Premium
Changes in LTCC protein expression and channel function in A7r5 cells by a truncated β subunit
Author(s) -
Telemaque Sabine,
Fausther Michel,
Flynn Shawn E,
Liu Meeiyueh,
Singleton Terrie,
Dranoff Jonathan A,
Marsh James D
Publication year - 2012
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.26.1_supplement.lb638
Subject(s) - protein subunit , green fluorescent protein , microbiology and biotechnology , gi alpha subunit , intracellular , chemistry , biology , biochemistry , gene
Cytosolic Ca 2+ concentration ([Ca 2+ ] i ) in vascular smooth muscle cells (VSMC) is primarily regulated by Ca 2+ entry through L‐type Ca 2+ channels (LTCC), via the pore‐forming α 1C subunit. The intracellular β 2a subunit chaperone interacts with the α 1C subunit to regulate membrane localization and channel function. The aim of this study was to determine whether a truncated β 2a subunit expressing the N‐terminus portion of the β subunit (N‐BID) can regulate expression of other LTCC subunits and affect channel function in VSMC. Adenoviruses were used to overexpress the truncated β subunit (N‐BID) or the native β 2a subunit (Full‐β 2a ) fused to GFP in A7r5 cells, a rat aortic smooth muscle cell line. After 48 hr, cells were visualized under fluorescent microscopy before whole cell lysates were collected to determine total protein levels. Immunoblot analysis showed that total protein levels of the α 1C subunit were significantly elevated in A7r5 cells expressing N‐BID when compared to GFP (adenovirus control). Similarly, total protein levels of the α 2 subunit, a membrane‐bound subunit of the LTCC, were also increased in N‐BID‐cells when compared to GFP. In contrast, overexpression of the Full‐β 2a subunit did not alter total protein expression of these membrane‐bound subunits. Importantly, neither constructs affected endogenous expression of the β 2 subunit. Agonist‐induced increase in [Ca 2+ ] i was determined by confocal microscopy and appeared to be attenuated in N‐BID cells. Our results suggest that N‐BID can act as a decoy to affect expression, processing or degradation of the LTCC subunits while decreasing LTCC function in vitro . It could potentially be used to modulate L‐type Ca 2+ channel function and ultimately regulate vascular tone. This study was partially funded by a Grant in Aid from the AHA.