Molecular simulation assisted identification of Ca(2+) binding residues in TMEM16A.
Calcium-activated chloride channels (CaCCs) play vital roles in a variety of physiological processes. Transmembrane protein 16A (TMEM16A) has been confirmed as the molecular counterpart of CaCCs which greatly pushes the molecular insights of CaCCs forward. However, the detailed mechanism of Ca(2+) binding and activating the channel is still obscure. Here, we utilized a combination of computational and electrophysiological approaches to discern the molecular mechanism by which Ca(2+) regulates the gating of TMEM16A channels. The simulation results show that the first intracellular loop serves as a Ca(2+) binding site including D439, E444 and E447. The experimental results indicate that a novel residue, E447, plays key role in Ca(2+) binding. Compared with WT TMEM16A, E447Y produces a 30-fold increase in EC50 of Ca(2+) activation and leads to a 100-fold increase in Ca(2+) concentrations that is needed to fully activate the channel. The following steered molecular dynamic (SMD) simulation data suggests that the mutations at 447 reduce the Ca(2+) dissociation energy. Our results indicated that both the electrical property and the size of the side-chain at residue 447 have significant effects on Ca(2+) dependent gating of TMEM16A.
Key laboratory of Molecular Biophysics, Tianjin, Hebei Province, China.|Institute of Biophysics, School of Sciences, Hebei University of Technology, Tianjin, China.
Chun-Li
CL
Pang
Key laboratory of Molecular Biophysics, Tianjin, Hebei Province, China.|Institute of Biophysics, School of Sciences, Hebei University of Technology, Tianjin, China.
Hong-Bo
HB
Yuan
Key laboratory of Molecular Biophysics, Tianjin, Hebei Province, China.|Institute of Biophysics, School of Sciences, Hebei University of Technology, Tianjin, China.
Tian-Guang
TG
Cao
School of Sciences, Yanshan University, Qinhuangdao, China.
Ji-Guo
JG
Su
Key laboratory of Molecular Biophysics, Tianjin, Hebei Province, China.|Institute of Biophysics, School of Sciences, Hebei University of Technology, Tianjin, China.
Ya-Fei
YF
Chen
Key laboratory of Molecular Biophysics, Tianjin, Hebei Province, China.|Institute of Biophysics, School of Sciences, Hebei University of Technology, Tianjin, China.
Hui
H
Liu
Key laboratory of Molecular Biophysics, Tianjin, Hebei Province, China.|Institute of Biophysics, School of Sciences, Hebei University of Technology, Tianjin, China.
Hui
H
Yu
Key Laboratory of Neural and Vascular Biology, Ministry of Education, Shijiazhuang, China.|The Key Laboratory of Pharmacology and Toxicology for New Drug, Shijiazhuang, Hebei Province, China.|Department of Pharmacology, Hebei Medical University, Shijiazhuang, China.
Hai-Ling
HL
Zhang
Key laboratory of Molecular Biophysics, Tianjin, Hebei Province, China. zhany@hebut.edu.cn.|Institute of Biophysics, School of Sciences, Hebei University of Technology, Tianjin, China. zhany@hebut.edu.cn.
Yong
Y
Zhan
Key laboratory of Molecular Biophysics, Tianjin, Hebei Province, China. hailong_an@hebut.edu.cn.|Institute of Biophysics, School of Sciences, Hebei University of Technology, Tianjin, China. hailong_an@hebut.edu.cn.
Hai-Long
HL
An
Key laboratory of Molecular Biophysics, Tianjin, Hebei Province, China.|Institute of Biophysics, School of Sciences, Hebei University of Technology, Tianjin, China.
Yue-Bin
YB
Han
D000596:Amino Acids / Q000737:chemistry; D000075369:Anoctamin-1; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism
C476213:ANO1 protein, human; D000596:Amino Acids; D000075369:Anoctamin-1; D018118:Chloride Channels; D009363:Neoplasm Proteins; D002118:Calcium
Calcium-binding site; Homology modeling; Molecular dynamics; Patch clamp; Site-directed mutagenesis; TMEM16A