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J Biol Chem. 2016 May 20;291(21):11030-41. doi: 10.1074/jbc.M115.695080. Epub 2016 Mar 9.

αB-Crystallin Interacts with Nav1.5 and Regulates Ubiquitination and Internalization of Cell Surface Nav1.5.

Author information

1
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.
2
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.
3
the Department of Cardiology, First Affiliated Hospital of Xiamen University, 55 Zhenhai Road, 361003 Xiamen, China.
4
the Center for Cardiovascular Genetics, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, chenq3@ccf.org.
5
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.

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.

KEYWORDS:

Nav1.5; SCN5A; cardiovascular disease; electrophysiology; patch clamp; sodium channel; ubiquitylation (ubiquitination); αB-crystallin

PMID:
26961874
PMCID:
PMC4900253
DOI:
10.1074/jbc.M115.695080
[Indexed for MEDLINE]
Free PMC Article

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