title: Permeation Mechanisms in the TMEM16B Calcium-Activated Chloride Channels.
abstract: TMEM16A and TMEM16B encode for Ca2+-activated Cl- channels (CaCC) and are expressed in many cell types and play a relevant role in many physiological processes. Here, I performed a site-directed mutagenesis study to understand the molecular mechanisms of ion permeation of TMEM16B. I mutated two positive charged residues R573 and K540, respectively located at the entrance and inside the putative channel pore and I measured the properties of wild-type and mutant TMEM16B channels expressed in HEK-293 cells using whole-cell and excised inside-out patch clamp experiments. I found evidence that R573 and K540 control the ion permeability of TMEM16B depending both on which side of the membrane the ion substitution occurs and on the level of channel activation. Moreover, these residues contribute to control blockage or activation by permeant anions. Finally, R573 mutation abolishes the anomalous mole fraction effect observed in the presence of a permeable anion and it alters the apparent Ca2+-sensitivity of the channel. These findings indicate that residues facing the putative channel pore are responsible both for controlling the ion selectivity and the gating of the channel, providing an initial understanding of molecular mechanism of ion permeation in TMEM16B.
authors.affiliation: Neurobiology Group, SISSA, Scuola Internazionale Superiore di Studi Avanzati, Trieste, Italy.
authors.forename: Simone
authors.identifier: http://orcid.org/0000-0002-3687-3984
authors.initials: S
authors.lastname: Pifferi
mesh_terms: D000838:Anions / Q000378:metabolism; D000075369:Anoctamin-1; D000075368:Anoctamins; D002118:Calcium / Q000378:metabolism*; D018118:Chloride Channels / Q000378:metabolism*; D004594:Electrophysiology; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008565:Membrane Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010539:Permeability
chemical_list: C476213:ANO1 protein, human; C521849:ANO2 protein, human; D000838:Anions; D000075369:Anoctamin-1; D000075368:Anoctamins; D018118:Chloride Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D002118:Calcium