Essential requirement for JPT2 in NAADP-evoked Ca²⁺ signaling

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a “clickable” NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry

Chapter 19 - Probing Ca2+ release mechanisms using sea urchin egg homogenates

Sea urchin eggs have been extensively used to study Ca2+ release through intracellular Ca2+-permeable channels. Their amenability to homogenization yields a robust, cell-free preparation that was central to establishing the Ca2+ mobilizing actions of cyclic ADP-ribose and NAADP. Egg homogenates have continued to provide insight into the basic properties and pharmacology of intracellular Ca2+ release channels. In this chapter, we describe methods for the preparation of egg homogenates and monitoring Ca2+ release using fluorimetry and radiotracer flux