sodium Channels / metabolism
Epitopes / chemistry*
Epitopes / metabolism
Gene Expression
HEK293 Cells
Humans
Hydrogen-Ion Concentration
Microscopy, Atomic Force
Patch-Clamp Techniques
Protein Multimerization
Proto-Oncogene Proteins c-myc / genetics
Proto-Oncogene Proteins c-myc / metabolism
Recombinant Fusion Proteins / chemistry*
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism

Substances

ASIC1 protein, human
Acid Sensing Ion Channels
Antibodies
Epithelial Sodium Channels
Epitopes
MYC protein, human
Proto-Oncogene Proteins c-myc
Recombinant Fusion Proteins
SCNN1A protein, human
SCNN1B protein, human
SCNN1G protein, human