title: Independent activation of ion conduction pores in the double-barreled calcium-activated chloride channel TMEM16A.
abstract: The TMEM16 proteins constitute a family of membrane proteins with unusual functional breadth, including lipid scramblases and Cl-channels. Members of both these branches are activated by Ca2+, acting from the intracellular side, and probably share a common architecture, which was defined in the recent structure of the lipid scramblase nhTMEM16. The structural features of subunits and the arrangement of Ca2+-binding sites in nhTMEM16 suggest that the dimeric protein harbors two locations for catalysis that are independent with respect to both activation and lipid conduction. Here, we ask whether a similar independence is observed in the Ca2+-activated Cl-channel TMEM16A. For this purpose, we generated concatenated constructs containing subunits with distinct activation and permeation properties. Our biochemical investigations demonstrate the integrity of concatemers after solubilization and purification. During investigation by patch-clamp electrophysiology, the functional behavior of constructs containing either two wild-type (WT) subunits or one WT subunit paired with a second subunit with compromised activation closely resembles TMEM16A. This resemblance extends to ion selectivity, conductance, and the concentration and voltage dependence of channel activation by Ca2+Constructs combining subunits with different potencies for Ca2+show a biphasic activation curve that can be described as a linear combination of the properties of its constituents. The functional independence is further supported by mutation of a putative pore-lining residue that changes the conduction properties of the mutated subunit. Our results strongly suggest that TMEM16A contains two ion conduction pores that are independently activated by Ca2+binding to sites that are embedded within the transmembrane part of each subunit.
authors: Department of Biochemistry, University of Zürich, CH-8057 Zürich, Switzerland.
authors: Novandy K
authors: NK
authors: Lim
authors: Department of Biochemistry, University of Zürich, CH-8057 Zürich, Switzerland.
authors: Andy K M
authors: AK
authors: Lam
authors: Department of Biochemistry, University of Zürich, CH-8057 Zürich, Switzerland dutzler@bioc.uzh.ch.
authors: Raimund
authors: 0000-0002-2193-6129
authors: R
authors: Dutzler
mesh_terms: D000818:Animals; D000075369:Anoctamin-1; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D051379:Mice; D009154:Mutation; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism
chemical_list: C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D018118:Chloride Channels; D021122:Protein Subunits