Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) is a messenger molecule that activates calcium release from intracellular acidic stores, such as lysosomes. Calcium release mediated by NAADP can result in both local and global signaling cascades within a cell, triggering other calcium signaling pathways. Calcium signaling is important in cellular functions such as fertilization, muscle contraction, and gene expression. Two-pore channels are a family of ion channels that have been identified in the NAADP pathway, however, the receptor protein for NAADP has not been identified. The purpose of this study is to synthesize biotinylated NAADP’s for purification of the NAADP receptor by affinity chromatography. Four biotinylated NAADP’s were successfully synthesized and characterized for their ability to mimic NAADP action in terms of binding and calcium release in the sea urchin system. All four biotinylated NAADP’s were able to successfully compete with 32P-NAADP in a competition binding assay with IC50 values 10 to 1000 fold higher than NAADP itself. These compounds also were able to release calcium with EC50 values 125 to 1200 fold higher than NAADP. These probes desensitized calcium release induced by 1µM NAADP with IC50 values similar to those observed in the competition binding assays. Immobilization of the biotinylated NAADPs on streptavidin-agarose for affinity purification of NAADP binding proteins resulted in the retention of several proteins compared to control columns. This approach might prove useful in the identification of the NAADP receptor.

Synthesis and use of biotinylated NAADP’s for the affinity purification of the NAADP receptor

251st National Meeting (2016)

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