Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [ ³²P-5-azido]nicotinic acid adenine dinucleotide phosphate ([ ³²P-5N₃]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [³²P-5N₃]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N ₃-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [³²P-5N ₃]NAADP binding was saturable and displayed high affinity (K _d ∼10 nM) in both binding and photolabeling experiments. [ ³²P-5N₃]NAADP photolabeling was irreversible in a high K⁺ buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [³²P-5N₃]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.