A Comprehensive Search for Calcium Binding Sites Critical for TMEM16A Calcium-Activated Chloride Channel Activity

Abstract

TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. 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. Our results thus demonstrate that direct binding of calcium to TMEM16A triggers channel activation independently of calmodulin, identify novel interaction sites between calcium ions and TMEM16A, and lay the groundwork for future studies examining the mechanism of calcium-dependent TMEM16 channel activation.