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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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Mutation of a single residue in the putative pore region inactivates TPC1. (A) Multiple sequence alignment of the two putative pore regions (P1 and P2) of several animal TPCs. Asterisks highlight residues conserved in both pores of all isoforms from different species. Predicted helical region is outlined by the cartoon. (B and C) Cytosolic calcium responses of individual fura-2–loaded SKBR3 cells microinjected with either 10 nM (B) or 10 µM (C) NAADP. Cells were from mock-transfected cultures or cultures expressing TPC1 mutated in the putative pore region (TPC1 L273P). (D) Pooled data quantifying the magnitude of cytosolic changes under the various experimental conditions are shown. Error bars indicate SEM.
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fig5: Mutation of a single residue in the putative pore region inactivates TPC1. (A) Multiple sequence alignment of the two putative pore regions (P1 and P2) of several animal TPCs. Asterisks highlight residues conserved in both pores of all isoforms from different species. Predicted helical region is outlined by the cartoon. (B and C) Cytosolic calcium responses of individual fura-2–loaded SKBR3 cells microinjected with either 10 nM (B) or 10 µM (C) NAADP. Cells were from mock-transfected cultures or cultures expressing TPC1 mutated in the putative pore region (TPC1 L273P). (D) Pooled data quantifying the magnitude of cytosolic changes under the various experimental conditions are shown. Error bars indicate SEM.

Mentions: An alignment of the amino acid sequences of putative pore regions of TPCs from several animals reveals two residues that are conserved in both pores and across species (Fig. 5 A, asterisks). One of these residues (leucine 273 in human TPC1) is in the putative helix of the pore-forming region (Fig. 5 A). Mutation of this residue to a helix-breaking proline residue abolished the potentiating effects of TPC1 overexpression on NAADP-mediated calcium signals (Fig. 5 B). The lack of response was not caused by mistargeting of TPC1 L273P because the mutant protein colocalized with wild-type TPC1 (Fig. S3 C). We also found that calcium signals, in response to higher concentrations of NAADP, were significantly reduced in cells overexpressing TPC1 L273P (Fig. 5 C), suggesting that this mutant acts in a dominant-negative manner. These data, summarized in Fig. 5 D, further confirm the specificity of the effect of overexpressing wild-type TPC1 on NAADP-mediated calcium signals, provide additional evidence that endogenous NAADP responses require TPCs, and show that TPC function is likely dependent on ion channel activity. Most ion channel pores are formed through assembly of at least three pore-forming subunits. Given the sequence similarity between TPCs and voltage-sensitive calcium and sodium channels (Ishibashi et al., 2000), which are composed of four repeats, it is tempting to speculate that TPCs are dimeric. Thus, the observed dominant-negative action of TPC1 L273P may result in oligomerization of the mutant with endogenous channels and should prove a powerful tool in future studies to inhibit NAADP signaling.


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)

Mutation of a single residue in the putative pore region inactivates TPC1. (A) Multiple sequence alignment of the two putative pore regions (P1 and P2) of several animal TPCs. Asterisks highlight residues conserved in both pores of all isoforms from different species. Predicted helical region is outlined by the cartoon. (B and C) Cytosolic calcium responses of individual fura-2–loaded SKBR3 cells microinjected with either 10 nM (B) or 10 µM (C) NAADP. Cells were from mock-transfected cultures or cultures expressing TPC1 mutated in the putative pore region (TPC1 L273P). (D) Pooled data quantifying the magnitude of cytosolic changes under the various experimental conditions are shown. Error bars indicate SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: Mutation of a single residue in the putative pore region inactivates TPC1. (A) Multiple sequence alignment of the two putative pore regions (P1 and P2) of several animal TPCs. Asterisks highlight residues conserved in both pores of all isoforms from different species. Predicted helical region is outlined by the cartoon. (B and C) Cytosolic calcium responses of individual fura-2–loaded SKBR3 cells microinjected with either 10 nM (B) or 10 µM (C) NAADP. Cells were from mock-transfected cultures or cultures expressing TPC1 mutated in the putative pore region (TPC1 L273P). (D) Pooled data quantifying the magnitude of cytosolic changes under the various experimental conditions are shown. Error bars indicate SEM.
Mentions: An alignment of the amino acid sequences of putative pore regions of TPCs from several animals reveals two residues that are conserved in both pores and across species (Fig. 5 A, asterisks). One of these residues (leucine 273 in human TPC1) is in the putative helix of the pore-forming region (Fig. 5 A). Mutation of this residue to a helix-breaking proline residue abolished the potentiating effects of TPC1 overexpression on NAADP-mediated calcium signals (Fig. 5 B). The lack of response was not caused by mistargeting of TPC1 L273P because the mutant protein colocalized with wild-type TPC1 (Fig. S3 C). We also found that calcium signals, in response to higher concentrations of NAADP, were significantly reduced in cells overexpressing TPC1 L273P (Fig. 5 C), suggesting that this mutant acts in a dominant-negative manner. These data, summarized in Fig. 5 D, further confirm the specificity of the effect of overexpressing wild-type TPC1 on NAADP-mediated calcium signals, provide additional evidence that endogenous NAADP responses require TPCs, and show that TPC function is likely dependent on ion channel activity. Most ion channel pores are formed through assembly of at least three pore-forming subunits. Given the sequence similarity between TPCs and voltage-sensitive calcium and sodium channels (Ishibashi et al., 2000), which are composed of four repeats, it is tempting to speculate that TPCs are dimeric. Thus, the observed dominant-negative action of TPC1 L273P may result in oligomerization of the mutant with endogenous channels and should prove a powerful tool in future studies to inhibit NAADP signaling.

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