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Leucine‐Rich Repeat Containing 10 (LRRC10) protein associates with and regulates the cardiac Ca v 1.2 L‐type calcium channels (LB693)
Author(s) -
August Benjamin,
Reynolds Courtney,
Balijepalli Ravi
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb693
Subject(s) - cav1.2 , protein subunit , immunoprecipitation , western blot , microbiology and biotechnology , hek 293 cells , myocyte , chemistry , point mutation , mutation , biology , gene , biochemistry
Leucine‐rich repeat containing 10 (LRRC10) is a cardiac‐specific protein expressed in embryonic and adult hearts and plays critical roles in cardiac development and function. Recently we have demonstrated that the Lrrc10 ‐null ( Lrrc10 ‐/‐ ) mice develop dilated cardiomyopathy and ventricular myocytes from Lrrc10 ‐/‐ mice exhibit reduced L‐type Ca 2+ channel (LTCC) current (I Ca,L ) density. However, the precise role of LRRC10 protein in the regulation of LTCC function in the cardiomyocytes is unknown. To investigate the regulatory role of LRRC10 on Ca v 1.2 channel function, we co‐expressed the Myc tagged LRRC10 (LRRC10‐Myc), heamagglutinin tagged Ca v 1.2 (Ca v 1.2‐HA) and auxiliary Ca v β 2CN4 subunit in HEK293 cells and performed co‐immunoprecipitation (co‐IP) on lysates using either anti‐HA, anti‐Myc antibody or control IgG. Western blot analysis demonstrated that Ca v 1.2 and LRRC10 associated with one another. Also, the auxiliary Ca v β 2C subunit and LRRC10 did not co‐IP with one another suggesting that the LRRC10 may directly interact with the Ca v 1.2 subunit. A single point mutation H150A or triple point mutations Y104A, W127A and H150A mutation in LRRC10 disrupted the LRRC10 protein association with Ca v 1.2. Co‐IP analysis using mouse ventricular homogenates demonstrated that LRRC10 and Ca v 1.2 subunit are associated with one another. Additionally, double immunogold labeling and electron microscopy analysis revealed that LRRC10 and Cav1.2 proteins are co‐localized at T‐tubules in the ventricular myocytes. Finally, whole‐cell patch clamp experiments performed in ventricular myocytes from Lrrc10 ‐/‐ mice demonstrated a significant reduction in the I Ca,L density (‐2.5 0.2 pA/pF) and compared to WT myocytes (‐6 0.6 pA/pF). Furthermore, the inactivation of the I Ca,L in Lrrc10 ‐/‐ myocytes was significantly delayed. We conclude that LRRC10 protein is a novel and essential regulator of the LTCC function in ventricular myocytes. Grant Funding Source : R01HL105713