title: TMEM16A-TMEM16B chimaeras to investigate the structure-function relationship of calcium-activated chloride channels.
abstract: TMEM16A and TMEM16B proteins are CaCCs (Ca2+-activated Cl- channels) with eight putative transmembrane segments. As shown previously, expression of TMEM16B generates CaCCs characterized by a 10-fold lower Ca2+ affinity and by faster activation and deactivation kinetics with respect to TMEM16A. To investigate the basis of the different properties, we generated chimaeric proteins in which different domains of the TMEM16A protein were replaced by the equivalent domains of TMEM16B. Replacement of the N-terminus, TMD (transmembrane domain) 1-2, the first intracellular loop and TMD3-4 did not change the channel's properties. Instead, replacement of intracellular loop 3 decreased the apparent Ca2+ affinity by nearly 8-fold with respect to wild-type TMEM16A. In contrast, the membrane currents derived from chimaeras containing TMD7-8 or the C-terminus of TMEM16B showed higher activation and deactivation rates without a change in Ca2+ sensitivity. Significantly accelerated kinetics were also found when the entire C-terminus of the TMEM16A protein (77 amino acid residues) was deleted. Our findings indicate that the third intracellular loop of TMEM16A and TMEM16B is the site involved in Ca2+-sensitivity, whereas the C-terminal part, including TMD7-8, affect the rate of transition between the open and the closed state.
authors.affiliation: U.O.C. Genetica Medica, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16147 Genova, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Scudieri
authors.forename: Elvira
authors.initials: E
authors.lastname: Sondo
authors.forename: Emanuela
authors.initials: E
authors.lastname: Caci
authors.forename: Roberto
authors.initials: R
authors.lastname: Ravazzolo
authors.forename: Luis J V
authors.initials: LJ
authors.lastname: Galietta
mesh_terms: D000075369:Anoctamin-1; D000075368:Anoctamins; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D013329:Structure-Activity Relationship
chemical_list: C476213:ANO1 protein, human; C521849:ANO2 protein, human; D000075369:Anoctamin-1; D000075368:Anoctamins; D018118:Chloride Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins