Protein kinase C activators block specific calcium and potassium current components in isolated hippocampal neurons (original) (raw)
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The Journal of physiology, 1992
1. Current or voltage clamp recordings from CA3 neurones of the adult rat hippocampal slice were performed to study the inactivation properties of a slow outward K+ current identified as the delayed rectifier (IK). 2. In current clamp experiments, burst firing evoked from resting membrane potential by intracellular current injection was reduced or blocked by conditioning hyperpolarizing pre-pulses of 20-40 mV amplitude. This effect was inhibited by tetraethylammonium (TEA; 20 mM) but was unaffected by Cs+ (3 mM), 4-aminopyridine (4-AP; 2 mM), carbachol (30-50 microM), mast cell degranulating peptide (MCDP; 300 nM), thyrotrophin releasing hormone (TRH; 1 microM) or by a Ca(2+)-free solution containing Mn2+ or Co2+ (2 mM). 3. Single-electrode voltage clamp experiments were carried out on neurones superfused with Ca(2+)-free solution, containing tetrodotoxin (TTX; 1 microM), Mn2+ or Co2+ (2 mM), 4-AP (2 mM), Cs+ (3 mM) and carbachol (30 microM). Step depolarizations from a holding pote...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1990
Despite their widespread use in investigations of protein kinase C (PKC), concern is often expressed regarding the specificity of action of phorbol esters. We have extensively compared the effects of PDBu, a phorbol ester that activates PKC, with those of its inactive analog, 4 alpha-PDBu, on calcium (Ca) channel regulation in acutely isolated guinea pig hippocampal neurons and found that PKC-dependent and -independent actions could be clearly distinguished. While both phorbol esters depressed whole-cell barium current through Ca channels (IBa), PDBu was approximately 100-fold more potent than 4 alpha-PDBu. PKC-independent effects began to appear in the range of 5-10 microM, doses that, while high, have been used in some investigations. Moreover, only PDBu (1) was active when applied intracellularly, (2) had effects that were blocked by the PKC inhibitor H-7, and (3) induced PKC translocation with potency similar to its potency in depressing IBa. The finding that 4 alpha-PDBu acted ...
Hippocampus, 2000
Exploring the principles that govern activity-dependent changes in excitability is an essential step to understand the function of the nervous system, because they act as a general postsynaptic control mechanism that modulates the flow of synaptic signals. We show an activity-dependent potentiation of the slow Ca 2؉ -activated K ؉ current (sI AHP ) which induces sustained decreases in the excitability in CA1 pyramidal neurons. We analyzed the sI AHP using the slice technique and voltage-clamp recordings with sharp or patch-electrodes. Using sharp electrodes-repeated activation with depolarizing pulses evoked a prolonged (8-min) potentiation of the amplitude (171%) and duration (208%) of the sI AHP . Using patch electrodes, early after entering the whole-cell configuration (<20 min), responses were as those reported above. However, although the sI AHP remained unchanged, its potentiation was markedly reduced in later recordings, suggesting that the underlying mechanisms were rapidly eliminated by intracellular dialysis. Inhibition of L-type Ca 2؉ current by nifedipine (20 M) markedly reduced the sI AHP (79%) and its potentiation (55%). Ryanodine (20 M) that blocks the release of intracellular Ca 2؉ also reduced sI AHP (29%) and its potentiation (25%). The potentiation of the sI AHP induced a marked and prolonged (>50%; Ϸ8 min) decrease in excitability. The results suggest that sI AHP is potentiated as a result of an increased intracellular Ca 2؉ concentration ([Ca 2؉ ] i ) following activation of voltage-gated L-type Ca 2؉ channels, aided by the subsequent release of Ca 2؉ from intracellular stores. Another possibility is that repeated activation increases the Ca 2؉ -binding capacity of the channels mediating the sI AHP . This potentiation of the sI AHP could be relevant in hippocampal physiology, because the changes in excitability it causes may regulate the induction threshold of the long-term potentiation of synaptic efficacy. Moreover, the potentiation would act as a protective mechanism by reducing excitability and preventing the accumulation of intracellular Ca 2؉ to toxic levels when intense synaptic activation occurs. Hippocampus 2000;10:198 -206.
Brain Research, 1992
Studies using phorbol esters imply that hippocampal Ca :+ channels are regulated by protein kinase C (PKC); however concerns have bee~ raised because in some circumstances phorbol esters have non-specific effects on ion channels. We have tested the hypothesis that PK(modulates Ca-" ÷ channel activity in hippocampal neurons by conducting a detailed comparison of the effects of the diacylglycerols, diC8 and OAG, with those of the phorbol ester, PDBu, on whole-cell and single-channel Ca-'+ currents. Close similarity of action of these differenl activators would support the hypothesis. We found that, like PDBu, the diacylglycerols (DAGs) suppressed whole-cell Ba '+ current (IBa) in dose-dependent and reversible manner and caused a hyperpolarizing shift in the voltage dependence of steady-state I n:, inactivation. Suppressior of ! n,, by diC8 and OAG was not mimicked by an enzymatically inactive diacylglycerol isomer, EGD. The effects of both PDBu and DAGs coul¢ be blocked by a specific peptide inhibitor of PKC, and both types of activator depressed I Ba when it was recorded in the nystatir perforated-patch mode. In single-channel recordings, DAGs enhanced L-type Ca z÷ channel activity in a manner indistinguishable from that ol PDBu. Finally, DAGs as well as PDBu markedly increased spontaneous synaptic activity in tissue-cultured hippocampal neurons. The numerou: similarities between the effects of DAGs and PDBu strongly support the general conclusion that PKC mediates the effects of these activators ant the specific conclusion that PKC modulates Ca" + channel activity in hippocampal neurons.
