An Allosteric Gating Mechanism of TMEM16A Calcium-Activated Chloride Channel

Abstract

The Ca2+-activated chloride channel (CaCC) TMEM16A is widely expressed throughout the body with diverse functions ranging from trans-epithelial fluid transport, smooth muscle contraction, to nociception. Activation of TMEM16A requires binding of intracellular Ca2+ whereas binding of the phosphatidylinositol PIP2 facilitates channel opening. Assembled as a homodimer, each TMEM16A monomer is consisted of ten transmembrane (TM) segments in which TMs 3 to 8 form an independent chloride-permeable pore. Owing to their direct contributions to the permeation pathway, TMs 3 to 8 have been extensively studied. Our recent work suggests that TMs 3-8 comprise of two functionally distinct modules in which TMs 6-8 constitute the Ca2+-binding module that controls Ca2+-dependent channel activation while TMs 3-5 form a regulatory module that mediates PIP2-dependent modulation of channel opening. However, it remains unknown whether the remaining TMs, namely TMs 1, 2, 9, and 10, all of which are distant from the permeation pathway, also contribute to channel gating. Here we combine electrophysiological analyses with mutagenesis to demonstrate that TM2 and TM10 interaction allosterically controls TMEM16A gating. These studies not only provide the molecular underpinnings of allosteric regulatory mechanism in TMEM16A but will also facilitate our mechanistic understanding of the gating mechanism in TMEM16 Ca2+-activated ion channels and lipid scramblases.