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Inorganic polyphosphate regulates neuronal excitability through modulation of voltage-gated channels.

Stotz SC, Scott LO, Drummond-Main C, Avchalumov Y, Girotto F, Davidsen J, Gómez-Gárcia MR, Rho JM, Pavlov EV, Colicos MA - Mol Brain (2014)

Bottom Line: Mechanistically, this is accomplished by shifting the voltage sensitivity of NaV channel activation toward the neuronal resting membrane potential, the block KV channels, and the activation of CaV channels.Next, using calcium imaging we found that polyP stimulates an increase in neuronal network activity and induces calcium influx in glial cells.We conclude that polyP release leads to increased excitability of the neuronal membrane through the modulation of voltage gated ion channels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology & Pharmacology and the Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada. mcolicos@ucalgary.ca.

ABSTRACT

Background: Inorganic polyphosphate (polyP) is a highly charged polyanion capable of interacting with a number of molecular targets. This signaling molecule is released into the extracellular matrix by central astrocytes and by peripheral platelets during inflammation. While the release of polyP is associated with both induction of blood coagulation and astrocyte extracellular signaling, the role of secreted polyP in regulation of neuronal activity remains undefined. Here we test the hypothesis that polyP is an important participant in neuronal signaling. Specifically, we investigate the ability of neurons to release polyP and to induce neuronal firing, and clarify the underlying molecular mechanisms of this process by studying the action of polyP on voltage gated channels.

Results: Using patch clamp techniques, and primary hippocampal and dorsal root ganglion cell cultures, we demonstrate that polyP directly influences neuronal activity, inducing action potential generation in both PNS and CNS neurons. Mechanistically, this is accomplished by shifting the voltage sensitivity of NaV channel activation toward the neuronal resting membrane potential, the block KV channels, and the activation of CaV channels. Next, using calcium imaging we found that polyP stimulates an increase in neuronal network activity and induces calcium influx in glial cells. Using in situ DAPI localization and live imaging, we demonstrate that polyP is naturally present in synaptic regions and is released from the neurons upon depolarization. Finally, using a biochemical assay we demonstrate that polyP is present in synaptosomes and can be released upon their membrane depolarization by the addition of potassium chloride.

Conclusions: We conclude that polyP release leads to increased excitability of the neuronal membrane through the modulation of voltage gated ion channels. Together, our data establishes that polyP could function as excitatory neuromodulator in both the PNS and CNS.

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DAPI stains polyP in distinct punta along axon-like projections. A) At 456 nm (emission) DAPI stains nucleic acids primarily in the nucleus but also in puncta. B) At 560 nm (emission) DAPI stains polyP. PolyP is in the perinuclear space and cytoplasm of the cell body, and is found in distinct puncta along axon-like projections. C) Overlay of A&B, showing polyP puncta on axon-like filaments across from dendritic structures. D) Gel electrophoreses demonstrating presence of and release from synaptic vesicles of polyP. Lanes: M - DNA marker; 1 - long polyP (average 130 chain length); 2 - medium polyP (average 60 chain length); 3 - short polyP (average 15 chain length); 4 - Synaptic vesicles + 70 mM KCl; 5 - Synaptic vesicles alone; 6 - Synaptic vesicles + 70 mM KCl + phosphatase (sample from lane 4); 7 - Synaptic vesicles + phosphatase (sample from lane 5). 70 mM KCl evoked vesicular depolarization. Phosphatase digested polyP. Live imaging of in vitro polyP release from and uptake into distinct puncta - E) T = 0. The arrows indicate distinct puncta to follow through F &G. F) Following a 10 second stimulation at 60Hz, a subpopulation (16% +/-5, SEM, n = 6) of polyP puncta disappear. Adjacent polyP puncta remain, indicating that there are no confounding focal plane shifts or spatial movements. G) 30 seconds later the diminished puncta have begun to refill with polyP.
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Figure 5: DAPI stains polyP in distinct punta along axon-like projections. A) At 456 nm (emission) DAPI stains nucleic acids primarily in the nucleus but also in puncta. B) At 560 nm (emission) DAPI stains polyP. PolyP is in the perinuclear space and cytoplasm of the cell body, and is found in distinct puncta along axon-like projections. C) Overlay of A&B, showing polyP puncta on axon-like filaments across from dendritic structures. D) Gel electrophoreses demonstrating presence of and release from synaptic vesicles of polyP. Lanes: M - DNA marker; 1 - long polyP (average 130 chain length); 2 - medium polyP (average 60 chain length); 3 - short polyP (average 15 chain length); 4 - Synaptic vesicles + 70 mM KCl; 5 - Synaptic vesicles alone; 6 - Synaptic vesicles + 70 mM KCl + phosphatase (sample from lane 4); 7 - Synaptic vesicles + phosphatase (sample from lane 5). 70 mM KCl evoked vesicular depolarization. Phosphatase digested polyP. Live imaging of in vitro polyP release from and uptake into distinct puncta - E) T = 0. The arrows indicate distinct puncta to follow through F &G. F) Following a 10 second stimulation at 60Hz, a subpopulation (16% +/-5, SEM, n = 6) of polyP puncta disappear. Adjacent polyP puncta remain, indicating that there are no confounding focal plane shifts or spatial movements. G) 30 seconds later the diminished puncta have begun to refill with polyP.

