title: Purified TMEM16A is sufficient to form Ca2+-activated Cl- channels.
abstract: Ca(2+)-activated Cl(-) channels (CaCCs) are key regulators of numerous physiological functions, ranging from electrolyte secretion in airway epithelia to cellular excitability in sensory neurons and muscle fibers. Recently, TMEM16A (ANO1) and -B were shown to be critical components of CaCCs. It is still unknown whether they are also sufficient to form functional CaCCs, or whether association with other subunits is required. Recent reports suggest that the Ca(2+) sensitivity of TMEM16A is mediated by its association with calmodulin, suggesting that functional CaCCs are heteromultimers. To test whether TMEM16A is necessary and sufficient to form functional CaCCs, we expressed, purified, and reconstituted human TMEM16A. The purified protein mediates Ca(2+)-dependent Cl(-) transport with submicromolar sensitivity to Ca(2+), consistent with what is seen in patch-clamp experiments. The channel is synergistically gated by Ca(2+) and voltage, so that opening is promoted by depolarizing potentials. Mutating two conserved glutamates in the TM6-7 intracellular loop selectively abolishes the Ca(2+) dependence of reconstituted TMEM16A, in a manner similar to what was reported for the heterologously expressed channel. Well-characterized CaCC blockers inhibit Cl(-) transport with Kis comparable to those measured for native and heterologously expressed CaCCs. Finally, direct physical interactions between calmodulin and TMEM16A could not be detected in copurification experiments or in functional assays. Our results demonstrate that purified TMEM16A is necessary and sufficient to recapitulate the biophysical and pharmacological properties of native and heterologously expressed CaCCs. Our results also show that association of TMEM16A with other proteins, such as calmodulin, is not required for function.
authors.affiliation: Departments of Anesthesiology, Physiology and Biophysics, and Biochemistry, Weill Cornell Medical College, Cornell University, New York, NY 10065.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Terashima
authors.forename: Alessandra
authors.initials: A
authors.lastname: Picollo
authors.forename: Alessio
authors.initials: A
authors.lastname: Accardi
mesh_terms: D000818:Animals; D000075369:Anoctamin-1; D001692:Biological Transport / Q000502:physiology; D002147:Calmodulin / Q000378:metabolism*; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D006801:Humans; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000502:physiology; D016296:Mutagenesis; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: C476213:ANO1 protein, human; D000075369:Anoctamin-1; D002147:Calmodulin; D018118:Chloride Channels; D009363:Neoplasm Proteins
keywords: anoctamin; ion channels; membrane protein; reconstitution