Hydrogen Peroxide Gates a Voltage-Dependent Cation Current inAplysiaNeuroendocrine Cells

Abstract

Nonselective cation channels promote persistent spiking in many neurons from a diversity of animals. In the hermaphroditic marinesnail, Aplysia californica, synaptic input to the neuroendocrine bag cell neurons triggers various cation channels, causing an ϳ30 min afterdischarge of action potentials and the secretion of egg-laying hormone. During the afterdischarge, protein kinase C is also activated, which in turn elevates hydrogen peroxide (H 2 O 2), likely by stimulating nicotinamide adenine dinucleotide phosphate oxidase. The present study investigated whether H 2 O 2 regulates cation channels to drive the afterdischarge. In single, cultured bag cell neurons, H 2 O 2 elicited a prolonged, concentration-and voltage-dependent inward current, associated with an increase in membrane conductance and a reversal potential of ϳϩ30 mV. Compared with normal saline, the presence of Ca 2ϩ-free, Na ϩ-free, or Na ϩ /Ca 2ϩ-free extracellular saline, lowered the current amplitude and left-shifted the reversal potential, consistent with a nonselective cationic conductance. Preventing H 2 O 2 reduction with the glutathione peroxidase inhibitor, mercaptosuccinate, enhanced the H 2 O 2-induced current, while boosting glutathione production with its precursor, N-acetylcysteine, or adding the reducing agent, dithiothreitol, lessened the response. Moreover, the current generated by the alkylating agent, N-ethylmaleimide, occluded the effect of H 2 O 2. The H 2 O 2-induced current was inhibited by tetrodotoxin as well as the cation channel blockers, 9-phenanthrol and clotrimazole. In current-clamp, H 2 O 2 stimulated burst firing, but this was attenuated or prevented altogether by the channel blockers. Finally, H 2 O 2 evoked an afterdischarge from whole bag cell neuron clusters recorded ex vivo by sharp-electrode. H 2 O 2 may regulate a cation channel to influence long-term changes in activity and ultimately reproduction.

Hydrogen peroxide and phosphoinositide metabolites synergistically regulate a cation current to influence neuroendocrine cell bursting

“…In keeping with gating a cation current, H 2 O 2 elicited prolonged action potential firing from whole‐cell current‐clamped cultured bag cell neurons bathed in nASW with K + ‐Asp‐based intracellular saline, that is, physiological conditions. We verified our past report (Chauhan & Magoski, 2019) that bath‐application of 1 mM H 2 O 2 to a neuron set to a resting potential of −40 mV, the average membrane potential during the afterdischarge, caused an ∼9 mV depolarization and consistently generated an afterdischarge‐like burst of spikes ( n = 10) (Fig. 1 and ).…”

“…In addition, we demonstrated that OAG enhances the activity of a different, voltage‐ dependent cation channel in cell‐free patches (Sturgeon & Magoski, 2018). Our prior work also indicates that the H 2 O 2 ‐induced macroscopic current is biophysically and pharmacologically the same as the voltage‐dependent cation conductance observed at the single‐channel level (Chauhan & Magoski, 2019). Thus, we tested whether the H 2 O 2 ‐induced cation current is similarly augmented by phosphoinositide metabolites in cultured bag cell neurons.…”

“…Our prior work showed that H 2 O 2 activates a non‐selective cation channel to elicit a long‐lasting voltage‐dependent cationic current in bag cell neurons (Chauhan & Magoski, 2019). This was confirmed here using single cultured bag cell neurons and whole‐cell voltage‐clamp (see Methods, ‘Whole‐cell voltage‐clamp and current‐clamp recording from cultured bag cell neurons’ for details).…”

“…Neurons were bathed in Na + ‐containing nASW (used throughout) and dialysed with Cs + ‐Asp‐based intracellular saline (to block K + channels as well as improve current resolution and stability) in the pipette for 15 min while holding at −60 or −30 mV. As per our earlier findings (Chauhan & Magoski, 2019), the voltage‐dependence of the H 2 O 2 ‐induced response resulted in minimal current at −60 mV (∼−50 pA; n = 6) and a significantly larger current at −30 mV (∼−120 pA; n = 9) (Fig. 1 and ); the latter holding potential was chosen for all subsequent voltage‐clamp experiments.…”

“…2009). We previously found that H 2 O 2 triggers this channel to evoke a voltage‐dependent inward current capable of eliciting afterdischarge‐like responses in cultured bag cell neurons or whole clusters (Chauhan & Magoski, 2019). Prior single‐channel recordings in excised, inside‐out patches showed that the cation channel is enhanced by exogenous DAG plus IP 3 , or direct activation of endogenous PLC (Sturgeon & Magoski, 2018).…”