Journal of neurophysiology, 1998
Effects of the protein kinase C activating phorbol ester, phorbol 12-myristate 13-acetate (PMA), were studied in whole cell recordings from layer V neurons in slices of mouse somatosensory neocortex. PMA was applied intracellularly (100 nM to 1 microM) to restrict its action to the cell under study. In current-clamp recordings, it enhanced neuronal excitability by inducing a 10- to 20-mV decrease in voltage threshold for action-potential generation. Because spike threshold in neocortical neurons critically depends on the properties of persistent Na+ current (INaP), effects of PMA on this current were studied in voltage clamp. After blocking K+ and Ca2+ currents, INaP was revealed by applying slow depolarizing voltage ramps from -70 to 0 mV. Intracellular PMA induced a decrease in INaP at very depolarized membrane potentials. It also shifted activation of INaP in the hyperpolarizing direction, however, such that there was a significant increase in persistent inward current at potenti...
The effects of protein kinase C activity on synaptic transmission in two areas of rat hippocampus
Brain Research, 2003
The effects of three protein kinase C (PKC) agonists (phorbol ester, ingenol and indolactam-V) and two PKC antagonists (D-erythrosphingosine and chelerythrine) on input-output (I-O) relations in the Schaffer collateral pathway to CA1 (SC-CA1) and mossy fiber pathway to CA3 (MF-CA3) were determined in rat hippocampus brain slices. In the SC-CA1 pathway, phorbol esters and indolactam-V had only small effects on field excitatory post-synaptic potentials (fEPSP) in slices from 60-day animals, although ingenol, an activator of novel PKC isozymes, caused a significant decrease of the field excitatory post-synaptic potentials amplitude in 60-day animals, but not in 30-day animals. In contrast, in the MF-CA3 pathway, PKC agonists induced a significant increase in the field excitatory postsynaptic potentials. PKC antagonists depressed the field excitatory post-synaptic potentials in the SC-CA1 pathway, but had no significant effect in the MF-CA3 pathway. In the MF-CA3 pathway, paired-pulse facilitation was abolished by PKC agonists and unaffected by antagonists. In SC-CA1, it was depressed by agonists to levels below control, whereas it was significantly increased by chelerythine. We conclude that PKC plays important but different roles in both regions. In the SC-CA1 pathway, PKC is almost maximally active under control circumstances, and PKC antagonists significantly reduce synaptic responses. In contrast, in the MF-CA3 pathway, there is no apparent activation under resting circumstances, but significant potentiation of synaptic transmission is induced when PKC is activated. There are developmental changes in the pattern of PKC isozymes, and both pre-and post-synaptic actions are important.
Neuroscience, 2011
In the early neonatal period activation of GABA B receptors attenuates calcium current through Ntype calcium channels while enhancing current through L-type calcium channels in rat hippocampal neurons. The attenuation of N-type calcium current has been previously demonstrated to occur through direct interactions of the βγ subunits of G i/o G-proteins, but the signal transduction pathway for the enhancement of L-type calcium channels in mammalian neurons remains unknown. In the present study, calcium currents were elicited in acute cultures from postnatal day 6-8 rat hippocampi in the presence of various modulators of protein kinase A (PKA) and protein kinase C (PKC) pathways. Overnight treatment with an inhibitor of G i/o (pertussis toxin, 200 ng/ml) abolished the attenuation of calcium current by the GABA B agonist, baclofen (10 μM) with no effect on the enhancement of calcium current. These data indicate that while the attenuation of N-type calcium current is mediated by the G i/o subtype of G-protein, the enhancement of L-type calcium current requires activation of a different G-protein. The enhancement of the sustained component of calcium current by baclofen was blocked by PKC inhibitors, GF-109203X (500 nM), chelerythrine chloride (5 μM), and PKC fragment 19-36 (2 μM) and mimicked by the PKC activator phorbol-12-myristate-13-acetate (1 μM). The enhancement of the sustained component of calcium current was blocked by PKA inhibitors H-89 (1 μM) and PKA fragment 6-22 (500 nM) but not Rp-cAMPS (30 μM) and it was not mimicked by the PKA activator, 8-Br-cAMP (500 μM-1 mM). The data suggest that activation of PKC alone is sufficient to enhance L-type calcium current but that PKA may also be involved in the GABA B receptor mediated effect.