Katja Rietdorf

NAADP mobilizes calcium from acidic organelles through two-pore channels

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). 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 controversial, although evidence indicates that NAADP mobilizes Ca2+ from lysosome-related acidic compartments. 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.

Phosphorylation of inositol 1,4,5-trisphosphate receptors by protein kinase B/Akt inhibits Ca2+ release and apoptosis

Imbalance of signals that control cell survival and death results in pathologies, including cancer and neurodegeneration. Two pathways that are integral to setting the balance between cell survival and cell death are controlled by lipid-activated protein kinase B (PKB)/Akt and Ca2+. PKB elicits its effects through the phosphorylation and inactivation of proapoptotic factors. Ca2+ stimulates many prodeath pathways, among which is mitochondrial permeability transition. We identified Ca2+ release through inositol 1,4,5-trisphosphate receptor (InsP3R) intracellular channels as a prosurvival target of PKB. We demonstrated that in response to survival signals, PKB interacts with and phosphorylates InsP3Rs, significantly reducing their Ca2+ release activity. Moreover, phosphorylation of InsP3Rs by PKB reduced cellular sensitivity to apoptotic stimuli through a mechanism that involved diminished Ca2+ flux from the endoplasmic reticulum to the mitochondria. In glioblastoma cells that exhibit hyperactive PKB, the same prosurvival effect of PKB on InsP3R was found to be responsible for the insensitivity of these cells to apoptotic stimuli. We propose that PKB-mediated abolition of InsP3-induced Ca2+ release may afford tumor cells a survival advantage.

Purified TPC Isoforms Form NAADP Receptors with Distinct Roles for Ca2+ Signaling and Endolysosomal Trafficking

Summary Intracellular 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].

TPC2 Is a Novel NAADP-sensitive Ca2+ Release Channel, Operating as a Dual Sensor of Luminal pH and Ca2+

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.

NAADP as an intracellular messenger regulating lysosomal calcium-release channels.

Recent studies into the mechanisms of action of the Ca(2+)-mobilizing messenger NAADP (nicotinic acid-adenine dinucleotide phosphate) have demonstrated that a novel family of intracellular Ca(2+)-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 Ca(2+) stores rather than the endoplasmic reticulum, the major site of action of the other two principal Ca(2+)-mobilizing messengers, InsP(3) 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 Ca(2+) stores which also affects the regulation of the endolysosomal system.

Calcium-sensing receptor antagonists abrogate airway hyperresponsiveness and inflammation in allergic asthma

Calcilytics reduce airway hyperresponsiveness and inflammation and may represent effective asthma therapeutics. Calcilytics may help asthmatics breathe easier Calcium may help to build strong bones. However, Yarova et al. now show that extracellular calcium may contribute to inflammation and airway hyperresponsiveness in allergic asthma. They show that elevated extracellular calcium can activate airway smooth muscle cells through the calcium-sensing receptor (CaSR). Asthmatic patients express higher levels of CaSR in their airways than do healthy individuals, as does a mouse model of allergic asthma. Indeed, extracellular calcium and other asthma-associated activators of CaSR increased airway hyperreactivity. What’s more, calcilytics—CaSR antagonists—can prevent these effects both in vitro and in vivo, supporting clinical testing of these drugs for asthmatics. Airway hyperresponsiveness and inflammation are fundamental hallmarks of allergic asthma that are accompanied by increases in certain polycations, such as eosinophil cationic protein. Levels of these cations in body fluids correlate with asthma severity. We show that polycations and elevated extracellular calcium activate the human recombinant and native calcium-sensing receptor (CaSR), leading to intracellular calcium mobilization, cyclic adenosine monophosphate breakdown, and p38 mitogen-activated protein kinase phosphorylation in airway smooth muscle (ASM) cells. These effects can be prevented by CaSR antagonists, termed calcilytics. Moreover, asthmatic patients and allergen-sensitized mice expressed more CaSR in ASMs than did their healthy counterparts. Indeed, polycations induced hyperreactivity in mouse bronchi, and this effect was prevented by calcilytics and absent in mice with CaSR ablation from ASM. Calcilytics also reduced airway hyperresponsiveness and inflammation in allergen-sensitized mice in vivo. These data show that a functional CaSR is up-regulated in asthmatic ASM and targeted by locally produced polycations to induce hyperresponsiveness and inflammation. Thus, calcilytics may represent effective asthma therapeutics.

Reconstituted Human TPC1 Is a Proton-Permeable Ion Channel and Is Activated by NAADP or Ca2+

Stimuli that increase calcium or NAADP may promote proton release from the endosomes and lysosomes by activating TPC1. Showing a Preference for Protons Protons (H+) and calcium (Ca2+) produce a variety of different effects in cells. One way to determine which channels allow each of these ions to pass is to isolate the proteins and incorporate them into artificial bilayers. With this approach, Pitt et al. found that H+ was the preferred ion that passed through the human two-pore channel 1 (TPC1), which in cells is located in acidic membrane-bound compartments called endosomes and lysosomes. They also identified intracellular signaling messengers that stimulated TPC1 and signals that changed the relative ability of different positively charged ions to flow through the channel. The exact function of the released H+ remains an open question. NAADP potently triggers Ca2+ release from acidic lysosomal and endolysosomal Ca2+ stores. Human two-pore channels (TPC1 and TPC2), which are located on these stores, are involved in this process, but there is controversy over whether TPC1 and TPC2 constitute the Ca2+ release channels. We therefore examined the single-channel properties of human TPC1 after reconstitution into bilayers of controlled composition. We found that TPC1 was permeable not only to Ca2+ but also to monovalent cations and that permeability to protons was the highest (relative permeability sequence: H+ >> K+ > Na+ ≥ Ca2+). NAADP or Ca2+ activated TPC1, and the presence of one of these ligands was required for channel activation. The endolysosome-located lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] had no effect on TPC1 open probability but significantly increased the relative permeability of Na+ to Ca2+ and of H+ to Ca2+. Furthermore, our data showed that, although both TPC1 and TPC2 are stimulated by NAADP, these channels differ in ion selectivity and modulation by Ca2+ and pH. We propose that NAADP triggers H+ release from lysosomes and endolysomes through activation of TPC1, but that the Ca2+-releasing ability of TPC1 will depend on the ionic composition of the acidic stores and may be influenced by other regulators that affect TPC1 ion permeation.

