Tpcn2 knockout mice have improved insulin sensitivity and are protected against high-fat diet-induced weight gain.

Type 2 diabetes is a complex disorder affected by multiple genes and the environment. Our laboratory has shown that in response to a glucose challenge, two-pore channel 2 ( Tpcn2) knockout mice exhibit a decreased insulin response but normal glucose clearance, suggesting they have improved insulin sensitivity compared with wild-type mice. We tested the hypothesis that improved insulin sensitivity in Tpcn2 knockout mice would protect against the negative effects of a high fat diet. Male and female Tpcn2 knockout (KO), heterozygous (Het), and wild-type (WT) mice were fed a low-fat (LF) or high-fat (HF) diet for 24 wk. HF diet significantly increases body weight in WT mice relative to those on the LF diet; this HF diet-induced increase in body weight is blunted in the Het and KO mice. Despite the protection against diet-induced weight gain, however, Tpcn2 KO mice are not protected against HF-diet-induced changes in glucose or insulin area under the curve during glucose tolerance tests in female mice, while HF diet has no significant effect on glucose tolerance in the male mice, regardless of genotype. Glucose disappearance during an insulin tolerance test is augmented in male KO mice, consistent with our previous findings suggesting enhanced insulin sensitivity in these mice. Male KO mice exhibit increased fasting plasma total cholesterol and triglyceride concentrations relative to WT mice on the LF diet, but this difference disappears in HF diet-fed mice where there is increased cholesterol and triglycerides across all genotypes. These data demonstrate that knockout of Tpcn2 may increase insulin action in male, but not female, mice. In addition, both male and female KO mice are protected against diet-induced weight gain, but this protection is likely independent from glucose tolerance, insulin sensitivity, and plasma lipid levels.

PLCζ is the physiological trigger of the Ca2+ oscillations that induce embryogenesis in mammals but conception can occur in its absence.

Activation of the egg by the sperm is the first, vital stage of embryogenesis. The sperm protein PLCζ has been proposed as the physiological agent that triggers the Ca2+ oscillations that normally initiate embryogenesis. Consistent with this, recombinant PLCζ induces Ca2+ oscillations in eggs and debilitating mutations in the PLCZ1 gene are associated with infertility in men. However, there has been no evidence that knockout of the gene encoding PLCζ abolishes the ability of sperm to induce Ca2+ oscillations in eggs. Here, we show that sperm derived from Plcz1-/- male mice fail to trigger Ca2+ oscillations in eggs, cause polyspermy and thus demonstrate that PLCζ is the physiological trigger of these Ca2+ oscillations. Remarkably, some eggs fertilized by PLCζ-null sperm can develop, albeit at greatly reduced efficiency, and after a significant time-delay. In addition, Plcz1-/- males are subfertile but not sterile, suggesting that in the absence of PLCζ, spontaneous egg activation can eventually occur via an alternative route. This is the first demonstration that in vivo fertilization without the normal physiological trigger of egg activation can result in offspring. PLCζ-null sperm now make it possible to resolve long-standing questions in fertilization biology, and to test the efficacy and safety of procedures used to treat human infertility.

Synthesis of the Ca2+-mobilizing messengers NAADP and cADPR by intracellular CD38 enzyme in the mouse heart: Role in ß-adrenoceptor signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are Ca2+-mobilizing messengers important for modulating cardiac excitation-contraction coupling and pathophysiology. CD38, which belongs to the ADP-ribosyl cyclase family, catalyzes synthesis of both NAADP and cADPR in vitro However, it remains unclear whether this is the main enzyme for their production under physiological conditions. Here we show that membrane fractions from WT but not CD38-/- mouse hearts supported NAADP and cADPR synthesis. Membrane permeabilization of cardiac myocytes with saponin and/or Triton X-100 increased NAADP synthesis, indicating that intracellular CD38 contributes to NAADP production. The permeabilization also permitted immunostaining of CD38, with a striated pattern in WT myocytes, whereas CD38-/- myocytes and nonpermeabilized WT myocytes showed little or no staining, without striation. A component of ß-adrenoreceptor signaling in the heart involves NAADP and lysosomes. Accordingly, in the presence of isoproterenol, Ca2+ transients and contraction amplitudes were smaller in CD38-/- myocytes than in the WT. In addition, suppressing lysosomal function with bafilomycin A1 reduced the isoproterenol-induced increase in Ca2+ transients in cardiac myocytes from WT but not CD38-/- mice. Whole hearts isolated from CD38-/- mice and exposed to isoproterenol showed reduced arrhythmias. SAN4825, an ADP-ribosyl cyclase inhibitor that reduces cADPR and NAADP synthesis in mouse membrane fractions, was shown to bind to CD38 in docking simulations and reduced the isoproterenol-induced arrhythmias in WT hearts. These observations support generation of NAADP and cADPR by intracellular CD38, which contributes to effects of ß-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm.

