Cyclic Adenosine Diphosphate Ribose Activates Ryanodine Receptors, whereas NAADP Activates Two-pore Domain Channels

The mechanism by which cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize intracellular Ca2+ stores remains controversial. It is open to question whether cADPR regulates ryanodine receptors (RyRs) directly, as originally proposed, or indirectly by promoting Ca2+ uptake into the sarco/endoplasmic reticulum by sarco/endoplasmic reticulum Ca2+-ATPases. Conversely, although we have proposed that NAADP mobilizes endolysosomal Ca2+ stores by activating two-pore domain channels (TPCs), others suggest that NAADP directly activates RyRs. We therefore assessed Ca2+ signals evoked by intracellular dialysis from a patch pipette of cADPR and NAADP into HEK293 cells that stably overexpress either TPC1, TPC2, RyR1, or RyR3. No change in intracellular Ca2+ concentration was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in cells that stably overexpress TPC1 and TPC2, respectively. By contrast, a marked Ca2+ transient was triggered by cADPR in HEK293 cells that stably expressed RyR1 and RyR3. The Ca2+ transient was abolished following depletion of endoplasmic reticulum stores by thapsigargin and block of RyRs by dantrolene but not following depletion of acidic Ca2+ stores by bafilomycin. By contrast, NAADP failed to evoke a Ca2+ transient in HEK293 cells that expressed RyR1 or RyR3, but it induced robust Ca2+ transients in cells that stably overexpressed TPC1 or TPC2 and in a manner that was blocked following depletion of acidic stores by bafilomycin. We conclude that cADPR triggers Ca2+ release by activating RyRs but not TPCs, whereas NAADP activates TPCs but not RyRs.