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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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Activation of single Ca2+-permeable TPC2 channels by NAADP. A, typical recordings from excised inside-out patches from the plasma membrane of HEK cells expressing TPC2, TPC2ΔN, or TPC2AA and stimulated as indicated with NAADP (500 nm in the bathing solution). Cs+ was the charge carrier, and the holding potential was −60 mV; C denotes the closed state. Results are typical (from top to bottom) of 15, 17, 17, and 4 experiments, respectively. B, current (i)-voltage (V) relationship for the NAADP-activated channels in TPC2ΔN-expressing cells. Results are means ± S.E., n = 8. C, recording, typical of three experiments, from an excised inside-out patch from the plasma membrane of HEK cells expressing TPC2ΔN. NAADP (500 nm) was added and removed as indicated. C denotes the closed state. D, recording, typical of three similar records, from a patch excised from the plasma membrane of HEK cells expressing TPC2ΔN and treated with NAADP (500 nm) and trans-NED19 (100 nm) as indicated. Cs+ was the charge carrier, and the holding potential was −40 mV; C, O1, and O2 denote the closed state and openings of one and two channels, respectively. Note the presence of basal activity (NPo), which is increased ∼20-fold by the addition of NAADP; trans-NED19 inhibits both the basal and NAADP-evoked activity. In both C and D changes of media are accompanied by brief electrical spike artifacts. E, summary results showing NPo for excised patches of cells expressing TPC2ΔN or TPC2AA and stimulated with the indicated concentrations of NAADP (nanomolar) in the bathing solution. Results are means ± S.E., with n shown above each bar. F, record, typical of five similar records, from an excised patch expressing TPC2ΔN and with Ca2+ as the charge carrier. Pipette solution contained 500 nm NAADP, and the holding potential was −100 mV. C denotes the closed state. G, current-voltage relationship from records similar to those shown in F. Recordings were restricted to negative holding potentials to avoid activation of voltage-gated Ca2+ channels that may be endogenously expressed in HEK cells (34). Results are means ± S.E., n = 5.
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Figure 4: Activation of single Ca2+-permeable TPC2 channels by NAADP. A, typical recordings from excised inside-out patches from the plasma membrane of HEK cells expressing TPC2, TPC2ΔN, or TPC2AA and stimulated as indicated with NAADP (500 nm in the bathing solution). Cs+ was the charge carrier, and the holding potential was −60 mV; C denotes the closed state. Results are typical (from top to bottom) of 15, 17, 17, and 4 experiments, respectively. B, current (i)-voltage (V) relationship for the NAADP-activated channels in TPC2ΔN-expressing cells. Results are means ± S.E., n = 8. C, recording, typical of three experiments, from an excised inside-out patch from the plasma membrane of HEK cells expressing TPC2ΔN. NAADP (500 nm) was added and removed as indicated. C denotes the closed state. D, recording, typical of three similar records, from a patch excised from the plasma membrane of HEK cells expressing TPC2ΔN and treated with NAADP (500 nm) and trans-NED19 (100 nm) as indicated. Cs+ was the charge carrier, and the holding potential was −40 mV; C, O1, and O2 denote the closed state and openings of one and two channels, respectively. Note the presence of basal activity (NPo), which is increased ∼20-fold by the addition of NAADP; trans-NED19 inhibits both the basal and NAADP-evoked activity. In both C and D changes of media are accompanied by brief electrical spike artifacts. E, summary results showing NPo for excised patches of cells expressing TPC2ΔN or TPC2AA and stimulated with the indicated concentrations of NAADP (nanomolar) in the bathing solution. Results are means ± S.E., with n shown above each bar. F, record, typical of five similar records, from an excised patch expressing TPC2ΔN and with Ca2+ as the charge carrier. Pipette solution contained 500 nm NAADP, and the holding potential was −100 mV. C denotes the closed state. G, current-voltage relationship from records similar to those shown in F. Recordings were restricted to negative holding potentials to avoid activation of voltage-gated Ca2+ channels that may be endogenously expressed in HEK cells (34). Results are means ± S.E., n = 5.

Mentions: In 17 of 38 recordings from inside-out patches from the plasma membrane of cells expressing TPC2ΔN, NAADP (200–500 nm, in the bathing solution) stimulated bursts of channel openings (Fig. 4A). Detection of channels in ∼40% of patches suggests an average of ∼0.5 randomly distributed channels/patch and is consistent with the relative scarcity (∼8%) of patches in which we detected more than one channel (supplemental text). The current-voltage relationship for these single-channel openings was, like the whole-cell currents (Fig. 3B), slightly inwardly rectifying. The unitary Cs+ conductance (γCs, measured between 0 and −60 mV) was 128 ± 4 pS (Fig. 4B). In continuous recordings, NAADP rapidly and reversibly stimulated this channel activity (Fig. 4C), and trans-NED19 rapidly reversed the activity (Fig. 4D). Channels with indistinguishable conductance (γCs = 138 ± 15 pS, n = 4, data not shown) and NPo (Fig. 4, A and E) were detected in excised patches from cells expressing TPC2AA. We detected no such channels in parallel recordings from either mock-transfected cells (n = 20; data not shown) or from cells expressing TPC2 (Fig. 4A). Some patches from TPC2ΔN-expressing cells (3 of 17 recordings) had detectable activity in the absence of added NAADP, although most were quiescent. In each, the activity was massively increased by addition of 200 or 500 nm NAADP (Fig. 4E).


