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Essential requirement for two-pore channel 1 in NAADP-mediated calcium signaling.

Brailoiu E, Churamani D, Cai X, Schrlau MG, Brailoiu GC, Gao X, Hooper R, Boulware MJ, Dun NJ, Marchant JS, Patel S - J. Cell Biol. (2009)

Bottom Line: However, the molecular identity of the target protein is unclear.In this study, we show that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP.Thus, TPC1 is critical for NAADP action and is likely the long sought after target channel for NAADP.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140, USA.

ABSTRACT
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a widespread and potent calcium-mobilizing messenger that is highly unusual in activating calcium channels located on acidic stores. However, the molecular identity of the target protein is unclear. In this study, we show that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP. Thus, TPC1 is critical for NAADP action and is likely the long sought after target channel for NAADP.

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An expanded family of TPC genes in animals. (A) Maximum likelihood tree constructed using the conserved regions of TPC sequences from the representative organisms listed in Table S1. Shading highlights TPC isoforms in plants, protists, and animals, with the latter subdivided into three distinct groupings. Bootstrap values >50 are shown at the branches. (B) End point RT-PCR analysis showing expression of transcripts for TPC isoforms in the indicated cell type/embryo (prism stage). The expected sizes of the amplicons were 152 (SpuTPC1), 93 (SpuTPC2), 107 (SpuTPC3), 473 (RnoTPC1), 575 (RnoTPC2), 250 (HsaTPC1), and 250 bp (HsaTPC2). Black lines indicate that intervening lanes have been spliced out. (C) Quantitative RT-PCR of TPC isoforms. Data were normalized to the expression level of the indicated housekeeping gene. Error bars indicate SEM.
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fig1: An expanded family of TPC genes in animals. (A) Maximum likelihood tree constructed using the conserved regions of TPC sequences from the representative organisms listed in Table S1. Shading highlights TPC isoforms in plants, protists, and animals, with the latter subdivided into three distinct groupings. Bootstrap values >50 are shown at the branches. (B) End point RT-PCR analysis showing expression of transcripts for TPC isoforms in the indicated cell type/embryo (prism stage). The expected sizes of the amplicons were 152 (SpuTPC1), 93 (SpuTPC2), 107 (SpuTPC3), 473 (RnoTPC1), 575 (RnoTPC2), 250 (HsaTPC1), and 250 bp (HsaTPC2). Black lines indicate that intervening lanes have been spliced out. (C) Quantitative RT-PCR of TPC isoforms. Data were normalized to the expression level of the indicated housekeeping gene. Error bars indicate SEM.

Mentions: TPCs are almost completely uncharacterized in animals. Comparative genomic analysis reveals that the TPC gene is widespread in the animal kingdom and has undergone multiplication with most species possessing either two (human and rat) or three (sea urchin) TPC genes (Fig. 1 A and Table S1). These genes are predicted to encode for proteins that display ∼35% sequence similarity (Table S2). Duplication of the TPC gene likely began early during animal evolution given the presence of multiple TPC genes in the sea anemone (Nematostella vectensis; Fig. 1 A), a member of the Cnidarian phylum considered the oldest of the eumetazoan lineage. Indeed, multiple TPC genes are also evident in the choanoflagellate Monosiga brevicollis, which is a close unicellular relative of animals (Fig. 1 A). Intriguingly, a single copy of the TPC gene is present in the protists Thalassiosira pseudonana (marine phytoplankton) and Phytophthora ramorum (sudden oak death pathogen; Fig. 1 A), suggesting that TPCs are ancient proteins that likely originated in the common ancestor of animals and plants. During the course of metazoan evolution, we note lineage-specific gene loss. This is most striking in mammals, where three genes are present in some species such as dog, whereas only two are present in other species such as human and rat (Fig. 1 A and Table S1). Semiquantitative RT-PCR analysis demonstrated the presence of transcripts for all three TPC isoforms in sea urchin eggs in which the effects of NAADP were first described (Lee and Aarhus, 1995), although levels of TPC3 were low compared with prism-stage embryos (Fig. 1 B). TPC1 and TPC2 transcripts were also detectable in rat PC12 and human SKBR3 cells (Fig. 1 B), which are cell lines that have both been shown to respond to NAADP (Brailoiu et al., 2006; Schrlau et al., 2008). Quantitative PCR indicated that TPC1 was the major isoform expressed in sea urchin eggs, PC12, and SKBR3 cells (Fig. 1 C). Therefore, TPCs comprise a ubiquitous family of novel uncharacterized ion channels in animals.


