Two-pore channel protein 2-mediated calcium release promotes angiopoietin 2 secretion by regulating Rab46-dependent Weibel-Palade body trafficking

Angiopoietin2 (Ang2), a regulator of angiogenesis, is stored with other pro-inflammatory and pro-thrombotic mediators, in endothelial-specific vesicles called Weibel-Palade bodies (WPBs). The WPB secretagogue, histamine, delays Ang2 secretion by activating Rab46-specific trafficking of Ang2-positive WPBs to the microtubule organising centre (MTOC), where they persist until Ca2+ binds to the EF-hand of Rab46, enabling detachment. Here, using Ca2+ imaging and high-resolution light microscopy, we pharmacologically investigated the contribution of endolysosomal two-pore channels proteins (TPC) to the Ca2+ signal necessary for Ang2 secretion. We show an increase in the histamine-evoked clustering of Rab46 (and thus WPBs) at the MTOC in the presence of TPC inhibitors Ned19 and tetrandrine, and a decrease in the presence of a TPC2 agonist, TPC2-A1-N. Histamine-evoked secretion of Ang2 was decreased by pharmacological inhibition of TPC channels but potentiated in the presence of an TPC2 agonist. These data suggest that histamine-mediated Ca2+ release via TPC2 channels is necessary for the Rab46-dependent detachment of Ang2-positive WPBs from the MTOC and thus Ang2 secretion. SummaryCa2+ binding to the EF-hand of Rab46 in endothelial cells has previously been reported but the molecular mechanisms and functional relevance is unclear. Here the authors show that Ca2+ released from TPC2 regulates the detachment of Rab46 from the MTOC and thereby allows secretion of Ang2 from WPBs.