Calcium influx and release controls neuroendocrine cell secretion and excitability
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Ca2+ dynamics affect many critical cellular processes. In the bag cell neurons of Aplysia californica, intracellular Ca2+ is elevated during a prolonged period of firing known as the afterdischarge. This consists of a fast and slow phase of firing, which triggers peptide secretion and culminates in egg-laying. The present study examines how Ca2+ influx and release shape neurosecretion and membrane activity. Using capacitance tracking as an index of secretion, a 5 Hz, 1 min train, to mimic the fast phase, induced a clear elevation in the membrane surface area of cultured bag cell neurons. The capacitance change was abolished by replacing external Ca2+ with Ba2+ or addition of the Ca2+ channel blocker, Ni2+. Additionally, the response was reduced by either strong buffering of intracellular Ca2+ or pretreatment with N-ethylmaleimide, an alkylating agent that disrupts vesicular transport. Depleting mitochondrial Ca2+ with the protonophore, carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP), also elevated capacitance, while depleting endoplasmic reticulum Ca2+ with the Ca2+-ATPase inhibitor, cyclopiazonic acid, did not. Similarly, FCCP alone depolarized bag cell neurons. In a concentration-dependent manner, FCCP elicited an inward current that was insensitive to Ni2+, associated with an increase in conductance, and a linear current/voltage relationship that reversed around -40 mV. Removal of extracellular Ca2+ reduced the current and left-shifted the reversal, consistent with opening a Ca2+-permeable, voltage-independent, non-selective cation channel. The current was decreased when intracellular Ca2+ was strongly buffered, while fura-imaging demonstrated that FCCP elevated intracellular Ca2+ with a similar time course, suggesting a dependence on intracellular Ca2+. Although both oligomycin A and bafilomycin A, inhibitors of mitochondrial ATP sythetase and V-type H+-ATPase, respectively, gradually increased Ca2+, neither produced a current. The FCCP-induced Ca2+ elevation and the current were also diminished by disabling the mitochondrial permeability transition pore with N-ethylmaleimide. The data suggests that a cation current is preferentially gated by Ca2+ released from the mitochondria, rather than disruption of ATP production. This current could provide depolarizing drive for the afterdischarge. While Ca2+ entry appears to be responsible for initiating neurosecretion, mitochondrial Ca2+ may support prolonged peptide release during and subsequent to the afterdischarge.