Modification of store-operated channel coupling and inositol trisphosphate receptor function by 2-aminoethoxydiphenyl borate in DT40 lymphocytes
Modification of store-operated channel coupling and inositol trisphosphate receptor function by 2-aminoethoxydiphenyl borate in DT40 lymphocytes
复制标题
DOI：
10.1074/jbc.m107755200
复制
发表时间：
2002-03-01
影响因子：
4.8
通讯作者：
Gill, DL
中科院分区：
文献类型：
--
作者：
Ma, HT;Venkatachalam, K;Gill, DL
钙储存操纵通道 (SOC) 为介导长期 Ca2+ 信号和补充多种细胞类型中的 Ca2+ 储存提供了重要手段。然而，内质网储存激活质膜 SOC 之间的耦合机制仍不清楚。在 DT40 鸡 B 淋巴细胞中，渗透性肌醇三磷酸受体 (InSP3R) 修饰剂 2-氨基乙氧基二苯硼酸盐 (2-APB) 是 1-10 muM 范围内钙池操纵的 Ca2+ 内流的强大激活剂。 2-APB 激活了真实的 SOC，因为该进入对 Ca2+ 具有完全选择性（没有检测到 Ba2+ 或 Sr2+ 离子的进入），并且对 La3+ 离子高度敏感（IC50 30-100 nM）。为了评估 InSP(3)R 在此反应中的作用，我们使用了 DT40 三重 InsP(3)R 敲除 (ko) 细胞系 DT40InSP(3)R-ko，其中使用与所有三种鸡 InSP3R 亚型的 N 端表位交叉反应的抗体通过蛋白质印迹分析验证了全长 InSP(3)R 或 InsP(3)R 片段的缺失。 2-APB 诱导的 SOC 激活与 DT40InSP(3)R-ko 中的细胞相同，表明 InSP(3)R 不参与其中。对于野生型 (wt) 和 ko DT40 细胞，2-APB 对钙池充足细胞中的 Ca2+ 进入没有影响，表明其作用仅限于处于池耦合状态的 SOC。 2-APB 诱导完整 DT40wt 细胞中储存的 Ca2+ 释放的强烈激活，但在 DT40InSp(3)R-ko 细胞中则不然，表明 InsP3R 介导的作用。相反，2-APB 阻断透化 DT40wt 细胞中的 InsP(3)Rs，表明 2-APB 的刺激作用仅限于完整细胞中功能偶联的 InSP(3)Rs。通过花萼蛋白 A 诱导的细胞骨架重排，完整细胞中 ER/PM 相互作用的解偶联通过储存清空和 2-APB 阻止了 SOC 激活；这种治疗完全阻止了 2-APB 诱导的 InSP3R 激活，但没有改变磷脂酶 C 偶联受体刺激介导的 InsP3R 激活。结果表明，2-APB 对 SOC 和 InSP(3)R 的强大双功能作用取决于这些通道的耦合状态，并表明 2-APB 可能以参与介导钙池操作的 Ca2+ 内流的耦合机制为目标。
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.