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Expanding the neuron's calcium signaling repertoire: intracellular calcium release via voltage-induced PLC and IP3R activation.

Ryglewski S, Pflueger HJ, Duch C - PLoS Biol. (2007)

Bottom Line: Specific and independent regulation of these vital cellular processes is achieved by a rich bouquet of different calcium signaling mechanisms within the neuron, which either can operate independently or may act in concert.This study demonstrates the existence of a novel calcium signaling mechanism by simultaneous patch clamping and calcium imaging from acutely isolated central neurons.This allows neurons to monitor activity by intracellular calcium release without relying on calcium as the input signal and opens up new insights into intracellular signaling, developmental regulation, and information processing in neuronal compartments lacking calcium channels.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology/Neurobiology, Free University of Berlin, Berlin, Germany.

ABSTRACT
Neuronal calcium acts as a charge carrier during information processing and as a ubiquitous intracellular messenger. Calcium signals are fundamental to numerous aspects of neuronal development and plasticity. Specific and independent regulation of these vital cellular processes is achieved by a rich bouquet of different calcium signaling mechanisms within the neuron, which either can operate independently or may act in concert. This study demonstrates the existence of a novel calcium signaling mechanism by simultaneous patch clamping and calcium imaging from acutely isolated central neurons. These neurons possess a membrane voltage sensor that, independent of calcium influx, causes G-protein activation, which subsequently leads to calcium release from intracellular stores via phospholipase C and inositol 1,4,5-trisphosphate receptor activation. This allows neurons to monitor activity by intracellular calcium release without relying on calcium as the input signal and opens up new insights into intracellular signaling, developmental regulation, and information processing in neuronal compartments lacking calcium channels.

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Voltage-Induced Intracellular Calcium Signals Depend on IP3R and PLC Activation(A) (i) In heparin-loaded (500 nM), acutely isolated DUM neurons in TEA- and TTX-containing saline, a voltage step from −90 mV to 0 mV causes a calcium inward and a calcium-activated potassium outward current accompanied by an intracellular calcium signal. (ii) Exposing the heparin-loaded cell for 3 min to zero-calcium saline causes a complete loss of all membrane currents and the calcium signal. (iii) Switching back to calcium-containing saline for 3 min restores the original currents and the calcium signal.(B) Same representative experiment as in (A), but the neuron is loaded with the PLC blocker U73122. Pharmacological block of IP3Rs and of PLC both abolish voltage-induced calcium signals in the absence of calcium inward current.
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pbio-0050066-g004: Voltage-Induced Intracellular Calcium Signals Depend on IP3R and PLC Activation(A) (i) In heparin-loaded (500 nM), acutely isolated DUM neurons in TEA- and TTX-containing saline, a voltage step from −90 mV to 0 mV causes a calcium inward and a calcium-activated potassium outward current accompanied by an intracellular calcium signal. (ii) Exposing the heparin-loaded cell for 3 min to zero-calcium saline causes a complete loss of all membrane currents and the calcium signal. (iii) Switching back to calcium-containing saline for 3 min restores the original currents and the calcium signal.(B) Same representative experiment as in (A), but the neuron is loaded with the PLC blocker U73122. Pharmacological block of IP3Rs and of PLC both abolish voltage-induced calcium signals in the absence of calcium inward current.

Mentions: In contrast, intracellular application of the IP3R-blocker heparin completely abolished voltage-dependent calcium release from internal stores in the absence of extracellular calcium (Figure 4A). Heparin has been shown to block insect DUM neuron IP3Rs at micromolar concentrations [19]. We have further demonstrated the effectiveness of heparin in our experiments by demonstrating that it blocks intracellular calcium release induced by bath application of the PLC agonist m-3M3FBS (25 μM; Calbiochem, San Diego, California, United States; see Figure S2). Loading the neurons intracellularly with heparin (0.5 μM) via the patch pipette had no effect on calcium currents recorded in voltage clamp (Figure 4Ai). Under zero extracellular calcium conditions, heparin-loaded cells showed no increase in [Ca2+]i as a response to membrane depolarizations. After 3 min in calcium-free saline, no voltage-activated calcium inward current and no increases in fluorescence were observed (Figure 4Aii). Washing for 3 min in calcium-containing saline completely restored the initial voltage-activated calcium current and the resulting increases in fluorescence (Figure 4Aiii). These data showed that membrane depolarization–induced intracellular calcium release without calcium influx depended on IP3R activation. These pharmacological data were further substantiated by the finding that voltage-induced intracellular calcium signals in the absence of calcium influx could be blocked by bath application of the membrane-permeable IP3R blocker 2-aminoethoxydiphenyl borate (2-APB). However, the effect of 2-APB was only partially reversible in locust DUM neurons (see Figure S3).