Carvedilol inhibits cADPR- and IP3-induced Ca2+ release.

Spontaneous Ca2+ waves, also termed store-overload-induced Ca2+ release (SOICR), in cardiac cells can trigger ventricular arrhythmias especially in failing hearts. SOICR occurs when RyRs are activated by an increase in sarcoplasmic reticulum (SR) luminal Ca2+. Carvedilol is one of the most effective drugs for preventing arrhythmias in patients with heart failure. Furthermore, carvedilol analogues with minimal ß-blocking activity also block SOICR showing that SOICR-inhibiting activity is distinct from that for ß-block. We show here that carvedilol is a potent inhibitor of cADPR-induced Ca2+ release in sea urchin egg homogenate. In addition, the carvedilol analog VK-II-86 with minimal ß-blocking activity also suppresses cADPR-induced Ca2+ release. Carvedilol appeared to be a non-competitive antagonist of cADPR and could also suppress Ca2+ release by caffeine. These results are consistent with cADPR releasing Ca2+ in sea urchin eggs by sensitizing RyRs to Ca2+ involving a luminal Ca2+ activation mechanism. In addition to action on the RyR, we also observed inhibition of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release by carvedilol suggesting a common mechanism between these evolutionarily related and conserved Ca2+ release channels.

Ebolavirus Glycoprotein Directs Fusion through NPC1+ Endolysosomes.

Ebolavirus, a deadly hemorrhagic fever virus, was thought to enter cells through endolysosomes harboring its glycoprotein receptor, Niemann-Pick C1. However, an alternate model was recently proposed in which ebolavirus enters through a later NPC1-negative endosome that contains two-pore Ca(2+) channel 2 (TPC2), a newly identified ebolavirus entry factor. Here, using live cell imaging, we obtained evidence that in contrast to the new model, ebolavirus enters cells through endolysosomes that contain both NPC1 and TPC2.

Two-Pore Channel 2 activity is required for slow muscle cell-generated Ca(2+) signaling during myogenesis in intact zebrafish.

We have recently characterized essential inositol 1,4,5-trisphosphate receptor (IP 3R) and ryanodine receptor (RyR)-mediated Ca(2+) signals generated during the differentiation of slow muscle cells (SMCs) in intact zebrafish embryos. Here, we show that the lysosomal two-pore channel 2 (TPC2) also plays a crucial role in generating, and perhaps triggering, these essential Ca(2+) signals, and thus contributes to the regulation of skeletal muscle myogenesis. We used a transgenic line of zebrafish that expresses the bioluminescent Ca(2+) reporter, aequorin, specifically in skeletal muscle, in conjunction with morpholino (MO)-based and pharmacological inhibition of TPC2, in both intact embryos and isolated SMCs. MO-based knock-down of TPC2 resulted in a dramatic attenuation of the Ca(2+) signals, whereas the introduction of TPCN2-MO and TPCN2 mRNA together partially rescued the Ca(2+) signaling signature. Embryos treated with trans-ned-19 or bafilomycin A1, a specific NAADP receptor inhibitor and vacuolar-type H(+)ATPase inhibitor, respectively, also displayed a similar disruption of SMC Ca(2+) signaling. TPC2 and lysosomes were shown via immunohistochemistry and confocal laser scanning microscopy to be localized in perinuclear and striated cytoplasmic domains of SMCs, coincident with patterns of IP 3R and RyR expression. These data together imply that TPC2-mediated Ca(2+) release from lysosomes acts upstream from RyR- and IP 3R-mediated Ca(2+) release, suggesting that the former might act as a sensitive trigger to initiate the SR-mediated Ca(2+)-induced-Ca(2+)-release essential for SMC myogenesis and function.

