Abstract 537: Rgulation of ALG-2 in NAADP-induced Ca2+ Release via TRP-ML1 Channel in Mouse Coronary Arterial Myocytes
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Abstract
We have demonstrated that nicotinic acid adenine dinucleotide phosphate (NAADP) sensitive Ca2+ release channel is characteristic of lysosomal TRP-ML1 in coronary arterial myocytes (CAMs). The present study hypothesized that NAADP induces Ca2+ release from TRP-ML1 channels via apoptosis-linked gene 2 (ALG-2), which was reported to act as a Ca2+ sensor that modulates the function of TRP-ML1. By RT-PCR and Western blot analysis, we demonstrated that ALG-2 is abundantly expressed in the cytosol of mouse CAMs. Confocal microscopy found that FasL and ET-1, well-known NAADP stimulators, increased the colocalization coefficient of ALG-2-GFP with TRP-ML1-RFP in CAMs from 0.17±0.033 to 0.3±0.044 and 0.3±0.09, respectively. In addition, the TRP-ML1 level was significantly increased in FasL or ET-1-treated CAMs after immunoprecipitation with anti-ALG-2, confirming that the interaction of ALG-2 with TRP-ML1 was enhanced in response to NAADP production. Furthermore, NAADP competitively inhibited recombinant ALG-2 protein binding with tis-NED-19, which fluorescently labels NAADP receptors. Importantly, fluorescent microscopic image analysis showed that NAADP-induced Ca2+ release was reduced from 380±42.03 nM to 216.7±14.7 nM after the transfection with ALG-2 siRNA in CAMs, which was also found after the treatment with FasL or ET-1. Our findings support the view that ALG-2 can directly bind with NAADP, which mediates the action of NAADP through lysosomal TRP-ML1 channels. Such results throw light on NAADP-TRP-ML1 working model in vascular cells.
Author Disclosures: M. Xu: None. X. Li: None. M. Xia: None. Y. Zhang: None. P. Li: None.
- © 2014 by American Heart Association, Inc.
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- Abstract 537: Rgulation of ALG-2 in NAADP-induced Ca2+ Release via TRP-ML1 Channel in Mouse Coronary Arterial MyocytesMing Xu, Xiao-Xue Li, Min Xia, Yang Zhang and Pin-Lan LiHypertension. 2014;64:A537, originally published February 5, 2015
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