Essential requirement for two-pore channel 1 in NAADP-mediated calcium signaling.

Brailoiu E, Churamani D, Cai X, Schrlau MG, Brailoiu GC, Gao X, Hooper R, Boulware MJ, Dun NJ, Marchant JS, Patel S - J. Cell Biol. (2009)

An expanded family of TPC genes in animals. (A) Maximum likelihood tree constructed using the conserved regions of TPC sequences from the representative organisms listed in Table S1. Shading highlights TPC isoforms in plants, protists, and animals, with the latter subdivided into three distinct groupings. Bootstrap values >50 are shown at the branches. (B) End point RT-PCR analysis showing expression of transcripts for TPC isoforms in the indicated cell type/embryo (prism stage). The expected sizes of the amplicons were 152 (SpuTPC1), 93 (SpuTPC2), 107 (SpuTPC3), 473 (RnoTPC1), 575 (RnoTPC2), 250 (HsaTPC1), and 250 bp (HsaTPC2). Black lines indicate that intervening lanes have been spliced out. (C) Quantitative RT-PCR of TPC isoforms. Data were normalized to the expression level of the indicated housekeeping gene. Error bars indicate SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2717647&req=5

fig1: An expanded family of TPC genes in animals. (A) Maximum likelihood tree constructed using the conserved regions of TPC sequences from the representative organisms listed in Table S1. Shading highlights TPC isoforms in plants, protists, and animals, with the latter subdivided into three distinct groupings. Bootstrap values >50 are shown at the branches. (B) End point RT-PCR analysis showing expression of transcripts for TPC isoforms in the indicated cell type/embryo (prism stage). The expected sizes of the amplicons were 152 (SpuTPC1), 93 (SpuTPC2), 107 (SpuTPC3), 473 (RnoTPC1), 575 (RnoTPC2), 250 (HsaTPC1), and 250 bp (HsaTPC2). Black lines indicate that intervening lanes have been spliced out. (C) Quantitative RT-PCR of TPC isoforms. Data were normalized to the expression level of the indicated housekeeping gene. Error bars indicate SEM.
Mentions: TPCs are almost completely uncharacterized in animals. Comparative genomic analysis reveals that the TPC gene is widespread in the animal kingdom and has undergone multiplication with most species possessing either two (human and rat) or three (sea urchin) TPC genes (Fig. 1 A and Table S1). These genes are predicted to encode for proteins that display ∼35% sequence similarity (Table S2). Duplication of the TPC gene likely began early during animal evolution given the presence of multiple TPC genes in the sea anemone (Nematostella vectensis; Fig. 1 A), a member of the Cnidarian phylum considered the oldest of the eumetazoan lineage. Indeed, multiple TPC genes are also evident in the choanoflagellate Monosiga brevicollis, which is a close unicellular relative of animals (Fig. 1 A). Intriguingly, a single copy of the TPC gene is present in the protists Thalassiosira pseudonana (marine phytoplankton) and Phytophthora ramorum (sudden oak death pathogen; Fig. 1 A), suggesting that TPCs are ancient proteins that likely originated in the common ancestor of animals and plants. During the course of metazoan evolution, we note lineage-specific gene loss. This is most striking in mammals, where three genes are present in some species such as dog, whereas only two are present in other species such as human and rat (Fig. 1 A and Table S1). Semiquantitative RT-PCR analysis demonstrated the presence of transcripts for all three TPC isoforms in sea urchin eggs in which the effects of NAADP were first described (Lee and Aarhus, 1995), although levels of TPC3 were low compared with prism-stage embryos (Fig. 1 B). TPC1 and TPC2 transcripts were also detectable in rat PC12 and human SKBR3 cells (Fig. 1 B), which are cell lines that have both been shown to respond to NAADP (Brailoiu et al., 2006; Schrlau et al., 2008). Quantitative PCR indicated that TPC1 was the major isoform expressed in sea urchin eggs, PC12, and SKBR3 cells (Fig. 1 C). Therefore, TPCs comprise a ubiquitous family of novel uncharacterized ion channels in animals.

Bottom Line: However, the molecular identity of the target protein is unclear.In this study, we show that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP.Thus, TPC1 is critical for NAADP action and is likely the long sought after target channel for NAADP.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140, USA.

ABSTRACT
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a widespread and potent calcium-mobilizing messenger that is highly unusual in activating calcium channels located on acidic stores. However, the molecular identity of the target protein is unclear. In this study, we show that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP. Thus, TPC1 is critical for NAADP action and is likely the long sought after target channel for NAADP.

Show MeSH