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An NAADP-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying Ca2+ signals.

Brailoiu E, Rahman T, Churamani D, Prole DL, Brailoiu GC, Hooper R, Taylor CW, Patel S - J. Biol. Chem. (2010)

Bottom Line: It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca(2+) stores, fuelling speculation that archetypal intracellular Ca(2+) channels are the primary targets of NAADP.Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca(2+) influx independent of ryanodine receptors and that it activates a Ca(2+)-permeable channel whose conductance is reduced by mutation of a residue within a putative pore.We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca(2+) channel.

View Article: PubMed Central - PubMed

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

ABSTRACT
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca(2+) release from acidic organelles via two-pore channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca(2+) stores, fuelling speculation that archetypal intracellular Ca(2+) channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca(2+) influx independent of ryanodine receptors and that it activates a Ca(2+)-permeable channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca(2+) channel.

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TPC2 is the pore-forming subunit of an NAADP-gated channel. A, depiction of TPC2 showing location of a putative pore residue (Leu-265, red). B, confocal fluorescence images of SKBR3 cells expressing GFP-tagged TPC2 in which Leu-265 was replaced by proline (TPC2L265P, left) or in which this was combined with removal of the N terminus (TPC2ΔNL265P, right). Images are typical of those from 6–10 cells. Scale bar, 5 μm. Similar results with HEK cells are shown in supplemental Fig. S2E. C, cytosolic Ca2+ signals from individual fura-2-loaded SKBR3 cells transiently transfected with the indicated C-terminally GFP-tagged TPC2 constructs and microinjected with NAADP (10 nm, arrowheads). Results are means ± S.E. of the indicated number (n) of cells. D, recording, typical of four similar records, from excised inside-out patches from the plasma membrane of HEK cells expressing TPC2ΔNL265P and stimulated with 500 nm NAADP in the bathing solution with Cs+ as the charge carrier. C denotes the closed state. E, current-voltage relationship from records similar to those shown in D. Results are means ± S.E., n = 4.
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Figure 5: TPC2 is the pore-forming subunit of an NAADP-gated channel. A, depiction of TPC2 showing location of a putative pore residue (Leu-265, red). B, confocal fluorescence images of SKBR3 cells expressing GFP-tagged TPC2 in which Leu-265 was replaced by proline (TPC2L265P, left) or in which this was combined with removal of the N terminus (TPC2ΔNL265P, right). Images are typical of those from 6–10 cells. Scale bar, 5 μm. Similar results with HEK cells are shown in supplemental Fig. S2E. C, cytosolic Ca2+ signals from individual fura-2-loaded SKBR3 cells transiently transfected with the indicated C-terminally GFP-tagged TPC2 constructs and microinjected with NAADP (10 nm, arrowheads). Results are means ± S.E. of the indicated number (n) of cells. D, recording, typical of four similar records, from excised inside-out patches from the plasma membrane of HEK cells expressing TPC2ΔNL265P and stimulated with 500 nm NAADP in the bathing solution with Cs+ as the charge carrier. C denotes the closed state. E, current-voltage relationship from records similar to those shown in D. Results are means ± S.E., n = 4.

Mentions: An outstanding question is whether TPC2 is itself the pore-forming subunit of the channel. To address this issue, we mutated a conserved residue (Leu-265) within a putative pore region (4) of TPC2 and TPC2ΔN (Fig. 5A). The mutant, TPC2L265P, showed an intracellular distribution similar to that of TPC2, whereas TPC2ΔNL265P, like TPC2ΔN, localized to the plasma membrane in both SKBR3 (Fig. 5B) and HEK (supplemental Fig. S2E) cells. Microinjection of NAADP into SKBR3 cells expressing either TPC2L265P or TPC2ΔNL265P failed to evoke Ca2+ signals (Fig. 5C). We recorded Cs+ currents in excised patches from cells expressing TPC2ΔNL265P. NAADP (500 nm) activated channels in these patches (Fig. 5D) that had similar activity (NPo = 0.32 ± 0.04, n = 3) to those of TPC2ΔN, but their γCs was massively reduced to 9.5 ± 0.41 pS (Fig. 5E). These results, showing that a mutation within the putative pore of TPC2 affects the conductance of the NAADP-activated channel, establish that TPC2 is a pore-forming subunit of the NAADP-activated cation channel. While our work was under review, others reported NAADP-evoked Ca2+ currents from enlarged lysosomes of cells overexpressing mouse TPC2 (32) and single-channel recordings from immunopurified human TPC2 incorporated into artificial lipid bilayers (33). Both studies reported dramatic effects of luminal pH on channel activity (32, 33). Our study is consistent with that of Pitt et al. (33) in demonstrating NAADP-stimulated channel activity at neutral pH, although in our experiments the effects of NAADP were more rapid in onset, reversible, and blocked by lower concentrations of trans-NED19. Our results and those from Pitt et al. (33) differ, however, from the study of Scheider et al. (32), who detected NAADP-mediated currents only at acidic luminal pH.


