Characterization of the Oligomeric Structure of the Ca2+-activated Cl− Channel Ano1/TMEM16A

Kuai Yu,Robert Tarran,John T. Sheridan,Erin N. Worthington,H. Criss Hartzell,Sherif E. Gabriel

doi:10.1074/jbc.m110.174847

Abstract

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.

Highlights

Until recently when three laboratories independently identified members of the Anoctamin 1 (Ano1) family, known as the transmembrane protein 16 (TMEM16) family, as essential subunits of Ca2؉-activated chloride channel (CaCC) [11,12,13]
The currents from both constructs exhibited outward rectification and time dependence that was typical of CaCC currents at this Ca2ϩ concentration and was identical to untagged Ano1 (Fig. 1, B–E) [35]
These results demonstrate that attaching eGFP or mCherry had no obvious effect on Ano1 function

Summary

Introduction

Until recently when three laboratories independently identified members of the Anoctamin 1 (Ano1) family, known as the transmembrane protein 16 (TMEM16) family, as essential subunits of CaCCs [11,12,13]. Ano1-eGFP-transfected HEK293 cells were lysed in cross-linking buffer (PBS, pH 8). A, total RNA was collected from untransfected HEK293 cells and RT-PCR was performed to determine which Ano proteins are endogenously expressed.

Results

Conclusion