Loss of activity mutations in phospholipase C zeta (PLCζ) abolishes calcium oscillatory ability of human recombinant protein in mouse oocytes

BACKGROUND Mammalian oocyte activation occurs via a series of intracellular calcium (Ca(2+)) oscillations thought to be induced by a sperm-specific phospholipase C zeta (PLCζ). There is now strong evidence to indicate that certain types of human male infertility are caused by failure of the sperm to activate the oocyte in an appropriate manner. Molecular analysis of the PLCζ gene of a male patient with oocyte activation deficiency has previously identified a point mutation causing a histidine to proline substitution at PLCζ residue 398 (PLCζ(H398P)), leading to abnormal Ca(2+) release profiles and reduced oocyte activation efficiency. METHODS AND RESULTS In the present study, we used HEK293T cells to produce recombinant human wild-type PLCζ (PLCζ(WT)) protein which, upon microinjection into mouse oocytes, induced Ca(2+) oscillations characteristic of oocyte activation. Injection of recombinant PLCζ(H398P) was unable to elicit Ca(2+) oscillations in mouse oocytes. Loss of activity mutations, such as PLCζ(H398P) and an artificially induced frameshift mutation (PLCζ(ΔYC2)) did not affect Ca(2+) release when over-expressed in HEK293T cells, whereas PLCζ(WT) inhibited adenosine triphosphate-activated Ca(2+) release. Confocal imaging of fluorescently tagged PLCζ isoforms in HEK293T cells suggested a cytoplasmic pattern of localization, while quantitative analysis of fluorescence levels showed that PLCζ(WT) > PLCζ(H398P) > PLCζ(ΔYC2), indicating that loss of activity mutations may lead to protein instability. This was further indicated by the low proportion of sperm and the lower levels of total PLCζ immunofluorescence from the patient exhibiting PLCζ(H398P) compared with fertile controls. CONCLUSIONS We demonstrate, for the first time, the production of active recombinant human PLCζ protein which retained the ability to elicit characteristic Ca(2+) oscillations in mouse oocytes, an ability which was eliminated by an infertility-linked mutation. These findings advance our understanding of PLCζ, and provide a critical step forward in obtaining purified PLCζ protein as a potential therapeutic agent for oocyte activation deficiency.

Two-pore channels form homo- and heterodimers.

Background: Two-pore channels (TPCs) are part of the NAADP-receptor complex, but how and whether they dimerize are unclear. Results: Human TPCs form homo- and heteromeric complexes. Conclusion: Multimerization can regulate function and localization of TPCs. Significance: Multimerization of TPCs is likely to affect fusion events in the endolysosomal system, disturbances of which can lead to the development of lysosomal storage diseases. Two-pore channels (TPCs) have been recently identified as NAADP-regulated Ca2+ release channels, which are localized on the endolysosomal system. TPCs have a 12-transmembrane domain (TMD) structure and are evolutionary intermediates between the 24-TMD α-subunits of Na+ or Ca2+ channels and the transient receptor potential channel superfamily, which have six TMDs in a single subunit and form tetramers with 24 TMDs as active channels. Based on this relationship, it is predicted that TPCs dimerize to form functional channels, but the dimerization of human TPCs has so far not been studied. Using co-immunoprecipitation studies and a mass spectroscopic analysis of the immunocomplex, we show the presence of homo- and heteromeric complexes for human TPC1 and TPC2. Despite their largely distinct localization, we identified a discrete number of endosomes that coexpressed TPC1 and TPC2. Homo- and heteromerization were confirmed by a FRET study, showing that both proteins interacted in a rotational (N- to C-terminal/head-to-tail) symmetry. This is the first report describing the presence of homomultimeric TPC1 channels and the first study showing that TPCs are capable of forming heteromers.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells

Background: TPCs are regulated by NAADP and other factors. Results: NAADP-induced Ca2+ release from acidic stores evokes depolarizing currents in pancreatic β cells. Inhibition of NAADP signaling or TPC knock out attenuates Ca2+ signaling and insulin secretion. Conclusion: NAADP-evoked Ca2+ release enhances β cell excitability and insulin secretion in response to glucose or sulfonylureas. Significance: NAADP signaling pathways offer novel therapeutic targets for diabetes treatment. Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca2+ action potentials due to the activation of voltage-dependent Ca2+ channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca2+ release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca2+ from the endolysosomal system, resulting in localized Ca2+ signals. We show here that NAADP-mediated Ca2+ release from endolysosomal Ca2+ stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca2+ release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca2+ signals, and insulin secretion. Our findings implicate NAADP-evoked Ca2+ release from acidic Ca2+ storage organelles in stimulus-secretion coupling in β cells.