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

• Published in: Biochemical and biophysical research communications Vol. 464; no. 1; pp. 38 - 44
• Main Authors: Jeggle, Pia, Smith, Ewan St. J., Stewart, Andrew P., Haerteis, Silke, Korbmacher, Christoph, Edwardson, J. Michael
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 14.08.2015
• Subjects: 60 APPLIED LIFE SCIENCES; Acid Sensing Ion Channels - chemistry; Acid Sensing Ion Channels - genetics; Acid Sensing Ion Channels - metabolism; Acid-sensing ion channel (ASIC); Animals; ANTIBODIES; Antibodies - chemistry; ATOMIC FORCE MICROSCOPY; BIOMEDICAL RADIOGRAPHY; Cell Line, Transformed; Channel assembly; CHO Cells; Cricetulus; DETERGENTS; Epithelial Na+ channel (ENaC); Epithelial Sodium Channels - chemistry; Epithelial Sodium Channels - genetics; Epithelial Sodium Channels - metabolism; Epitopes - chemistry; Epitopes - metabolism; FLUORESCEIN; Gene Expression; HAMSTERS; HEK293 Cells; Humans; HYBRIDIZATION; Hydrogen-Ion Concentration; ISOTHIOCYANATES; KIDNEYS; Microscopy, Atomic Force; MUSCLES; OVARIES; Patch-Clamp Techniques; Protein Multimerization; PROTEINS; Proto-Oncogene Proteins c-myc - genetics; Proto-Oncogene Proteins c-myc - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Atomic force microscopy; ASIC; FITC; Acid-sensing ion channel (ASIC); AFM; Epithelial Na+ channel (ENaC); HA; CHO; ENaC; VSMC; Channel assembly; HEK; Epithelial Na(+) channel (ENaC)
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2015.05.091

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. •There is evidence for a close association between ASIC and ENaC.•We used AFM to test whether ASIC1a and ENaC subunits form cross-clade ion channels.•Isolated proteins were incubated with subunit-specific antibodies and Fab fragments.•Some proteins were doubly decorated at ∼120° by an antibody and a Fab fragment.•Our results indicate the formation of ASIC1a/ENaC heterotrimers.