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic ß-Adrenoceptor Signaling in the Heart.

Ca(2+)-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca(2+) release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca(2+) responses in cardiac myocytes from Tpcn2(-/-) mice, unlike myocytes from wild-type (WT) mice. Ca(2+)/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca(2+) transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of ß-adrenoreceptor stimulation were reduced in myocytes from Tpcn2(-/-) mice. Increases in amplitude of L-type Ca(2+) currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2(-/-) mice showing no loss of ß-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2(-/-) mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute ß-adrenoreceptor stimulation. Hearts from Tpcn2(-/-) mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼ 25 nm). We propose that Ca(2+)-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in ß-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of ß-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of ß-adrenoceptors to hypertrophy and associated arrhythmias.

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

Pancreatic ß cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca(2+) action potentials due to the activation of voltage-dependent Ca(2+) channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca(2+) release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic ß cells. NAADP-regulated Ca(2+) release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca(2+) from the endolysosomal system, resulting in localized Ca(2+) signals. We show here that NAADP-mediated Ca(2+) release from endolysosomal Ca(2+) stores activates inward membrane currents and depolarizes the ß 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 Ca(2+) release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca(2+) signals, and insulin secretion. Our findings implicate NAADP-evoked Ca(2+) release from acidic Ca(2+) storage organelles in stimulus-secretion coupling in ß cells.

TPC: the NAADP discovery channel?

The Ca2+-mobilizing second messenger, NAADP (nicotinic acid adenine dinucleotide phosphate), has been with us for nearly 20 years and yet we still cannot fully agree on the identity of its target Ca2+-release channel. In spite of some recent robust challenges to the idea that two-pore channels (TPCs) represent the elusive "NAADP receptor", evidence continues to accumulate that TPCs are important for NAADP-mediated responses. This article will briefly outline the background and review more recent work pertaining to the TPC story.

Expression of Ca²âº-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells.

The second messenger NAADP triggers Ca(2+) release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2(-/-)), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca(2+) responses as assessed by single-cell Ca(2+) imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2(-/-) cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca(2+)-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca(2+) release. High-affinity [(32)P]NAADP binding still occurs in Tpcn1/2(-/-) tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca(2+)-permeable channels indispensable for NAADP signalling.

Two-Pore Channels: Lessons from Mutant Mouse Models.

Recent interest in two-pore channels (TPCs) has resulted in a variety of studies dealing with the functional role and mechanism of action of these endo-lysosomal proteins in diverse physiological processes. With the availability of mouse lines harbouring mutant alleles for Tpcnl and/or Tpcn2 genes, several studies have made use of them to validate, consolidate and discover new roles for these channels not only at the cellular level but, importantly, also at the level of the whole organism. The different mutant mouse lines that have been used were derived from distinct genetic manipulation strategies, with the aim of knocking out expression of TPC proteins. However, the expression of different residual TPC sequences predicted to occur in these mutant mouse lines, together with the varied degree to which the effects on Tpcn expression have been studied, makes it important to assess the true knockout status of some of the lines. In this review we summarize these Tpcn mutant mouse lines with regard to their predicted effect on Tpcn expression and the extent to which they have been characterized. Additionally, we discuss how results derived from studies using these Tpcn mutant mouse lines have consolidated previously proposed roles for TPCs, such as mediators of NAADP signalling, endo-lysosomal functions, and pancreatic ß cell physiology. We will also review how they have been instrumental in the assignment of new physiological roles for these cation channels in processes such as membrane electrical excitability, neoangiogenesis, viral infection and brown adipose tissue and heart function, revealing, in some cases, a specific contribution of a particular TPC isoform.

Synthesis of NAADP-AM as a membrane-permeant NAADP analog.

Nicotinic acid adenine dinucleotide phosphate (NAADP), like the other major messengers for Ca²âº mobilization, is passively membrane-impermeant. Instead, a cell-permeant acetoxymethyl ester derivative of NAADP (NAADP-AM) can be synthesized as described here and used to study NAADP-mediated Ca²âº release.

Measurement of luminal pH of acidic stores as a readout for NAADP action.

In addition to mobilizing Ca²âº, NAADP plays a role in modulating the luminal pH (pHL) of acidic stores of the endolysosomal system. The effects of NAADP on pHL have been most extensively studied in the sea urchin egg, both in the intact egg and in egg homogenates. Related observations have also been made in mammalian systems (e.g., guinea pig atrial myocytes and pancreatic acinar cells). Although the connection between Ca²âº mobilization and increase in pHL is not understood, pHL can be a useful parameter to measure when studying NAADP-mediated signaling. This protocol describes the fluorescent measurement of pHL of acidic stores. It relies on the use of acridine orange (AO), a standard dye for pHL. AO selectively accumulates to high concentrations in the lumen of organelles as a function of acidity; at these high concentrations it self-quenches. When pHL increases, some AO is lost from the vesicle. As a result, the lower luminal AO concentration relieves the quenching and fluorescence increases in the lumen.

Synthesis of caged NAADP.

Caged derivatives of Ca²âº-mobilizing messengers, such as nicotinic acid adenine dinucleotide phosphate (NAADP), are particularly useful for establishing the effects of these messengers on Ca²âº signaling. Caged NAADP is no longer commercially available but can be synthesized in house, as described here. In brief, a stable precursor of the caging reagent is made and converted to an unstable reactive reagent immediately before addition to the compound to be caged.

VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2-dependent Ca2+ signaling.

Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca(2+) signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca(2+) mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca(2+) stores, resulting in Ca(2+) release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2(-/-) mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca(2+) release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca(2+) release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2(-/-) mice, but was unaffected in Tpcn1(-/-) animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca(2+) signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.

Preparation and use of sea urchin egg homogenates for studying NAADP-mediated Ca²âº release.

NAADP and other Ca(2+)-mobilizing messengers are membrane impermeant and thus must be added directly to cell-free or broken-cell preparations to effect Ca(2+) release. The sea urchin egg homogenate, where the biological activity of NAADP was first reported, remains the gold standard cell-free system for studying NAADP-mediated Ca(2+) release. Here we describe how to prepare sea urchin egg homogenate and use it to measure NAADP-mediated Ca(2+) release.

Synthesis of [³²P]NAADP for the radioreceptor binding assay.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a major messenger for Ca(2+) mobilization in cells. NAADP-binding proteins are highly selective and have a strong affinity for NAADP. This is the basis of the radioreceptor binding assay, which is used to measure NAADP levels in cells and tissues and to identify cellular stimuli that use NAADP as an intracellular messenger. In the radioreceptor binding assay, radiolabeled NAADP ([(32)P]NAADP) competes with endogenous NAADP present in samples for binding to their receptors. Here, we describe the synthesis of [(32)P]NAADP for use in the radioreceptor binding assay.

TPC1 has two variant isoforms, and their removal has different effects on endo-lysosomal functions compared to loss of TPC2.

Organelle ion homeostasis within the endo-lysosomal system is critical for physiological functions. Two-pore channels (TPCs) are cation channels that reside in endo-lysosomal organelles, and overexpression results in endo-lysosomal trafficking defects. However, the impact of a lack of TPC expression on endo-lysosomal trafficking is unknown. Here, we characterize Tpcn1 expression in two transgenic mouse lines (Tpcn1(XG716) and Tpcn1(T159)) and show expression of a novel evolutionarily conserved Tpcn1B transcript from an alternative promoter, raising important questions regarding the status of Tpcn1 expression in mice recently described to be Tpcn1 knockouts. We show that the transgenic Tpcn1(T159) line lacks expression of both Tpcn1 isoforms in all tissues analyzed. Using mouse embryonic fibroblasts (MEFs) from Tpcn1(-/-) and Tpcn2(-/-) animals, we show that a lack of Tpcn1 or Tpcn2 expression has no significant impact on resting endo-lysosomal pH or morphology. However, differential effects in endo-lysosomal function were observed upon the loss of Tpcn1 or Tpcn2 expression; thus, while Tpcn1(-/-) MEFs have impaired trafficking of cholera toxin from the plasma membrane to the Golgi apparatus, Tpcn2(-/-) MEFs show slower kinetics of ligand-induced platelet-derived growth factor receptor ß (PDGFRß) degradation, which is dependent on trafficking to lysosomes. Our findings indicate that TPC1 and TPC2 have important but distinct roles in the endo-lysosomal pathway.

Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ∼10 nM) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.