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)

Activation of single Ca2+-permeable TPC2 channels by NAADP. A, typical recordings from excised inside-out patches from the plasma membrane of HEK cells expressing TPC2, TPC2ΔN, or TPC2AA and stimulated as indicated with NAADP (500 nm in the bathing solution). Cs+ was the charge carrier, and the holding potential was −60 mV; C denotes the closed state. Results are typical (from top to bottom) of 15, 17, 17, and 4 experiments, respectively. B, current (i)-voltage (V) relationship for the NAADP-activated channels in TPC2ΔN-expressing cells. Results are means ± S.E., n = 8. C, recording, typical of three experiments, from an excised inside-out patch from the plasma membrane of HEK cells expressing TPC2ΔN. NAADP (500 nm) was added and removed as indicated. C denotes the closed state. D, recording, typical of three similar records, from a patch excised from the plasma membrane of HEK cells expressing TPC2ΔN and treated with NAADP (500 nm) and trans-NED19 (100 nm) as indicated. Cs+ was the charge carrier, and the holding potential was −40 mV; C, O1, and O2 denote the closed state and openings of one and two channels, respectively. Note the presence of basal activity (NPo), which is increased ∼20-fold by the addition of NAADP; trans-NED19 inhibits both the basal and NAADP-evoked activity. In both C and D changes of media are accompanied by brief electrical spike artifacts. E, summary results showing NPo for excised patches of cells expressing TPC2ΔN or TPC2AA and stimulated with the indicated concentrations of NAADP (nanomolar) in the bathing solution. Results are means ± S.E., with n shown above each bar. F, record, typical of five similar records, from an excised patch expressing TPC2ΔN and with Ca2+ as the charge carrier. Pipette solution contained 500 nm NAADP, and the holding potential was −100 mV. C denotes the closed state. G, current-voltage relationship from records similar to those shown in F. Recordings were restricted to negative holding potentials to avoid activation of voltage-gated Ca2+ channels that may be endogenously expressed in HEK cells (34). Results are means ± S.E., n = 5.
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Figure 4: Activation of single Ca2+-permeable TPC2 channels by NAADP. A, typical recordings from excised inside-out patches from the plasma membrane of HEK cells expressing TPC2, TPC2ΔN, or TPC2AA and stimulated as indicated with NAADP (500 nm in the bathing solution). Cs+ was the charge carrier, and the holding potential was −60 mV; C denotes the closed state. Results are typical (from top to bottom) of 15, 17, 17, and 4 experiments, respectively. B, current (i)-voltage (V) relationship for the NAADP-activated channels in TPC2ΔN-expressing cells. Results are means ± S.E., n = 8. C, recording, typical of three experiments, from an excised inside-out patch from the plasma membrane of HEK cells expressing TPC2ΔN. NAADP (500 nm) was added and removed as indicated. C denotes the closed state. D, recording, typical of three similar records, from a patch excised from the plasma membrane of HEK cells expressing TPC2ΔN and treated with NAADP (500 nm) and trans-NED19 (100 nm) as indicated. Cs+ was the charge carrier, and the holding potential was −40 mV; C, O1, and O2 denote the closed state and openings of one and two channels, respectively. Note the presence of basal activity (NPo), which is increased ∼20-fold by the addition of NAADP; trans-NED19 inhibits both the basal and NAADP-evoked activity. In both C and D changes of media are accompanied by brief electrical spike artifacts. E, summary results showing NPo for excised patches of cells expressing TPC2ΔN or TPC2AA and stimulated with the indicated concentrations of NAADP (nanomolar) in the bathing solution. Results are means ± S.E., with n shown above each bar. F, record, typical of five similar records, from an excised patch expressing TPC2ΔN and with Ca2+ as the charge carrier. Pipette solution contained 500 nm NAADP, and the holding potential was −100 mV. C denotes the closed state. G, current-voltage relationship from records similar to those shown in F. Recordings were restricted to negative holding potentials to avoid activation of voltage-gated Ca2+ channels that may be endogenously expressed in HEK cells (34). Results are means ± S.E., n = 5.
Mentions: In 17 of 38 recordings from inside-out patches from the plasma membrane of cells expressing TPC2ΔN, NAADP (200–500 nm, in the bathing solution) stimulated bursts of channel openings (Fig. 4A). Detection of channels in ∼40% of patches suggests an average of ∼0.5 randomly distributed channels/patch and is consistent with the relative scarcity (∼8%) of patches in which we detected more than one channel (supplemental text). The current-voltage relationship for these single-channel openings was, like the whole-cell currents (Fig. 3B), slightly inwardly rectifying. The unitary Cs+ conductance (γCs, measured between 0 and −60 mV) was 128 ± 4 pS (Fig. 4B). In continuous recordings, NAADP rapidly and reversibly stimulated this channel activity (Fig. 4C), and trans-NED19 rapidly reversed the activity (Fig. 4D). Channels with indistinguishable conductance (γCs = 138 ± 15 pS, n = 4, data not shown) and NPo (Fig. 4, A and E) were detected in excised patches from cells expressing TPC2AA. We detected no such channels in parallel recordings from either mock-transfected cells (n = 20; data not shown) or from cells expressing TPC2 (Fig. 4A). Some patches from TPC2ΔN-expressing cells (3 of 17 recordings) had detectable activity in the absence of added NAADP, although most were quiescent. In each, the activity was massively increased by addition of 200 or 500 nm NAADP (Fig. 4E).

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