Three signalling molecules cause increases in intracellular Ca2+ levels by triggering release of Ca2+ from intracellular stores due to their action on specific Ca2+-permeable receptors: inositol-1,4,5-trisphosphate binds to and opens the sarcoplasmic reticulum InsP3 receptor; cyclic ADP ribose activates the endoplasmic reticulum ryanodine receptor; but the molecular identity and location of the nicotinic acid adenine dinucleotide phosphate (NAADP) receptor is unknown. Here Calcraft et al. show that the lysosomal two-pore channel, TPC2, is the molecular target of NAADP. Ca2+ mobilization from intracellular stores represents an important cell signalling process that is regulated, in mammalian cells, by inositol-1,4,5-trisphosphate (InsP3), cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). While the nature of the receptors for InsP3 and cyclic ADP ribose are known, here the lysosomal two-pore channel, TPC2, is shown to be the molecular target of NAADP. Ca2+ mobilization from intracellular stores represents an important cell signalling process1 that is regulated, in mammalian cells, by inositol-1,4,5-trisphosphate (InsP3), cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). InsP3 and cyclic ADP ribose cause the release of Ca2+ from sarcoplasmic/endoplasmic reticulum stores by the activation of InsP3 and ryanodine receptors (InsP3Rs and RyRs). In contrast, the nature of the intracellular stores targeted by NAADP and the molecular identity of the NAADP receptors remain controversial1,2, although evidence indicates that NAADP mobilizes Ca2+ from lysosome-related acidic compartments3,4. Here we show that two-pore channels (TPCs) comprise a family of NAADP receptors, with human TPC1 (also known as TPCN1) and chicken TPC3 (TPCN3) being expressed on endosomal membranes, and human TPC2 (TPCN2) on lysosomal membranes when expressed in HEK293 cells. Membranes enriched with TPC2 show high affinity NAADP binding, and TPC2 underpins NAADP-induced Ca2+ release from lysosome-related stores that is subsequently amplified by Ca2+-induced Ca2+ release by InsP3Rs. Responses to NAADP were abolished by disrupting the lysosomal proton gradient and by ablating TPC2 expression, but were only attenuated by depleting endoplasmic reticulum Ca2+ stores or by blocking InsP3Rs. Thus, TPCs form NAADP receptors that release Ca2+ from acidic organelles, which can trigger further Ca2+ signals via sarcoplasmic/endoplasmic reticulum. 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.

Research into the biological role of the Ca2+-releasing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate) has been hampered by a lack of chemical probes. To find new chemical probes for exploring NAADP signaling, we turned to virtual screening, which can evaluate millions of molecules rapidly and inexpensively. We used NAADP as the query ligand to screen the chemical library ZINC for compounds with similar three-dimensional shape and electrostatic properties. We tested the top-ranking hits in a sea urchin egg bioassay and found that one hit, Ned-19, blocks NAADP signaling at nanomolar concentrations. In intact cells, Ned-19 blocked NAADP signaling and fluorescently labeled NAADP receptors. Moreover, we show the utility of Ned-19 as a chemical probe by using it to demonstrate that NAADP is a key causal link between glucose sensing and Ca2+ increases in mouse pancreatic beta cells.

Intracellular Ca(2+) signals constitute key elements in signal transduction. Of the three major Ca(2+) 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 Ca(2+) 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 Ca(2+) release, but the subsequent amplification of this trigger Ca(2+) by IP(3)Rs is more tightly coupled for TPC2. In contrast, TPC3 expression suppressed NAADP-induced Ca(2+) 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 Ca(2+) storage and release via TPCs and coordinates endoplasmic reticulum Ca(2+) release in a role that impacts on Ca(2+) signaling in health and disease [6].

