Studies of TMEM16A Calcium Activated Chloride Channel Structure and Function

TMEM16A is a novel calcium-activated chloride channel first cloned in 2008. It is responsible for regulating secretions from the epithelium, excitability of smooth muscle, membrane potential of neurons, and cellular proliferation. This work describes two studies investigating the structure and function of TMEM16A ion channels. In the first, I report a series of experiments that show that TMEM16A channels have a homodimeric architecture facilitated by its cytoplasmic N-terminus. This dimerization domain is important for channel assembly in eukaryotic cells, and the in vitro association of peptides containing the dimerization domain is consistent with a homotypic protein-protein interaction. Amino acid substitutions in the dimerization domain affect functional TMEM16A-CaCC channel expression as expected from its critical role in channel subunit assembly. In the second, I describe the identification of four acidic amino acid residues as putative calcium-binding sites. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, gating of TMEM16A mutants containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility