In plant cells, Al ion plays dual roles as an inducer and an inhibitor of Ca2+ influx depending on the concentration. Here, the effects of Al on Ca2+ signaling were assessed in tobacco BY-2 cells expressing aequorin and a putative plant Ca2+ channel from Arabidopsis thaliana, AtTPC1 (two-pore channel 1). In wild-type cells (expressing only aequorin), Al treatment induced the generation of superoxide, and Ca2+ influx was secondarily induced by superoxide. Higher Al concentrations inhibited the Al-stimulated and superoxide-mediated Ca2+ influx, indicating that Ca2+ channels responsive to reactive oxygen species (ROS) are blocked by high concentration of Al. H2O2-induced Ca2+ influx was also inhibited by Al. Thus, inhibitory action of Al against ROS-induced Ca2+ influx was confirmed. Similarly, known Ca2+ channel blockers such as ions of La and Gd inhibited the H2O2-induced Ca2+ influx. While La also inhibited the hypoosmotically induced Ca2+ influx, Al showed no inhibitory effect against the hypoosmotic Ca2+ influx. The effects of Al and La on Ca2+ influx were also tested in the cell line overexpressing AtTPC1 and the cell line At TPC1-dependently cosuppressing the endogenous TPC1 equivalents. Notably, responsiveness to H2O2 was lost in the cosuppression cell line, thus TPC1 channels are required for ROS-responsive Ca2+ influx. Data also suggested that hypoosmotic shock induces TPC1-independent Ca2+ influx and Al shows no inhibitory action against the TPC1-independent event. In addition, AtTPC1 overexpression resulted in a marked increase in Al-sensitive Ca2+ influx, indicating that TPC1 channels participate in osmotic Ca2+ influx only when overexpressed. We concluded that members of TPC1 channel family are the only ROS-responsive Ca2+ channels and are the possible targets of Al-dependent inhibition. (C) 2004 Elsevier Inc. All rights reserved.