Mitochondrial Ca²⁺Activates a Cation Current inAplysiaBag Cell Neurons

Abstract

Ion channels may be gated by Ca(2+) entering from the extracellular space or released from intracellular stores--typically the endoplasmic reticulum. The present study examines how Ca(2+) impacts ion channels in the bag cell neurons of Aplysia californica. These neuroendocrine cells trigger ovulation through an afterdischarge involving Ca(2+) influx from Ca(2+) channels and Ca(2+) release from both the mitochondria and endoplasmic reticulum. Liberating mitochondrial Ca(2+) with the protonophore, carbonyl cyanide-4-trifluoromethoxyphenyl-hydrazone (FCCP), depolarized bag cell neurons, whereas depleting endoplasmic reticulum Ca(2+) with the Ca(2+)-ATPase inhibitor, cyclopiazonic acid, did not. In a concentration-dependent manner, FCCP elicited an inward current associated with an increase in conductance and a linear current/voltage relationship that reversed near -40 mV. The reversal potential was unaffected by changing intracellular Cl(-), but left-shifted when extracellular Ca(2+) was removed and right-shifted when intracellular K(+) was decreased. Strong buffering of intracellular Ca(2+) decreased the current, although the response was not altered by blocking Ca(2+)-dependent proteases. Furthermore, fura imaging demonstrated that FCCP elevated intracellular Ca(2+) with a time course similar to the current itself. Inhibiting either the V-type H(+)-ATPase or the ATP synthetase failed to produce a current, ruling out acidic Ca(2+) stores or disruption of ATP production as mechanisms for the FCCP response. Similarly, any involvement of reactive oxygen species potentially produced by mitochondrial depolarization was mitigated by the fact that dialysis with xanthine/xanthine oxidase did not evoke an inward current. However, both the FCCP-induced current and Ca(2+) elevation were diminished by disabling the mitochondrial permeability transition pore with the alkylating agent, N-ethylmaleimide. The data suggest that mitochondrial Ca(2+) gates a voltage-independent, nonselective cation current with the potential to drive the afterdischarge and contribute to reproduction. Employing Ca(2+) from mitochondria, rather than the more common endoplasmic reticulum, represents a diversification of the mechanisms that influence neuronal activity.

Diacylglycerol‐mediated regulation of Aplysia bag cell neuron excitability requires protein kinase C

“…; Hickey et al . ; White & Magoski, ). There was an ∼200% increase in the current during the step at peak I OAG vs .…”

“…; Hickey et al . ; White & Magoski, ), OAG increases membrane conductance and reverses close to −20 mV, suggesting it too carries cations non‐selectively. In addition, activation of I OAG also causes a marked elevation in [Ca 2+ ] i , punctuated by sharp increases in the fura ratio synchronized with large spikes of I OAG .…”

Ca2+removal by the plasma membrane Ca2+-ATPase influences the contribution of mitochondria to activity-dependent Ca2+dynamics inAplysianeuroendocrine cells

“…Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 21857, Sigma-Aldrich), carboxyeosin (C-22803, Invitrogen), ryanodine (559276, Calbiochem, San Diego, CA), and cyclopiazonic acid (CPA; C1530, Sigma-Aldrich or 239805, Calbiochem) all required DMSO as a vehicle. The maximal final concentration of DMSO was 0.5% (vol/vol), which in control experiments as well as prior work from our laboratory had no effect on membrane potential, various macroscopic or single-channel currents, resting intracellular Ca 2ϩ , or Ca 2ϩ transients evoked by a train of action potentials (Gardam et al 2008;Geiger and Magoski 2008;Hickey et al 2010;Hung and Magoski 2007;Kachoei et al 2006;Lupinsky and Magoski 2006;Tam et al 2009Tam et al , 2011. Tetraphenylphosphonium chloride (TPP; 218790, Sigma-Aldrich) was prepared in water.…”

“…Prior work has established that the mitochondria contribute prominently to both Ca 2ϩ removal and CICR after voltage-gated Ca 2ϩ influx in cultured bag cell neurons (Geiger and Magoski 2008;Groten et al 2013). Consequently, the mitochondria are implicated in sustaining peptide release and membrane excitability during the afterdischarge (Fisher et al 1994;Hickey et al 2010Hickey et al , 2013Michel and Wayne 2002;Wayne et al 1998).…”

Electrical coupling betweenAplysiabag cell neurons: characterization and role in synchronous firing

“…The maximal final concentration of DMSO or ethanol ranged from 0.05 to 0.5% (vol/vol), which in control experiments had no effect on holding current, membrane conductance, or junctional current. Prior work from our laboratory has found that these vehicles do not alter bag cell neuron macroscopic or single-channel currents, membrane potential, membrane capacitance, or intracellular Ca 2ϩ (Magoski et al 2000;Magoski and Kaczmarek 2005;Kachoei et al 2006;Lupinsky and Magoski 2006;Hung and Magoski 2007;Gardam et al 2008;Geiger and Magoski 2008;Tam et al 2009Tam et al , 2011Hickey et al 2010Hickey et al , 2013. We also attempted to employ carbenoxolone (C4790; Sigma-Aldrich) but were unable to dissolve it into nASW from either a DMSO-or ethanol-based stock.…”