Identification Of Critical Residues For Anion Conductance In The Tmem16a Channel

TMEM16A (also called DOG-1 and Anoctamin-1) is a transmembrane-localized, homodimeric protein that makes up the pore forming subunit of a calcium-activated chloride channel found in epithelial cells, tumor cells and a subset of DRG neurons. However, the structural and biophysical properties of this channel remain poorly understood. Here, we aimed to identify residues that determine the conductance properties of TMEM16A, by using site-directed mutagenesis and heterologous expression in HEK293 cells followed by patch clamp electrophysiology. Using a recently crystallized fungal orthologue of the TMEM16 family as a model, we identified several residues clustered around the putative pore region that are critical for normal channel conductance of anions. Mutations at these residues also influenced the binding properties of several known channel inhibitors. We conclude that the identified residues are likely to be directly involved in dictating the physical properties of the anion conduction pathway, and may also help to confirm the as yet unknown location of the TMEM16A channelu0027s anion pore.