Kawano Tomonori, Kadono Takashi, Fumoto Katsumi, Lapeyrie Frédéric, Kuse Masaki, Isobe Minoru, Furuichi Takuya, Muto Shoshi
Graduate School of Environmental Engineering, The University of Kitakyushu, Kitakyushu 808-0135, Japan.
Biochem Biophys Res Commun. 2004 Nov 5;324(1):40-5. doi: 10.1016/j.bbrc.2004.09.015.
In plant cells, Al ion plays dual roles as an inducer and an inhibitor of Ca(2+) influx depending on the concentration. Here, the effects of Al on Ca(2+) signaling were assessed in tobacco BY-2 cells expressing aequorin and a putative plant Ca(2+) channel from Arabidopsis thaliana, AtTPC1 (two-pore channel 1). In wild-type cells (expressing only aequorin), Al treatment induced the generation of superoxide, and Ca(2+) influx was secondarily induced by superoxide. Higher Al concentrations inhibited the Al-stimulated and superoxide-mediated Ca(2+) influx, indicating that Ca(2+) channels responsive to reactive oxygen species (ROS) are blocked by high concentration of Al. H(2)O(2)-induced Ca(2+) influx was also inhibited by Al. Thus, inhibitory action of Al against ROS-induced Ca(2+) influx was confirmed. Similarly, known Ca(2+) channel blockers such as ions of La and Gd inhibited the H(2)O(2)-induced Ca(2+) influx. While La also inhibited the hypoosmotically induced Ca(2+) influx, Al showed no inhibitory effect against the hypoosmotic Ca(2+) influx. The effects of Al and La on Ca(2+) influx were also tested in the cell line overexpressing AtTPC1 and the cell line AtTPC1-dependently cosuppressing the endogenous TPC1 equivalents. Notably, responsiveness to H(2)O(2) was lost in the cosuppression cell line, thus TPC1 channels are required for ROS-responsive Ca(2+) influx. Data also suggested that hypoosmotic shock induces TPC1-independent Ca(2+) influx and Al shows no inhibitory action against the TPC1-independent event. In addition, AtTPC1 overexpression resulted in a marked increase in Al-sensitive Ca(2+) influx, indicating that TPC1 channels participate in osmotic Ca(2+) influx only when overexpressed. We concluded that members of TPC1 channel family are the only ROS-responsive Ca(2+) channels and are the possible targets of Al-dependent inhibition.
在植物细胞中，铝离子根据浓度不同，对钙离子内流起着诱导剂和抑制剂的双重作用。在此，我们在表达水母发光蛋白和拟南芥一种假定的植物钙离子通道AtTPC1（双孔通道1）的烟草BY-2细胞中评估了铝对钙离子信号传导的影响。在野生型细胞（仅表达水母发光蛋白）中，铝处理诱导了超氧化物的产生，并且超氧化物继而诱导了钙离子内流。更高浓度的铝抑制了铝刺激的和超氧化物介导的钙离子内流，这表明对活性氧（ROS）有反应的钙离子通道被高浓度的铝所阻断。过氧化氢诱导的钙离子内流也被铝抑制。因此，证实了铝对ROS诱导的钙离子内流具有抑制作用。同样，已知的钙离子通道阻滞剂如镧离子和钆离子也抑制了过氧化氢诱导的钙离子内流。虽然镧也抑制了低渗诱导的钙离子内流，但铝对低渗钙离子内流没有抑制作用。我们还在过表达AtTPC1的细胞系和AtTPC1依赖性共抑制内源性TPC1等同物的细胞系中测试了铝和镧对钙离子内流的影响。值得注意的是，共抑制细胞系中对过氧化氢的反应性丧失了，因此TPC1通道是ROS反应性钙离子内流所必需的。数据还表明，低渗休克诱导了不依赖TPC1的钙离子内流，并且铝对不依赖TPC1的事件没有抑制作用。此外，AtTPC1的过表达导致对铝敏感的钙离子内流显著增加，表明TPC1通道仅在过表达时参与渗透性钙离子内流。我们得出结论，TPC1通道家族成员是唯一对ROS有反应的钙离子通道，并且是铝依赖性抑制的可能靶点。
