Characterization of the oligomeric structure of the Ca(2+)-activated Cl- channel Ano1/TMEM16A.
Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca(2+)-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl(-) secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca(2+), suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.
Department of Cell and Molecular Physiology, University of North Carolina, North Carolina, Chapel Hill 27599, USA.
John T
JT
Sheridan
Erin N
EN
Worthington
Kuai
K
Yu
Sherif E
SE
Gabriel
H Criss
HC
Hartzell
Robert
R
Tarran
D008841:Actin Cytoskeleton / Q000502:physiology; D000818:Animals; D000075369:Anoctamin-1; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018408:Patch-Clamp Techniques; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D020545:Respiratory Mucosa / Q000166:cytology; D014162:Transfection
C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D018118:Chloride Channels; D049452:Green Fluorescent Proteins; D002118:Calcium