Diazirine-AIOC-NAADP, a Clickable-Photoactive NAADP Analog for Sea Urchin NAADP Binding Proteins.

Calcium ions (Ca(2+)) play a vital role as intracellular messengers, regulating essential cellular processes. Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as a potent second messenger, responsible for releasing Ca(2+) in both mammals and echinoderms. Despite identification of two human NAADP receptor proteins, their counterparts in sea urchins remain elusive. Sea urchin NAADP binding proteins are important due to their unique identities and NAADP binding properties which may illuminate new signaling modalities in other species. Consequently, the development of new photoactive and clickable NAADP analogs with specificity for binding targets in sea urchin egg homogenates is a priority. We designed and synthesized diazirine-AIOC-NAADP, a photoactive and "clickable" NAADP analog, to specifically label and identify sea urchin NAADP receptors. This analog, synthesized using a chemo-enzymatic approach, induced Ca(2+) release from sea urchin egg homogenates at low-micromolar concentrations. The ability of diazirine-AIOC-NAADP to mobilize Ca(2+) in cultured human cells was investigated by microinjection of the probe into U2OS cells. Microinjected NAADP elicited a robust Ca(2+) release, but even 6000-fold higher concentrations of diazirine-AIOC-NAADP were unable to release Ca(2+). Our results indicate that our new probe is specifically recognized at low concentration by sea urchin egg NAADP receptors but not by the NAADP receptors in a human cultured cell line.