MASTER-NAADP: a membrane permeable precursor of the Ca²⁺ mobilizing second messenger NAADP

Upon stimulation of membrane receptors, nicotinic acid adenine dinucleotide phosphate (NAADP) is formed as second messenger within seconds and evokes Ca²⁺ signaling in many different cell types. Here, to directly stimulate NAADP signaling, MASTER-NAADP, a Membrane permeAble, STabilized, bio-rEversibly pRotected precursor of NAADP is synthesized and release of its active NAADP mimetic, benzoic acid C-nucleoside, 2'-phospho-3'F-adenosine-diphosphate, by esterase digestion is confirmed. In the presence of NAADP receptor HN1L/JPT2 (hematological and neurological expressed 1-like protein, HN1L, also known as Jupiter microtubule-associated homolog 2, JPT2), this active NAADP mimetic releases Ca²⁺ and increases the open probability of type 1 ryanodine receptor. When added to intact cells, MASTER-NAADP initially evokes single local Ca²⁺ signals of low amplitude. Subsequently, also global Ca²⁺ signaling is observed in T cells, natural killer cells, and Neuro2A cells. In contrast, control compound MASTER-NADP does not stimulate Ca²⁺ signaling. Likewise, in cells devoid of HN1L/JPT2, MASTER-NAADP does not affect Ca²⁺ signaling, confirming that the product released from MASTER-NAADP is a bona fide NAADP mimetic.