title: αB-Crystallin Interacts with Nav1.5 and Regulates Ubiquitination and Internalization of Cell Surface Nav1.5.
abstract: Nav1.5, the pore-forming α subunit of the cardiac voltage-gated Na(+) channel complex, is required for the initiation and propagation of the cardiac action potential. Mutations in Nav1.5 cause cardiac arrhythmias and sudden death. The cardiac Na(+) channel functions as a protein complex; however, its complete components remain to be fully elucidated. A yeast two-hybrid screen identified a new candidate Nav1.5-interacting protein, αB-crystallin. GST pull-down, co-immunoprecipitation, and immunostaining analyses validated the interaction between Nav1.5 and αB-crystallin. Whole-cell patch clamping showed that overexpression of αB-crystallin significantly increased peak sodium current (INa) density, and the underlying molecular mechanism is the increased cell surface expression level of Nav1.5 via reduced internalization of cell surface Nav1.5 and ubiquitination of Nav1.5. Knock-out of αB-crystallin expression significantly decreased the cell surface expression level of Nav1.5. Co-immunoprecipitation analysis showed that αB-crystallin interacted with Nedd4-2; however, a catalytically inactive Nedd4-2-C801S mutant impaired the interaction and abolished the up-regulation of INa by αB-crystallin. Nav1.5 mutation V1980A at the interaction site for Nedd4-2 eliminated the effect of αB-crystallin on reduction of Nav1.5 ubiquitination and increases of INa density. Two disease-causing mutations in αB-crystallin, R109H and R151X (nonsense mutation), eliminated the effect of αB-crystallin on INa This study identifies αB-crystallin as a new binding partner for Nav1.5. αB-Crystallin interacts with Nav1.5 and increases INa by modulating the expression level and internalization of cell surface Nav1.5 and ubiquitination of Nav1.5, which requires the protein-protein interactions between αB-crystallin and Nav1.5 and between αB-crystallin and functionally active Nedd4-2.
authors.affiliation: From the Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Center, College of Life Science and Technology and Center for Human Genome Research, 1037 Luoyu Road, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: From the Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Center, College of Life Science and Technology and Center for Human Genome Research, 1037 Luoyu Road, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Zhijie
authors.initials: Z
authors.lastname: Wang
authors.affiliation: From the Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Center, College of Life Science and Technology and Center for Human Genome Research, 1037 Luoyu Road, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Yinan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: From the Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Center, College of Life Science and Technology and Center for Human Genome Research, 1037 Luoyu Road, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Xiong
authors.affiliation: From the Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Center, College of Life Science and Technology and Center for Human Genome Research, 1037 Luoyu Road, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: the Center for Cardiovascular Genetics, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, the Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, and Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, Ohio 44106, and.
authors.forename: Ling
authors.initials: L
authors.lastname: Wu
authors.affiliation: From the Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Center, College of Life Science and Technology and Center for Human Genome Research, 1037 Luoyu Road, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Yuan
authors.affiliation: From the Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Center, College of Life Science and Technology and Center for Human Genome Research, 1037 Luoyu Road, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Longfei
authors.initials: L
authors.lastname: Wang
authors.affiliation: the Department of Cardiology, First Affiliated Hospital of Xiamen University, 55 Zhenhai Road, 361003 Xiamen, China.
authors.forename: Yuxi
authors.initials: Y
authors.lastname: Hou
authors.affiliation: From the Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Center, College of Life Science and Technology and Center for Human Genome Research, 1037 Luoyu Road, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Yu
authors.affiliation: the Department of Cardiology, First Affiliated Hospital of Xiamen University, 55 Zhenhai Road, 361003 Xiamen, China.
authors.forename: Zhengrong
authors.initials: Z
authors.lastname: Huang
authors.affiliation: From the Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Center, College of Life Science and Technology and Center for Human Genome Research, 1037 Luoyu Road, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Chengqi
authors.initials: C
authors.lastname: Xu
authors.affiliation: the Center for Cardiovascular Genetics, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, chenq3@ccf.org.
authors.forename: Qiuyun
authors.initials: Q
authors.lastname: Chen
authors.affiliation: From the Key Laboratory of Molecular Biophysics of the Ministry of Education, Cardio-X Center, College of Life Science and Technology and Center for Human Genome Research, 1037 Luoyu Road, Huazhong University of Science and Technology, Wuhan 430074, China, the Center for Cardiovascular Genetics, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, qkwang@hust.edu.cn.
authors.forename: Qing K
authors.initials: QK
authors.lastname: Wang
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000075702:Nedd4 Ubiquitin Protein Ligases; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D021381:Protein Transport; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: C059215:CRYAB protein, human; D056827:Endosomal Sorting Complexes Required for Transport; D050505:Mutant Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D011994:Recombinant Proteins; C568320:SCN5A protein, human; D038203:alpha-Crystallin B Chain; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases
keywords: Nav1.5; SCN5A; cardiovascular disease; electrophysiology; patch clamp; sodium channel; ubiquitylation (ubiquitination); αB-crystallin