Edited by Roger J. ColbranSynaptic inhibition depends on a transmembrane gradient of chloride, which is set by the neuron-specific K ؉ -Cl ؊ co-transporter KCC2. Reduced KCC2 levels in the neuronal membrane contribute to the generation of epilepsy, neuropathic pain, and autism spectrum disorders; thus, it is important to characterize the mechanisms regulating KCC2 expression. In the present study, we determined the role of KCC2-protein interactions in regulating total and surface membrane KCC2 expression. Using quantitative immunofluorescence in cultured mouse hippocampal neurons, we discovered that the kainate receptor subunit GluK2 and the auxiliary subunit Neto2 significantly increase the total KCC2 abundance in neurons but that GluK2 exclusively increases the abundance of KCC2 in the surface membrane. Using a live cell imaging assay, we further determined that KCC2 recycling primarily occurs within 1-2 h and that GluK2 produces an ϳ40% increase in the amount of KCC2 recycled to the membrane during this time period. This GluK2-mediated increase in surface recycling translated to a significant increase in KCC2 expression in the surface membrane. Moreover, we found that KCC2 recycling is enhanced by protein kinase C-mediated phosphorylation of the GluK2 C-terminal residues Ser-846 and Ser-868. Lastly, using gramicidin-perforated patch clamp recordings, we found that the GluK2-mediated increase in KCC2 recycling to the surface membrane translates to a hyperpolarization of the reversal potential for GABA (E GABA ). In conclusion, our results have revealed a mechanism by which kainate receptors regulate KCC2 expression in the hippocampus.The classic fast hyperpolarizing inhibition of the mature brain results primarily from the activation of GABA A receptors.These receptors are Cl Ϫ -permeable ion channels, and inhibition results from the influx of Cl Ϫ into the neuron (1). This inward gradient for Cl Ϫ is set by the neuron-specific K ϩ /Cl Ϫ co-transporter KCC2 (2, 3). Despite the requirement of KCC2 for hyperpolarizing inhibition, the mechanisms that regulate KCC2 expression and function are still under intense investigation. In addition to the critical role KCC2 plays in synaptic inhibition, KCC2 is highly localized to excitatory synapses (4, 5), where it plays important roles in the development (6) and the function of glutamatergic synapses (7,8). In fact, single-particle tracking revealed that KCC2 is tightly confined to excitatory synapses (5), which may result from local protein interactions. Thus, understanding how proteins associated with excitatory synapses regulate KCC2 function may provide critical insight to the function of KCC2.KCC2 exists in a multiprotein complex and is regulated by components of excitatory synaptic transmission (9 -11). Specifically, KCC2 interacts with both the kainate-type ionotropic glutamate receptor subunit GluK2 (9) and its auxiliary subunit Neto2 (10). Neto2 regulates KCC2-mediated Cl Ϫ extrusion by binding to the active oligomeric form of KCC2 (10), whereas the GluK2-KCC2 i...
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.
After Ca(2+) influx, mitochondria can sequester Ca(2+) and subsequently release it back into the cytosol. This form of Ca(2+)-induced Ca(2+) release (CICR) prolongs Ca(2+) signaling and can potentially mediate activity-dependent plasticity. As Ca(2+) is required for its subsequent release, Ca(2+) removal systems, like the plasma membrane Ca(2+)-ATPase (PMCA), could impact CICR. Here we examine such a role for the PMCA in the bag cell neurons of Aplysia californica CICR is triggered in these neurons during an afterdischarge and is implicated in sustaining membrane excitability and peptide secretion. Somatic Ca(2+) was measured from fura-PE3-loaded cultured bag cell neurons recorded under whole cell voltage clamp. Voltage-gated Ca(2+) influx was elicited with a 5-Hz, 1-min train, which mimics the fast phase of the afterdischarge. PMCA inhibition with carboxyeosin or extracellular alkalization augmented the effectiveness of Ca(2+) influx in eliciting mitochondrial CICR. A Ca(2+) compartment model recapitulated these findings and indicated that disrupting PMCA-dependent Ca(2+) removal increases CICR by enhancing mitochondrial Ca(2+) loading. Indeed, carboxyeosin augmented train-evoked mitochondrial Ca(2+) uptake. Consistent with their role on Ca(2+) dynamics, cell labeling revealed that the PMCA and mitochondria overlap with Ca(2+) entry sites. Finally, PMCA-dependent Ca(2+) extrusion did not impact endoplasmic reticulum-dependent Ca(2+) removal or release, despite the organelle residing near Ca(2+) entry sites. Our results demonstrate that Ca(2+) removal by the PMCA influences the propensity for stimulus-evoked CICR by adjusting the amount of Ca(2+) available for mitochondrial Ca(2+) uptake. This study highlights a mechanism by which the PMCA could impact activity-dependent plasticity in the bag cell neurons.
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