title: Anion permeation in calcium-activated chloride channels formed by TMEM16A from Xenopus tropicalis.
abstract: Calcium-activated chloride channels (CaCC) formed by anoctamin1/TMEM16A subunits are ubiquitously expressed, and these channels are known to prevent polyspermy in amphibian oocytes. Here, we describe a TMEM16A clone isolated from Xenopus tropicalis oocytes (xtTMEM16A) and how the anion permeation properties are modified in single-site mutants of the ion pore. The anion permeability sequence was SCN(-) > I(-) > Br(-) > Cl(-) > gluconate (relative permeabilities 5.6:3.0:2.1:1:0.2, respectively). Dose-response curves indicated that the voltage-dependent half-maximal concentration for Ca(2+) activation (K d of the Hill equation at +100 mV) was 120 nM in normal external Cl(-), whereas it was displaced leftward to 75 nM Ca(2+), when I(-) replaced Cl(-). The I(-):Cl(-) mole fraction (MF) of the external solution was varied in order to gain insight into the permeation mechanism of the pore. No anomaly in MF behavior was observed for conductance, but it was observed for current reversal potential, which deviated from the prediction of the Goldman-Hodgkin-Katz equation. Mutations of positively charged amino acids in the pore, R646 and R761, to glutamate resulted in reduction of the relative permeability to I(-). Data from the wild type and mutants could be well fitted by a three-barrier, two-site permeation model. This suggests a multi-ion pore with at least two binding sites for anions, with R646 mole fraction closer to the extracellular membrane surface--being important for the stability of both sites--and R761--located deeper within the membrane--mainly affecting the innermost binding site. Considerations of xtTMEM16A putative pore region topology are discussed in the light of two alternative topological models of the protein.
authors.affiliation: Departamento de Neurobiología Celular y Molecular, Laboratorio de Neurobiología Molecular y Celular, Instituto de Neurobiología, Campus UNAM Juriquilla, Santiago de Querétaro, CP 76230, Querétaro, México.
authors.forename: J P
authors.initials: JP
authors.lastname: Reyes
authors.forename: A
authors.initials: A
authors.lastname: López-Rodríguez
authors.forename: A E
authors.initials: AE
authors.lastname: Espino-Saldaña
authors.forename: A
authors.initials: A
authors.lastname: Huanosta-Gutiérrez
authors.forename: R
authors.initials: R
authors.lastname: Miledi
authors.forename: A
authors.initials: A
authors.lastname: Martínez-Torres
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions / Q000378:metabolism; D018118:Chloride Channels / Q000502:physiology*; D003001:Cloning, Molecular / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010539:Permeability; D014162:Transfection; D014981:Xenopus; D029867:Xenopus Proteins / Q000502:physiology*
chemical_list: D000838:Anions; D018118:Chloride Channels; D029867:Xenopus Proteins; C559904:xANO2 protein, Xenopus