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Mapping the in vitro interactome of cardiac sodium (Na + )‐calcium (Ca 2+ ) exchanger 1 (NCX1)
Author(s) -
Lubelwana Hafver Tandekile,
Wanichawan Pimthanya,
Manfra Ornella,
Souza Gustavo Antonio,
Lunde Marianne,
Martinsen Marita,
Louch William Edward,
Sejersted Ole Mathias,
Carlson Cathrine Rein
Publication year - 2017
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201600417
Subject(s) - interactome , sodium calcium exchanger , antiporter , cytosol , chemistry , hek 293 cells , microbiology and biotechnology , calcium binding protein , calcium , biology , biochemistry , gene , intracellular , membrane , enzyme , organic chemistry
Abstract The sodium (Na + )‐calcium (Ca 2+ ) exchanger 1 (NCX1) is an antiporter membrane protein encoded by the SLC8A1 gene. In the heart, it maintains cytosolic Ca 2+ homeostasis, serving as the primary mechanism for Ca 2+ extrusion during relaxation. Dysregulation of NCX1 is observed in end‐stage human heart failure. In this study, we used affinity purification coupled with MS in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: (1) anti‐NCX1 against endogenous NCX1 and (2) anti‐His (where His is histidine) with His‐trigger factor‐NCX1 cyt recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature, and 29 occurred in both screens. Ten novel protein partners (DYRK1A, PPP2R2A, SNTB1, DMD, RABGGTA, DNAJB4, BAG3, PDE3A, POPDC2, STK39) were validated for binding to NCX1, and two partners (DYRK1A, SNTB1) increased NCX1 activity when expressed in HEK293 cells. A cardiac NCX1 protein–protein interaction map was constructed. The map was highly connected, containing distinct clusters of proteins with different biological functions, where “cell communication” and “signal transduction” formed the largest clusters. The NCX1 interactome was also significantly enriched with proteins/genes involved in “cardiovascular disease” which can be explored as novel drug targets in future research.