title: Regulation of bestrophins by Ca2+: a theoretical and experimental study.
abstract: Bestrophins are a recently discovered family of Cl(-) channels, for which no structural information is available. Some family members are activated by increased intracellular Ca2+ concentration. Bestrophins feature a well conserved Asp-rich tract in their COOH terminus (Asp-rich domain), which is homologous to Ca2+-binding motifs in human thrombospondins and in human big-conductance Ca2+- and voltage-gated K+ channels (BK(Ca)). Consequently, the Asp-rich domain is also a candidate for Ca2+ binding in bestrophins. Based on these considerations, we constructed homology models of human bestrophin-1 (Best1) Asp-rich domain using human thrombospondin-1 X-ray structure as a template. Molecular dynamics simulations were used to identify Asp and Glu residues binding Ca2+ and to predict the effects of their mutations to alanine. We then proceeded to test selected mutations in the Asp-rich domain of the highly homologous mouse bestrophin-2. The mutants expressed in HEK-293 cells were investigated by electrophysiological experiments using the whole-cell voltage-clamp technique. Based on our molecular modeling results, we predicted that Asp-rich domain has two defined binding sites and that D301A and D304A mutations may impact the binding of the metal ions. The experiments confirmed that these mutations do actually affect the function of the protein causing a large decrease in the Ca2+-activated Cl(-) current, fully consistent with our predictions. In addition, other studied mutations (E306A, D312A) did not decrease Ca2+-activated Cl(-) current in agreement with modeling results.
authors.affiliation: International School for Advanced Studies (SISSA/ISAS), Trieste, Italy.
authors.forename: Agata
authors.initials: A
authors.lastname: Kranjc
authors.forename: Federico W
authors.initials: FW
authors.lastname: Grillo
authors.forename: Juraj
authors.initials: J
authors.lastname: Rievaj
authors.forename: Anna
authors.initials: A
authors.lastname: Boccaccio
authors.forename: Fabio
authors.initials: F
authors.lastname: Pietrucci
authors.forename: Anna
authors.initials: A
authors.lastname: Menini
authors.forename: Paolo
authors.initials: P
authors.lastname: Carloni
authors.forename: Claudio
authors.initials: C
authors.lastname: Anselmi
mesh_terms: D000818:Animals; D000075002:Bestrophins; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D003198:Computer Simulation; D055724:Electrophysiological Phenomena; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D018408:Patch-Clamp Techniques; D017386:Sequence Homology, Amino Acid; D019700:Thrombospondin 1 / Q000737:chemistry
chemical_list: C113133:BEST1 protein, human; C497463:Best2 protein, mouse; D000075002:Bestrophins; D018118:Chloride Channels; D005136:Eye Proteins; D019700:Thrombospondin 1; D002118:Calcium