Changes in LTCC protein expression and channel function in A7r5 cells by a truncated β subunit

Abstract

Cytosolic Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC) is primarily regulated by Ca2+ entry through L‐type Ca2+ channels (LTCC), via the pore‐forming α1C subunit. The intracellular β2a subunit chaperone interacts with the α1C subunit to regulate membrane localization and channel function. The aim of this study was to determine whether a truncated β2a 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 β2a subunit (Full‐β2a) 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 α1C subunit were significantly elevated in A7r5 cells expressing N‐BID when compared to GFP (adenovirus control). Similarly, total protein levels of the α2 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‐β2a subunit did not alter total protein expression of these membrane‐bound subunits. Importantly, neither constructs affected endogenous expression of the β2 subunit. Agonist‐induced increase in [Ca2+]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 Ca2+ channel function and ultimately regulate vascular tone. This study was partially funded by a Grant in Aid from the AHA.