Mentions: PolyP is a gliotransmitter, released by astrocytes to signal neighboring astrocytes [5]. Here we demonstrate that polyP is also released and taken up by neuronal synapses. PolyP can be imaged in live cells using DAPI stain and collecting emission wavelengths at 560 nm [1,20-22]. In our hippocampal cultures polyP is found in distinct puncta along axon-like projections and in the cell body perinuclear space and cytoplasm (Figure 5A). In contrast, at 456 nm emission, DAPI stains nuclear DNA and dendritic nucleic acids (Figure 5B). Overlap of the two emission signals clearly shows the differential compartmentalization of polyP versus nucleic acids (Figure 5C). Photoconductive stimulation can induce neuronal cultures grown on silicon wafers to fire in a high frequency, physiological manner [23]. Before stimulation, multiple polyP positive puncta are visible along axon-like projections (Figure 5E). Immediately following stimulation a subpopulation of the puncta disappear (Figure 5F), suggesting that the polyP is released concurrent with neuronal activity. As not all puncta vanish (~16% +/-5 of total puncta remain, n = 6 experiments), polyP may be in vesicles and other compartments outside of the readily released pool. The remaining puncta provide corroborative evidence that there was no shift in focal plane during the imaging. Interestingly, puncta reappeared within 30 seconds of the firing event (Figure 5G), suggesting that the polyP is rapidly replenished. Together, these data suggest polyP is can be released and replenished into neuronal puncta, similar to other neurotransmitters.


Inorganic polyphosphate regulates neuronal excitability through modulation of voltage-gated channels.

Stotz SC, Scott LO, Drummond-Main C, Avchalumov Y, Girotto F, Davidsen J, Gómez-Gárcia MR, Rho JM, Pavlov EV, Colicos MA - Mol Brain (2014)

