Hydrogen peroxide regulates a cation channel to control neuroendocrine cell excitability

Abstract

Non-selective cation channels promote neuronal spiking in many animals. For the hermaphroditic marine snail, Aplysia californica, synaptic input to neuroendocrine bag cell neurons triggers various cation channels, causing an ~30-min afterdischarge of action potentials and egg-laying hormone secretion. During the afterdischarge, protein kinase C (PKC) elevates hydrogen peroxide (H2O2), likely by stimulating nicotinamide adenine dinucleotide phosphate oxidase (NOX). Here, I show that in whole-cell voltage-clamped single, cultured bag cell neurons, H2O2 elicited a prolonged, concentration- and voltage-dependent inward current, associated with an increase in membrane conductance, an ~+30 mV reversal potential, and a requirement for extracellular cations. This current was inhibited by cation channel blockers, and boosted or lessened by preventing or enhancing cellular reduction, respectively. Under current-clamp, H2O2 stimulated bursting in single neurons and evoked afterdischarges from bag cell neurons in intact clusters. During the afterdischarge, phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3). Pharmacologically activating PLC, or adding a DAG analogue plus IP3, enhanced both the current as well as the frequency of firing brought about by H2O2. Stimulating PKC with phorbol ester caused both a voltage-dependent current, that reversed at ~+30 mV, and augmented H2O2-induced spiking. Because H2O2 is produced intracellularly, it may gate the cation channel at a cytoplasmic site. Consistent with this, the H2O2-induced current had a relatively slow activation latency and was sensitive to redox modifiers delivered intracellularly but not extracellularly. In addition, intracellular activation of NOX evoked a current that occluded the H2O2 response. Moreover, H2O2 enhanced cation channel activity in excised, inside-out patches, where the intracellular face is exposed. The mitochondria are a potential source of H2O2 as well as being a Ca2+ reservoir. Releasing mitochondrial Ca2+ with a protonophore augmented the H2O2-induced current, and this was prevented by buffering Ca2+ with high intracellular EGTA. Futhermore, the H2O2-induced current was lessened by a mitochondrial-targeted antioxidant. These results suggest that oxidation by H2O2 activates a voltage-dependent cation channel in bag cell neurons. The synergy of H2O2, phosphoinostide metabolites, and mitochondrial Ca2+ in promoting bursting has implications for neuroendocrine function in Aplysia and the wider animal kingdom.