title: Independent activation of distinct pores in dimeric TMEM16A channels.
abstract: The TMEM16 family encompasses Ca2+-activated Cl-channels (CaCCs) and lipid scramblases. These proteins are formed by two identical subunits, as confirmed by the recently solved crystal structure of a TMEM16 lipid scramblase. However, the high-resolution structure did not provide definitive information regarding the pore architecture of the TMEM16 channels. In this study, we express TMEM16A channels constituting two covalently linked subunits with different Ca2+affinities. The dose-response curve of the heterodimer appears to be a weighted sum of two dose-response curves-one corresponding to the high-affinity subunit and the other to the low-affinity subunit. However, fluorescence resonance energy transfer experiments suggest that the covalently linked heterodimeric proteins fold and assemble as one molecule. Together these results suggest that activation of the two TMEM16A subunits likely activate independently of each other. The Ca2+activation curve for the heterodimer at a low Ca2+concentration range ([Ca2+] < 5 µM) is similar to that of the wild-type channel-the Hill coefficients in both cases are significantly greater than one. This suggests that Ca2+binding to one subunit of TMEM16A is sufficient to activate the channel and that each subunit contains more than one Ca2+-binding site. We also take advantage of the I-V curve rectification that results from mutation of a pore residue to address the pore architecture of the channel. By introducing the pore mutation and the mutation that alters Ca2+affinity in the same or different subunits, we demonstrate that activation of different subunits appears to be associated with the opening of different pores. These results suggest that the TMEM16A CaCC may also adopt a "double-barrel" pore architecture, similar to that found in CLC channels and transporters.
authors: Center for Neuroscience, University of California, Davis, Davis, CA 95618.
authors: Grace
authors: 0000-0003-0604-3952
authors: G
authors: Jeng
authors: Center for Neuroscience, University of California, Davis, Davis, CA 95618.
authors: Muskaan
authors: M
authors: Aggarwal
authors: Center for Neuroscience, University of California, Davis, Davis, CA 95618.
authors: Wei-Ping
authors: WP
authors: Yu
authors: Center for Neuroscience, University of California, Davis, Davis, CA 95618 tycchen@ucdavis.edu.|Department of Neurology, University of California, Davis, Davis, CA 95618.
authors: Tsung-Yu
authors: TY
authors: Chen
mesh_terms: D000818:Animals; D000075369:Anoctamin-1; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D018118:Chloride Channels; D021122:Protein Subunits; D002118:Calcium