Figure 4.

Nicotinic acid adenine nucleotide diphosphate (NAADP)-mediated Ca²⁺ release in HEK293 cells expressing each of the three sea urchin two-pore channel (TPC) isoforms. Representative Ca²⁺ traces of cells dialyzed with NAADP (100 nm) and fura-2 via patch pipette in whole-cell configuration, in absence or presence of bafilomycin A1 (1 µm) or the IP₃R antagonist, heparin (200 µg/mL). Arrows indicate break-in. In wild-type cells, only a small endogenous response to NAADP was seen. In SpTPC1 and SpTPC2 cells, NAADP-evoked biphasic responses, the first component was from acidic stores (bafilomycin-sensitive), whereas the second phase, which requires the first to trigger it, is caused by the recruitment of IP₃Rs (heparin-sensitive). TPC3 expression suppresses the endogenous response. Models for NAADP-triggered Ca²⁺ responses, based on interaction between different organelles (circle, lysosome, and network, endoplasmic reticulum [ER]) are also shown above each series of traces.