Molecular mechanism of 2-APB-induced Ca2+ influx in external acidification in PC12

2-Aminoethoxydiphenyl borate (2-APB) is used as a pharmacological tool because it antagonizes inositol 1,4,5-trisphosphate receptors and store-operated Ca2+ (SOC) channels, and activates wsome TRP channels. Recently, we reported that 2-APB enhanced the increase in cytotoxic [Ca2+](i), resulting in cell death under external acidic conditions in rat pheochromocytoma cell line PC12. However, the molecular mechanism and functional role of the 2-APB-induced Ca2+ influx in PC12 have not been clarified. In this study, to identify the possible target for the action of 2-APB we examined the pharmacological and molecular properties of [Ca2+]; and secretory responses to 2-APB under extracellular low pH conditions. 2-APB dose-dependently induced a [Ca2+](i) increase and dopamine release, which were greatly enhanced by the external acidification (pH 6.5). [Ca2+](i) and secretory responses to 2-APB at pH 6.5 were inhibited by the removal of extracellular Ca2+ and SOC channel blockers such as SK&F96365, La3+ and Gd3+. PC12 expressed all SOC channel molecules, Orai 1, Oral 2 and Orai 3. When we used an siRNA system, downregulation of Oral 3, but not Orai 1 and Oral 2, attenuated both [Ca2+](i) and secretory responses to 2-APB. These results suggest that 2-APB evokes external acid-dependent increases of [Ca2+] and dopamine release in PC12 through the activation of Oral 3. The present results indicate that 2-APB may be a useful pharmacological tool for Orai channel-related signaling. (c) 2014 Elsevier Inc. All rights reserved.