DAPI stains polyP in distinct punta along axon-like projections. A) At 456 nm (emission) DAPI stains nucleic acids primarily in the nucleus but also in puncta. B) At 560 nm (emission) DAPI stains polyP. PolyP is in the perinuclear space and cytoplasm of the cell body, and is found in distinct puncta along axon-like projections. C) Overlay of A&B, showing polyP puncta on axon-like filaments across from dendritic structures. D) Gel electrophoreses demonstrating presence of and release from synaptic vesicles of polyP. Lanes: M - DNA marker; 1 - long polyP (average 130 chain length); 2 - medium polyP (average 60 chain length); 3 - short polyP (average 15 chain length); 4 - Synaptic vesicles + 70 mM KCl; 5 - Synaptic vesicles alone; 6 - Synaptic vesicles + 70 mM KCl + phosphatase (sample from lane 4); 7 - Synaptic vesicles + phosphatase (sample from lane 5). 70 mM KCl evoked vesicular depolarization. Phosphatase digested polyP. Live imaging of in vitro polyP release from and uptake into distinct puncta - E) T = 0. The arrows indicate distinct puncta to follow through F &G. F) Following a 10 second stimulation at 60Hz, a subpopulation (16% +/-5, SEM, n = 6) of polyP puncta disappear. Adjacent polyP puncta remain, indicating that there are no confounding focal plane shifts or spatial movements. G) 30 seconds later the diminished puncta have begun to refill with polyP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: DAPI stains polyP in distinct punta along axon-like projections. A) At 456 nm (emission) DAPI stains nucleic acids primarily in the nucleus but also in puncta. B) At 560 nm (emission) DAPI stains polyP. PolyP is in the perinuclear space and cytoplasm of the cell body, and is found in distinct puncta along axon-like projections. C) Overlay of A&B, showing polyP puncta on axon-like filaments across from dendritic structures. D) Gel electrophoreses demonstrating presence of and release from synaptic vesicles of polyP. Lanes: M - DNA marker; 1 - long polyP (average 130 chain length); 2 - medium polyP (average 60 chain length); 3 - short polyP (average 15 chain length); 4 - Synaptic vesicles + 70 mM KCl; 5 - Synaptic vesicles alone; 6 - Synaptic vesicles + 70 mM KCl + phosphatase (sample from lane 4); 7 - Synaptic vesicles + phosphatase (sample from lane 5). 70 mM KCl evoked vesicular depolarization. Phosphatase digested polyP. Live imaging of in vitro polyP release from and uptake into distinct puncta - E) T = 0. The arrows indicate distinct puncta to follow through F &G. F) Following a 10 second stimulation at 60Hz, a subpopulation (16% +/-5, SEM, n = 6) of polyP puncta disappear. Adjacent polyP puncta remain, indicating that there are no confounding focal plane shifts or spatial movements. G) 30 seconds later the diminished puncta have begun to refill with polyP.
Mentions: PolyP is a gliotransmitter, released by astrocytes to signal neighboring astrocytes [5]. Here we demonstrate that polyP is also released and taken up by neuronal synapses. PolyP can be imaged in live cells using DAPI stain and collecting emission wavelengths at 560 nm [1,20-22]. In our hippocampal cultures polyP is found in distinct puncta along axon-like projections and in the cell body perinuclear space and cytoplasm (Figure 5A). In contrast, at 456 nm emission, DAPI stains nuclear DNA and dendritic nucleic acids (Figure 5B). Overlap of the two emission signals clearly shows the differential compartmentalization of polyP versus nucleic acids (Figure 5C). Photoconductive stimulation can induce neuronal cultures grown on silicon wafers to fire in a high frequency, physiological manner [23]. Before stimulation, multiple polyP positive puncta are visible along axon-like projections (Figure 5E). Immediately following stimulation a subpopulation of the puncta disappear (Figure 5F), suggesting that the polyP is released concurrent with neuronal activity. As not all puncta vanish (~16% +/-5 of total puncta remain, n = 6 experiments), polyP may be in vesicles and other compartments outside of the readily released pool. The remaining puncta provide corroborative evidence that there was no shift in focal plane during the imaging. Interestingly, puncta reappeared within 30 seconds of the firing event (Figure 5G), suggesting that the polyP is rapidly replenished. Together, these data suggest polyP is can be released and replenished into neuronal puncta, similar to other neurotransmitters.

Bottom Line: Mechanistically, this is accomplished by shifting the voltage sensitivity of NaV channel activation toward the neuronal resting membrane potential, the block KV channels, and the activation of CaV channels.Next, using calcium imaging we found that polyP stimulates an increase in neuronal network activity and induces calcium influx in glial cells.We conclude that polyP release leads to increased excitability of the neuronal membrane through the modulation of voltage gated ion channels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology & Pharmacology and the Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada. mcolicos@ucalgary.ca.

ABSTRACT

Background: Inorganic polyphosphate (polyP) is a highly charged polyanion capable of interacting with a number of molecular targets. This signaling molecule is released into the extracellular matrix by central astrocytes and by peripheral platelets during inflammation. While the release of polyP is associated with both induction of blood coagulation and astrocyte extracellular signaling, the role of secreted polyP in regulation of neuronal activity remains undefined. Here we test the hypothesis that polyP is an important participant in neuronal signaling. Specifically, we investigate the ability of neurons to release polyP and to induce neuronal firing, and clarify the underlying molecular mechanisms of this process by studying the action of polyP on voltage gated channels.

Results: Using patch clamp techniques, and primary hippocampal and dorsal root ganglion cell cultures, we demonstrate that polyP directly influences neuronal activity, inducing action potential generation in both PNS and CNS neurons. Mechanistically, this is accomplished by shifting the voltage sensitivity of NaV channel activation toward the neuronal resting membrane potential, the block KV channels, and the activation of CaV channels. Next, using calcium imaging we found that polyP stimulates an increase in neuronal network activity and induces calcium influx in glial cells. Using in situ DAPI localization and live imaging, we demonstrate that polyP is naturally present in synaptic regions and is released from the neurons upon depolarization. Finally, using a biochemical assay we demonstrate that polyP is present in synaptosomes and can be released upon their membrane depolarization by the addition of potassium chloride.

Conclusions: We conclude that polyP release leads to increased excitability of the neuronal membrane through the modulation of voltage gated ion channels. Together, our data establishes that polyP could function as excitatory neuromodulator in both the PNS and CNS.

Show MeSH
Related in: MedlinePlus