Both RyRs and TPCs are required for NAADP-induced intracellular Ca<sup>2+</sup> release
Gerasimenko JV., Charlesworth RM., Sherwood MW., Ferdek PE., Mikoshiba K., Parrington J., Petersen OH., Gerasimenko OV.
© 2015 The Authors. Intracellular Ca 2+ release is mostly mediated by inositol trisphosphate, but intracellular cyclic-ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are important messengers in many systems. Whereas cADPR generally activates type 2 ryanodine receptors (RyR2s), the NAADP-activated Ca 2+ release mechanism is less clear. Using knockouts and antibodies against RyRs and Two-Pore Channels (TPCs), we have compared their relative importance for NAADP-induced Ca 2+ release from two-photon permeabilized pancreatic acinar cells. In these cells, cholecystokinin-elicited Ca 2+ release is mediated by NAADP. TPC2-KO reduced NAADP-induced Ca 2+ release by 64%, but the combination of TPC2-KO and an antibody against TPC1, significantly reduced Ca 2+ release by 86% (64% vs. 86%, p < 0.0002). In RyR3-KO, NAADP-evoked Ca 2+ release reduced by ~50% but, when combined with antibodies against RyR1, responses were 90% inhibited. Antibodies against RyR2 had practically no effect on NAADP-evoked Ca 2+ release, but reduced release in response to cADPR by 55%. Antibodies to RyR1 inhibited NAADP-induced Ca 2+ liberation by 81%, but only reduced cADPR responses by 30%. We conclude that full NAADP-mediated Ca 2+ release requires both TPCs and RyRs. The sequence of relative importance for NAADP-elicited Ca 2+ release from the all stores is RyR1 > TPC2 > RyR3 > TPC1 > > RyR2. However, when assessing NAADP-induced Ca 2+ release solely from the acidic stores (granules/endosomes/lysosomes), antibodies against TPC2 and TPC1 virtually abolished the Ca 2+ liberation as did antibodies against RyR1 and RyR3. Our results indicate that the primary, but very small, NAADP-elicited Ca 2+ release via TPCs from endosomes/lysosomes triggers the detectable Ca 2+ -induced Ca 2+ release via RyR1 and RyR3 occurring from the granules and the ER.