Ca2+ mobilization from intracellular stores represents an important cell signaling process 1 which is regulated, in mammalian cells, by inositol 1,4,5-trisphosphate (InsP3), cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). InsP3 and cADPR release Ca2+ from sarco / endoplasmic reticulum (S/ER) stores through activation of InsP3 and ryanodine receptors (InsP3Rs and RyRs). By contrast, the nature of the intracellular stores targeted by NAADP and molecular identity of the NAADP receptors remain controversial 1,2, although evidence indicates that NAADP mobilizes Ca2+ from lysosome-related acidic compartments 3,4. Here we show that two-pore channels (TPCs) comprise a family of NAADP receptors, with TPC1 and TPC3 being expressed on endosomal and TPC2 on lysosomal membranes. Membranes enriched with TPC2 exhibit high affinity NAADP binding and TPC2 underpins NAADP-induced Ca2+ release from lysosome-related stores that is subsequently amplified by Ca2+-induced Ca2+ release via InsP3Rs. Responses to NAADP were abolished by disrupting the lysosomal proton gradient and by ablating TPC2 expression, but only attenuated by depleting ER Ca2+ stores or blocking InsP3Rs. Thus, TPCs form NAADP receptors that release Ca2+ from acidic organelles, which can trigger additional Ca2+ signals via S/ER. TPCs therefore provide new insights into the regulation and organization of Ca2+ signals in animal cells and will advance our understanding of the physiological role of NAADP.

Egg activation, which is the first step in the initiation of embryo development, involves both completion of meiosis and progression into mitotic cycles. In mammals, the fertilizing sperm delivers the activating signal, which consists of oscillations in free cytosolic Ca 2+ concentration ([Ca 2+ ] i ). Intracytoplasmic sperm injection (ICSI) is a technique that in vitro fertilization clinics use to treat a myriad of male factor infertility cases. Importantly, some patients who repeatedly fail ICSI also fail to induce egg activation and are, therefore, sterile. Here, we have found that sperm from patients who repeatedly failed ICSI were unable to induce [Ca 2+ ] i oscillations in mouse eggs. We have also shown that PLC, zeta 1 (PLCZ1), the sperm protein thought to induce [Ca 2+ ] i oscillations, was localized to the equatorial region of wild-type sperm heads but was undetectable in sperm from patients who had failed ICSI. The absence of PLCZ1 in these patients was further confirmed by Western blot, although genomic sequencing failed to reveal conclusive PLCZ1 mutations. Using mouse eggs, we reproduced the failure of sperm from these patients to induce egg activation and rescued it by injection of mouse Plcz1 mRNA. Together, our results indicate that the inability of human sperm to initiate [Ca 2+ ] i oscillations leads to failure of egg activation and sterility and that abnormal PLCZ1 expression underlies this functional defect.

Our findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.

SummaryIntracellular Ca2+ signals constitute key elements in signal transduction. Of the three major Ca2+ mobilizing messengers described, the most potent, nicotinic acid adenine dinucleotide phosphate (NAADP) is the least well understood in terms of its molecular targets [1]. Recently, we showed that heterologous expression of two-pore channel (TPC) proteins enhances NAADP-induced Ca2+ release, whereas the NAADP response was abolished in pancreatic beta cells from Tpcn2 gene knockout mice [2]. However, whether TPCs constitute native NAADP receptors is unclear. Here we show that immunopurified endogenous TPC complexes possess the hallmark properties ascribed to NAADP receptors, including nanomolar ligand affinity [3–5]. Our study also reveals important functional differences between the three TPC isoforms. Thus, TPC1 and TPC2 both mediate NAADP-induced Ca2+ release, but the subsequent amplification of this trigger Ca2+ by IP3Rs is more tightly coupled for TPC2. In contrast, TPC3 expression suppressed NAADP-induced Ca2+ release. Finally, increased TPC expression has dramatic and contrasting effects on endolysosomal structures and dynamics, implicating a role for NAADP in the regulation of vesicular trafficking. We propose that NAADP regulates endolysosomal Ca2+ storage and release via TPCs and coordinates endoplasmic reticulum Ca2+ release in a role that impacts on Ca2+ signaling in health and disease [6].

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca2+ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca2+ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca2+ release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca2+ that will enable it to act as a Ca2+ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca2+] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca2+ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca2+ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μm but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.

Although ICSI results in average fertilization rates of 70%, complete or virtually complete fertilization failure still occurs in 1-5% of ICSI cycles. While oocyte activation failure can, in some cases, be overcome by artificial oocyte activators such as calcium ionophores, a more physiological oocyte activation agent might release Ca(2+) within the oocyte in a more efficient and controlled manner. As PLCζ is now widely considered to be the physiological agent responsible for activating mammalian oocytes, it represents both a novel diagnostic biomarker of oocyte activation capability and a possible mode of treatment for certain types of male infertility.

At fertilization in mammals, the sperm induces a characteristic series of Ca2+ oscillations in the egg which serve as the essential trigger for egg activation and early development of the embryo. It is not known how the sperm initiates this fundamental process, however, nor has any pathway linking sperm-egg membrane-receptor binding with intracellular Ca2+ release been demonstrated. Microinjection of sperm extracts into mammalian eggs elicits Ca2+ oscillations identical to those occurring at fertilization, which suggests that sperm may introduce a Ca2+ oscillation-inducing factor into the egg on gamete membrane fusion. Here we identify a soluble sperm protein that exhibits Ca2+ oscillation-inducing ('oscillogen') activity in eggs. Sperm oscillogen exists as an oligomer with a subunit of M(r) 33K and a specific intracellular localization at the equatorial segment of the sperm head. Cloning of the 33K oscillogen complementary DNA indicates similarity with a hexose phosphate isomerase found in prokaryotes. This sperm-derived oscillogen, termed oscillin, may represent the physiological trigger for development in mammals.

Significance The formation of new blood vessels (neoangiogenesis) accompanies tissue regeneration and healing, but is also crucial for tumor growth, hence understanding how capillaries are stimulated to grow in response to local cues is essential for the much sought-after aim of controlling this process. We have elucidated a Ca 2+ signaling pathway involving NAADP, TPCs, and lysosomal Ca 2+ release activated in vascular endothelial cells by VEGF, the main angiogenic growth factor, and we show that the angiogenic response can be abolished, in cultured cells and in vivo, by inhibiting components of this signaling cascade. The specificity of this pathway in terms of VEGF receptor subtype, intracellular messengers, target channels and Ca 2+ storage organelles, offers new targets for novel antiangiogenic therapeutic strategies.