| Abstract No.: | A-B1033 |
| Country: | Canada |
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| Title: | MITOCHONDRIAL DEPOLARIZATION ACTIVATES A PLASMA MEMBRANE CATION CHANNEL IN APLYSIA BAG CELL NEURONS |
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| Authors/Affiliations: | 1 Charlene Hickey*; 1 Neil Magoski;
1 Queens University, Kingston, ON, Canada
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| Content: | Objectives: Intracellular Ca2+ release can trigger a number of biochemical and biophysical events. The bag cell neurons of Aplysia californica provide a rare opportunity to observe the link between single neuron Ca2+ levels and a behaviour that is critical to the survival of the animal. Excitation of these neurons elicits an approximate 30 minute burst, known as the afterdischarge, which initiates reproduction through the release of intracellular Ca2+, activation of membrane channels, and peptide secretion. There is also evidence for the presence of store-operated Ca2+ channels in these neurons. However, little is known regarding the manner in which different Ca2+ sources in these cells or other neurons selectively activate membrane channels. Our objective was to characterize a cation channel that is activated by depolarizing the mitochondria and releasing its Ca2+.
Materials and Methods: Cultured bag cell neurons were obtained from 150-300 g Aplysia californica.
Voltage-clamp recordings were done using whole-cell with intracellular saline containing 300 nM Ca2+, 5 mM ATP and 5 mM EGTA. Current-clamp recordings were made using sharp microelectrodes filled with 2 M K+ acetate. The external solution used in both voltage-clamp and current-clamp was artificial seawater.
Results: Depolarizing the mitochondria with 20 µM of the protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) resulted in an inward current associated with an increase in membrane conductance. However, inhibiting the endoplasmic reticulum Ca2+-ATPase with 20 µM of cyclopiazonic acid (CPA) did not induce any current. CPA also had no effect on the current elicited by FCCP. The amplitude of the inward current decreased significantly when intracellular Ca2+ was buffered by high EGTA, suggesting that the channel responsible is Ca2+-dependent. The I/V relationship was largely linear and had a reversal potential of approximately -40 mV, which pointed to the likelihood that the channel non-selectively passes cations. When extracellular Ca2+ was removed, the inward current was significantly smaller and the reversal potential shifted to nearly -47 mV; therefore, Ca2+ may pass through this channel. Ni2+, Gd2+, and SKF-96365 are often effective blockers of store-operated Ca2+ influx, as well as some cation channels. However, the current activated by FCCP was not significantly blocked by any of these. Finally, the addition of FCCP caused a large depolarization (from -60 mV to approximately -20 mV). Afterdischarge-like spiking was observed in about half of the neurons treated with FCCP. This spiking was blocked by 10 mM Ni2+, but the depolarization itself was not.
Conclusions: This channel may be activated either directly by the Ca2+ that is released from mitochondrial stores or by some unknown signal generated by depolarization of the mitochondria. The potential Ca2+ permeability of the conductance makes possible a role in peptide secretion or induction of various Ca2+-dependent events that occur during the afterdischarge. Moreover, this channel could provide depolarizing drive for the afterdischarge itself.
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