An NAADP-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying Ca2+ signals.

Brailoiu E, Rahman T, Churamani D, Prole DL, Brailoiu GC, Hooper R, Taylor CW, Patel S - J. Biol. Chem. (2010)

TPC2 is the pore-forming subunit of an NAADP-gated channel. A, depiction of TPC2 showing location of a putative pore residue (Leu-265, red). B, confocal fluorescence images of SKBR3 cells expressing GFP-tagged TPC2 in which Leu-265 was replaced by proline (TPC2L265P, left) or in which this was combined with removal of the N terminus (TPC2ΔNL265P, right). Images are typical of those from 6–10 cells. Scale bar, 5 μm. Similar results with HEK cells are shown in supplemental Fig. S2E. C, cytosolic Ca2+ signals from individual fura-2-loaded SKBR3 cells transiently transfected with the indicated C-terminally GFP-tagged TPC2 constructs and microinjected with NAADP (10 nm, arrowheads). Results are means ± S.E. of the indicated number (n) of cells. D, recording, typical of four similar records, from excised inside-out patches from the plasma membrane of HEK cells expressing TPC2ΔNL265P and stimulated with 500 nm NAADP in the bathing solution with Cs+ as the charge carrier. C denotes the closed state. E, current-voltage relationship from records similar to those shown in D. Results are means ± S.E., n = 4.
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Figure 5: TPC2 is the pore-forming subunit of an NAADP-gated channel. A, depiction of TPC2 showing location of a putative pore residue (Leu-265, red). B, confocal fluorescence images of SKBR3 cells expressing GFP-tagged TPC2 in which Leu-265 was replaced by proline (TPC2L265P, left) or in which this was combined with removal of the N terminus (TPC2ΔNL265P, right). Images are typical of those from 6–10 cells. Scale bar, 5 μm. Similar results with HEK cells are shown in supplemental Fig. S2E. C, cytosolic Ca2+ signals from individual fura-2-loaded SKBR3 cells transiently transfected with the indicated C-terminally GFP-tagged TPC2 constructs and microinjected with NAADP (10 nm, arrowheads). Results are means ± S.E. of the indicated number (n) of cells. D, recording, typical of four similar records, from excised inside-out patches from the plasma membrane of HEK cells expressing TPC2ΔNL265P and stimulated with 500 nm NAADP in the bathing solution with Cs+ as the charge carrier. C denotes the closed state. E, current-voltage relationship from records similar to those shown in D. Results are means ± S.E., n = 4.
Mentions: An outstanding question is whether TPC2 is itself the pore-forming subunit of the channel. To address this issue, we mutated a conserved residue (Leu-265) within a putative pore region (4) of TPC2 and TPC2ΔN (Fig. 5A). The mutant, TPC2L265P, showed an intracellular distribution similar to that of TPC2, whereas TPC2ΔNL265P, like TPC2ΔN, localized to the plasma membrane in both SKBR3 (Fig. 5B) and HEK (supplemental Fig. S2E) cells. Microinjection of NAADP into SKBR3 cells expressing either TPC2L265P or TPC2ΔNL265P failed to evoke Ca2+ signals (Fig. 5C). We recorded Cs+ currents in excised patches from cells expressing TPC2ΔNL265P. NAADP (500 nm) activated channels in these patches (Fig. 5D) that had similar activity (NPo = 0.32 ± 0.04, n = 3) to those of TPC2ΔN, but their γCs was massively reduced to 9.5 ± 0.41 pS (Fig. 5E). These results, showing that a mutation within the putative pore of TPC2 affects the conductance of the NAADP-activated channel, establish that TPC2 is a pore-forming subunit of the NAADP-activated cation channel. While our work was under review, others reported NAADP-evoked Ca2+ currents from enlarged lysosomes of cells overexpressing mouse TPC2 (32) and single-channel recordings from immunopurified human TPC2 incorporated into artificial lipid bilayers (33). Both studies reported dramatic effects of luminal pH on channel activity (32, 33). Our study is consistent with that of Pitt et al. (33) in demonstrating NAADP-stimulated channel activity at neutral pH, although in our experiments the effects of NAADP were more rapid in onset, reversible, and blocked by lower concentrations of trans-NED19. Our results and those from Pitt et al. (33) differ, however, from the study of Scheider et al. (32), who detected NAADP-mediated currents only at acidic luminal pH.

Bottom Line: It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca(2+) stores, fuelling speculation that archetypal intracellular Ca(2+) channels are the primary targets of NAADP.Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca(2+) influx independent of ryanodine receptors and that it activates a Ca(2+)-permeable channel whose conductance is reduced by mutation of a residue within a putative pore.We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca(2+) channel.

View Article: PubMed Central - PubMed

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

ABSTRACT
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca(2+) release from acidic organelles via two-pore channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca(2+) stores, fuelling speculation that archetypal intracellular Ca(2+) channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca(2+) influx independent of ryanodine receptors and that it activates a Ca(2+)-permeable channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca(2+) channel.

Show MeSH