regulation of bestrophins by ca2+ a theoretical and experimental study监管bestrophins ca2 +的理论和实验研究.pdfVIP

Regulation of Bestrophins by Ca2+: A Theoretical and Experimental Study 1. 1. 1. 1,2¤a 1 Agata Kranjc , Federico W. Grillo , Juraj Rievaj , Anna Boccaccio , Fabio Pietrucci , Anna Menini1,2, Paolo Carloni1,2,3, Claudio Anselmi1,3¤b* 1 International School for Advanced Studies (SISSA/ISAS), Trieste, Italy, 2 Italian Institute of Technology (IIT), SISSA-Unit, Trieste, Italy, 3 CNR-INFM-DEMOCRITOS Modeling Center for Research in Atomistic Simulation, Trieste, Italy Abstract Bestrophins are a recently discovered family of Cl2 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 (BKCa). 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