LITAF Regulates Cardiac L-type Calcium Channels by Modulating NEDD4-1 Ubiquitin Ligase
- Moshal, K.S., Roder, K., Kabakov, A.Y., Werdich, A.A., Chiang, D.Y., Turan, N.N., Xie, A., Kim, T.Y., Cooper, L.L., Lu, Y., Zhong, M., Li, W., Terentyev, D., Choi, B.R., Karma, A., MacRae, C.A., Koren, G.
- Circulation. Genomic and precision medicine 12(9): 407-420 (Journal)
- Registered Authors
- MacRae, Calum A.
- lipopolysaccharide-induced tumor necrosis factor
- MeSH Terms
- Calcium Channels, L-Type/genetics
- Calcium Channels, L-Type/metabolism*
- Myocytes, Cardiac/enzymology
- Nedd4 Ubiquitin Protein Ligases/genetics
- Nedd4 Ubiquitin Protein Ligases/metabolism
- Nuclear Proteins/genetics
- Nuclear Proteins/metabolism
- Transcription Factors/genetics
- Transcription Factors/metabolism
- Zebrafish/growth & development
- 31462068 Full text @ Circ Genom Precis Med
Moshal, K.S., Roder, K., Kabakov, A.Y., Werdich, A.A., Chiang, D.Y., Turan, N.N., Xie, A., Kim, T.Y., Cooper, L.L., Lu, Y., Zhong, M., Li, W., Terentyev, D., Choi, B.R., Karma, A., MacRae, C.A., Koren, G. (2019) LITAF Regulates Cardiac L-type Calcium Channels by Modulating NEDD4-1 Ubiquitin Ligase. Circulation. Genomic and precision medicine. 12(9):407-420.
Background - The turnover of cardiac ion channels underlying action potential duration (APD) is regulated by ubiquitination. Genome-wide association studies (GWAS) 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 ventricular myocytes (ARbCM) to determine intracellular Ca2+handling. LTCC (L-type calcium channel) current was measured using whole cell recording. To study the effect of LITAF on Cav1.2 channel expression, surface biotinylation and westerns were performed. LITAF interactions were studied using co-immunoprecipitation and in situ proximity ligation assay (PLA). 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 ICa,L and Cavα1c abundance, whereas LITAF knockdown increased ICa,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in ARbCM, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed co-localization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by co-expressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on ICa,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of ICa,L current associated with LITAF overexpression simultaneously shortened APD 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.
Genes / Markers
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes