NAADP Activates Two-Pore Channels on T Cell Cytolytic Granules to Stimulate Exocytosis and Killing

Abstract

SummaryA cytotoxic T lymphocyte (CTL) kills an infected or tumorigenic cell by Ca2+-dependent exocytosis of cytolytic granules at the immunological synapse formed between the two cells. Although inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum activates the store-operated Ca2+-influx pathway that is necessary for exocytosis, it is not a sufficient stimulus [1–4]. Here we identify the Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and its recently identified molecular target, two-pore channels (TPCs) [5–7], as being important for T cell receptor signaling in CTLs. We demonstrate that cytolytic granules are not only reservoirs of cytolytic proteins but are also the acidic Ca2+ stores mobilized by NAADP via TPC channels on the granules themselves, so that TPCs migrate to the immunological synapse upon CTL activation. Moreover, NAADP activates TPCs to drive exocytosis in a way that is not mimicked by global Ca2+ signals induced by IP3 or ionomycin, suggesting that critical, local Ca2+ nanodomains around TPCs stimulate granule exocytosis. Hence, by virtue of the NAADP/TPC pathway, cytolytic granules generate Ca2+ signals that lead to their own exocytosis and to cell killing. This study highlights a selective role for NAADP in stimulating exocytosis crucial for immune cell function and may impact on stimulus-secretion coupling in wider cellular contexts.

Altered distribution and function of natural killer cells in murine and human Niemann-Pick disease type C1

“…33 In agreement with this, cytotoxic granules in cytotoxic T cells (CTLs) were recently shown to be a source of calcium for their exocytosis. 34 When NK cells contact their target, they form an immunologic synapse in an analogous manner to CTLs where the microtubule organizing center and Golgi apparatus become polarized. 35 Calcium influx from an extracellular source has been shown to be essential for granule exocytosis from CTLs 36 via store-operated calcium entry (SOCE), which requires the calcium-release-activated channel, ORAI1, 37 and the intracellularcalcium-depletion sensor, STIM1.…”

Two-pore channel 1 interacts with citron kinase, regulating completion of cytokinesis

“…10 TPCs have been shown to regulate differentiation, 11 smooth muscle contraction 12 and endothelial cell activation, 13 consistent with previous studies implicating nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca 2C release in these events, [14][15][16] and supported by several overexpression, knockdown and knockout models. 3,4,6,17 Regulation and affinity of TPC ion channels is a contentious issue. Literature suggest proton-permeable ion channels activated by NAADP or Ca 2C ; 18 although photoaffinity labeling studies suggest that NAADP does not directly bind TPCs.…”

NAADP-sensitive Сa(2+) stores in permeabilized rat hepatocytes

“…Recent studies demonstrate that two-pore channels (TPC) are located to endosomes and lysosomes and form Ca 2+ release channels that respond to the activation by NAADP mobilizing Ca 2+ from acidic stores [5,24]. Ned-19 itself inhibits both NAADPmediated Ca 2+ release and NAADP binding [25].…”

“…The first demonstration that NAADP levels increase in response to an extracellular stimulus arose from studying sea urchin fertilization (NAADP changed in both the eggs and sperm upon contact) [2]. Subsequently it was shown on different mammalian preparations that NAADP is a potent Ca 2+ -releasing second messenger [3][4][5].…”