NAADP-mediated Ca²⁺ signaling via type 1 ryanodine receptor in T cells revealed by a synthetic NAADP antagonist

The nucleotide NAADP was recently discovered as a second messenger involved in the initiation and propagation of Ca2+ signaling in lymphoma T cells, but its impact on primary T cell function is still unknown. An optimized, synthetic, small molecule inhibitor of NAADP action, termed BZ194, was designed and synthesized. BZ194 neither interfered with Ca²⁺ mobilization by D-myo-inositol 1,4,5-trisphosphate or cyclic ADP-ribose nor with capacitative Ca²⁺ entry. BZ194 specifically and effectively blocked NAADP-stimulated [³H]ryanodine binding to the purified type 1 ryanodine receptor. Further, in intact T cells, Ca²⁺ mobilization evoked by NAADP or by formation of the immunological synapse between primary effector T cells and astrocytes was inhibited by BZ194. Downstream events of Ca²⁺ mobilization, such as nuclear translocation of "nuclear factor of activated T cells" (NFAT), T cell receptor-driven interleukin-2 production, and proliferation in antigen-experienced CD4⁺ effector T cells, were attenuated by the NAADP antagonist. Taken together, specific inhibition of the NAADP signaling pathway constitutes a way to specifically and effectively modulate T-cell activation and has potential in the therapy of autoimmune diseases.