Cognitive dysfunctions such as mild cognitive impairment (MCI), Alzheimer’s disease (AD), and other forms of dementia are recognized as common comorbidities of type 2 diabetes mellitus (T2DM). Currently, there are no disease-modifying therapies or definitive clinical diagnostic and prognostic tools for dementia, and the mechanisms underpinning the link between T2DM and cognitive dysfunction remain equivocal. Some of the suggested pathophysiological mechanisms underlying cognitive decline in diabetes patients include hyperglycemia, insulin resistance and altered insulin signaling, neuroinflammation, cerebral microvascular injury, and buildup of cerebral amyloid and tau proteins. Given the skyrocketing global rates of diabetes and neurodegenerative disorders, there is an urgent need to discover novel biomarkers relevant to the co-morbidity of both conditions to guide future diagnostic approaches. This review aims to provide a comprehensive background of the potential risk factors, the identified biomarkers of diabetes-related cognitive decrements, and the underlying processes of diabetes-associated cognitive dysfunction. Aging, poor glycemic control, hypoglycemia and hyperglycemic episodes, depression, and vascular complications are associated with increased risk of dementia. Conclusive research studies that have attempted to find specific biomarkers are limited. However, the most frequent considerations in such investigations are related to C reactive protein, tau protein, brain-derived neurotrophic factor, advanced glycation end products, glycosylated hemoglobin, and adipokines.

The role of intracellular Ca²⁺ pools in oscillations of the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) triggered by Ca²⁺ influx was investigated in mouse pancreatic B-cells. [Ca²⁺]_c oscillations occurring spontaneously during glucose stimulation or repetitively induced by pulses of high K⁺ (in the presence of diazoxide) were characterized by a descending phase in two components. A rapid decrease in [Ca²⁺]_c coincided with closure of voltage-dependent Ca²⁺ channels and was followed by a slower phase independent of Ca²⁺ influx. Blocking the SERCA pump with thapsigargin or cyclopiazonic acid accelerated the rising phase of [Ca²⁺]_coscillations and increased their amplitude, which suggests that the endoplasmic reticulum (ER) rapidly takes up Ca²⁺. It also suppressed the slow [Ca²⁺]_c recovery phase, which indicates that this phase corresponds to the slow release of Ca²⁺ that was taken up by the ER during the upstroke of the [Ca²⁺]_c transient. Glucose promoted the buffering capacity of the ER and amplified the slow [Ca²⁺]_crecovery phase. The slow phase induced by high K⁺ pulses was not affected by modulators of Ca²⁺- or inositol 1,4,5-trisphosphate-induced Ca²⁺ release, did not involve a depolarization-induced Ca²⁺ release, and was also observed at the end of a rapid rise in [Ca²⁺]_c triggered from caged Ca²⁺. It is attributed to passive leakage of Ca²⁺ from the ER. We suggest that the ER displays oscillations of the Ca²⁺ concentration ([Ca²⁺]_ER) concomitant and parallel to [Ca²⁺]_c. The observation that thapsigargin depolarizes the membrane of B-cells supports the proposal that the degree of Ca²⁺ filling of the ER modulates the membrane potential. Therefore, [Ca²⁺]_ER oscillations occurring during glucose stimulation are likely to influence the bursting behavior of B-cells and eventually [Ca²⁺]_c oscillations.

Recent studies into the mechanisms of action of the Ca2+-mobilizing messenger NAADP (nicotinic acid–adenine dinucleotide phosphate) have demonstrated that a novel family of intracellular Ca2+-release channels termed TPCs (two-pore channels) are components of the NAADP receptor. TPCs appear to be exclusively localized to the endolysosomal system. These findings confirm previous pharmacological and biochemical studies suggesting that NAADP targets acidic Ca2+ stores rather than the endoplasmic reticulum, the major site of action of the other two principal Ca2+-mobilizing messengers, InsP3 and cADPR (cADP-ribose). Studies of the messenger roles of NAADP and the function of TPCs highlight the novel role of lysosomes and other organelles of the endocytic pathway as messenger-regulated Ca2+ stores which also affects the regulation of the endolysosomal system.