Modification of store-operated channel coupling and inositol trisphosphate receptor function by 2-aminoethoxydiphenyl borate in DT40 lymphocytes

被引:150
作者
Ma, HT
Venkatachalam, K
Parys, JB
Gill, DL
机构
[1] Univ Maryland, Sch Med, Dept Biochem & Mol Biol, Baltimore, MD 21201 USA
[2] Katholieke Univ Leuven, Fysiol Lab, B-3000 Louvain, Belgium
关键词
D O I
10.1074/jbc.M107755200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Store-operated channels (SOCs) provide an important means for mediating longer-term Ca2+ signals and replenishment of Ca2+ stores in a multitude of cell types. However, the coupling mechanism between endoplasmic reticulum stores to activate plasma membrane SOCs remains unknown. In DT40 chicken B lymphocytes, the permeant inositol trisphosphate receptor (InSP3R) modifier, 2-aminoethoxydiphenyl borate (2-APB), was a powerful activator of store-operated Ca2+ entry between 1-10 muM. 2-APB activated authentic SOCs because the entry was totally selective for Ca2+ (no detectable entry of Ba2+ or Sr2+ ions), and highly sensitive to La3+ ions (IC50 30-100 nM). To assess the role of InSP(3)Rs in this response, we used the DT40 triple InsP(3)R-knockout (ko) cell line, DT40InSP(3)R-ko, in which the absence of full-length InSP(3)Rs or InsP(3)R fragments was verified by Western analysis using antibodies cross-reacting with N-terminal epitopes of all three chicken InSP3R subtypes. The 2-APB-induced activation of SOCs was identical in the DT40InSP(3)R-ko, cells indicating InSP(3)Rs were not involved. With both wild type (wt) and ko DT40 cells, 2-APB had no effect on Ca2+ entry in store-replete cells, indicating that its action was restricted to SOCs in a store-coupled state. 2-APB induced a robust activation of Ca2+ release from stores in intact DT40wt cells but not in DT40InSp(3)R-ko cells, indicating an InsP3R-mediated effect. In contrast, 2-APB blocked InsP(3)Rs in permeabilized DT40wt cells, suggesting that the stimulatory action of 2-APB was restricted to functionally coupled InSP(3)Rs in intact cells. Uncoupling of ER/PM interactions in intact cells by calyculin A-induced cytoskeletal rearrangement prevented SOC activation by store-emptying and 2-APB; this treatment completely prevented 2-APB-induced InSP3R activation but did not alter InsP3R activation mediated by phospholipase C-coupled receptor stimulation. The results indicate that the robust bifunctional actions of 2-APB on both SOCs and InSP(3)Rs are dependent on the coupled state of these channels and suggest that 2-APB may target the coupling machinery involved in mediating store-operated Ca2+ entry.
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页码:6915 / 6922
页数:8
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