Expanding the neuron's calcium signaling repertoire: intracellular calcium release via voltage-induced PLC and IP3R activation.

Ryglewski S, Pflueger HJ, Duch C - PLoS Biol. (2007)

Voltage-Induced Intracellular Calcium Signals Depend on IP3R and PLC Activation(A) (i) In heparin-loaded (500 nM), acutely isolated DUM neurons in TEA- and TTX-containing saline, a voltage step from −90 mV to 0 mV causes a calcium inward and a calcium-activated potassium outward current accompanied by an intracellular calcium signal. (ii) Exposing the heparin-loaded cell for 3 min to zero-calcium saline causes a complete loss of all membrane currents and the calcium signal. (iii) Switching back to calcium-containing saline for 3 min restores the original currents and the calcium signal.(B) Same representative experiment as in (A), but the neuron is loaded with the PLC blocker U73122. Pharmacological block of IP3Rs and of PLC both abolish voltage-induced calcium signals in the absence of calcium inward current.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1808487&req=5

pbio-0050066-g004: Voltage-Induced Intracellular Calcium Signals Depend on IP3R and PLC Activation(A) (i) In heparin-loaded (500 nM), acutely isolated DUM neurons in TEA- and TTX-containing saline, a voltage step from −90 mV to 0 mV causes a calcium inward and a calcium-activated potassium outward current accompanied by an intracellular calcium signal. (ii) Exposing the heparin-loaded cell for 3 min to zero-calcium saline causes a complete loss of all membrane currents and the calcium signal. (iii) Switching back to calcium-containing saline for 3 min restores the original currents and the calcium signal.(B) Same representative experiment as in (A), but the neuron is loaded with the PLC blocker U73122. Pharmacological block of IP3Rs and of PLC both abolish voltage-induced calcium signals in the absence of calcium inward current.
Mentions: In contrast, intracellular application of the IP3R-blocker heparin completely abolished voltage-dependent calcium release from internal stores in the absence of extracellular calcium (Figure 4A). Heparin has been shown to block insect DUM neuron IP3Rs at micromolar concentrations [19]. We have further demonstrated the effectiveness of heparin in our experiments by demonstrating that it blocks intracellular calcium release induced by bath application of the PLC agonist m-3M3FBS (25 μM; Calbiochem, San Diego, California, United States; see Figure S2). Loading the neurons intracellularly with heparin (0.5 μM) via the patch pipette had no effect on calcium currents recorded in voltage clamp (Figure 4Ai). Under zero extracellular calcium conditions, heparin-loaded cells showed no increase in [Ca2+]i as a response to membrane depolarizations. After 3 min in calcium-free saline, no voltage-activated calcium inward current and no increases in fluorescence were observed (Figure 4Aii). Washing for 3 min in calcium-containing saline completely restored the initial voltage-activated calcium current and the resulting increases in fluorescence (Figure 4Aiii). These data showed that membrane depolarization–induced intracellular calcium release without calcium influx depended on IP3R activation. These pharmacological data were further substantiated by the finding that voltage-induced intracellular calcium signals in the absence of calcium influx could be blocked by bath application of the membrane-permeable IP3R blocker 2-aminoethoxydiphenyl borate (2-APB). However, the effect of 2-APB was only partially reversible in locust DUM neurons (see Figure S3).

Bottom Line: Specific and independent regulation of these vital cellular processes is achieved by a rich bouquet of different calcium signaling mechanisms within the neuron, which either can operate independently or may act in concert.This study demonstrates the existence of a novel calcium signaling mechanism by simultaneous patch clamping and calcium imaging from acutely isolated central neurons.This allows neurons to monitor activity by intracellular calcium release without relying on calcium as the input signal and opens up new insights into intracellular signaling, developmental regulation, and information processing in neuronal compartments lacking calcium channels.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology/Neurobiology, Free University of Berlin, Berlin, Germany.

ABSTRACT
Neuronal calcium acts as a charge carrier during information processing and as a ubiquitous intracellular messenger. Calcium signals are fundamental to numerous aspects of neuronal development and plasticity. Specific and independent regulation of these vital cellular processes is achieved by a rich bouquet of different calcium signaling mechanisms within the neuron, which either can operate independently or may act in concert. This study demonstrates the existence of a novel calcium signaling mechanism by simultaneous patch clamping and calcium imaging from acutely isolated central neurons. These neurons possess a membrane voltage sensor that, independent of calcium influx, causes G-protein activation, which subsequently leads to calcium release from intracellular stores via phospholipase C and inositol 1,4,5-trisphosphate receptor activation. This allows neurons to monitor activity by intracellular calcium release without relying on calcium as the input signal and opens up new insights into intracellular signaling, developmental regulation, and information processing in neuronal compartments lacking calcium channels.

Show MeSH
Related in: MedlinePlus