LITAF (Lipopolysaccharide-Induced Tumor Necrosis Factor) Regulates Cardiac L-Type Calcium Channels by Modulating NEDD (Neural Precursor Cell Expressed Developmentally Downregulated Protein) 4-1 Ubiquitin Ligase

Karni S. Moshal; Karim Roder; Anatoli Y. KabakovAndreas A. WerdichDavid Yi-Eng ChiangNilufer N. TuranAn Xie

摘要

BACKGROUND:The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation. METHODS: Optical mapping was performed in zebrafish hearts to determine Ca2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca2+handling. L-type calcium channel (LTCC) current (/CaL) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay. RESULTS: LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in /CaL and Cava1c abundance, whereas LITAF knockdown increased /CaL and Cava1c protein. LITAF-overexpressing decreases calciumtransients in adult rabbit cardiomyocytes, which was associated with lower Cava1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cava1c via increased Cava1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cava1c ubiquitination. Cava1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on /CaL and Cava1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of /CaL current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes. CONCLUSIONS: LITAF acts as an adaptor protein promoting NEDD4-1-mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.gggaction potential, calcium, rabbit, tumor necrosis factor, ubiquitination

机译：背景:心脏离子通道的营业额基本动作电位持续时间是监管泛素化。研究QT间隔确定几个单核苷酸多态性位于或附近的基因蛋白质泛素化。基因变异LITAF上游(lipopolysaccharide-induced肿瘤坏死因素)基因促使我们决定的作用调节心脏兴奋。在斑马鱼心脏执行映射确定Ca2 +瞬变。钙成像进行成年兔心肌细胞来确定细胞内Ca2 +处理。使用全细胞电流(/ CaL)测量录音。(1.2 l型电压门控钙通道)渠道表达,表面生物素酰化,西部片进行。研究了使用coimmunoprecipitation和原位接近结扎试验。击倒在斑马鱼导致一个健壮的增加钙瞬变。LITAF在质询兔心肌细胞导致减少/卡尔和Cava1c丰富,而LITAF击倒增加/卡尔和Cava1c蛋白质。减少calciumtransients在成年的兔子心肌细胞,较低有关Cava1c水平。LITAF表达下调和表面池总额通过增加Cava1c Cava1c泛素化其随后的溶酶体降解。在tsA201 colocalization LITAF与确立和心肌细胞。前驱细胞表达发育4 - 1表达下调的蛋白质),但不是它的催化地NEDD4-1-C867A活动形式,增加Cava1c泛素化。泛素化进一步增加了coexpressed LITAF NEDD4-1但不是NEDD4-1-C867A。负面影响的LITAF /卡尔和Cava1c水平的质询兔心肌细胞。计算机模拟表明,减少/加州当前与LITAF相关联超表达同时缩短行动潜在的持续时间和减少钙瞬变兔子心肌细胞。LITAF作为适配器蛋白质促进NEDD4-1-mediated泛素化和随后的退化的确立,从而控制确立膜水平和功能,因此心脏激发。肿瘤坏死因子,泛素化

LITAF (Lipopolysaccharide-Induced Tumor Necrosis Factor) Regulates Cardiac L-Type Calcium Channels by Modulating NEDD (Neural Precursor Cell Expressed Developmentally Downregulated Protein) 4-1 Ubiquitin Ligase

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