Evidence that Ca/calmodulin-dependent protein kinase mediates the modulation of the Ca2+-dependent K+ current,I AHP,by acetylcholine,but not by glutamate,in hippocampal neurons

Muscarinic and metabotropic glutamate receptor agonists increase the excitability of hippocampal and other cortical neurons by suppressing the Ca²⁺-activated K⁺current,I _AHP, which underlies the slow afterhyperpolarization (AHP) and spike frequency adaptation. We have examined the mechanism of action of a muscarinic agonist (carbachol) and a metabotropic glutamate receptor agonist (1-Aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) onI _AHP in hippocampal CA1 neurons in slices, by using highly specific protein kinase inhibitors. We found that inhibition of protein kinase A (PKA) with the adenosine 3,5-cyclic monophosphate (cAMP) analogue Rp-adenosine-3,5-cyclic phosphorothioate Rp-cAMPS, did not prevent the muscarinic and glutamatergic suppression ofI _AHP. In contrast, two specific peptide inhibitors of Ca²⁺/calmodulin-dependent protein kinase II (CaM-K II), each partially blocked the effect of carbachol, but not the effect of t-ACPD onI _AHP. We conclude that CaM-K II, but not PKA, is involved in mediating the muscarinic suppression ofI _AHP, although other pathways may also contribute. In contrast, neither CaM-K II nor PKA seems to mediate the metabotropic glutamate receptor action onI _AHP.     

Interaction Between α and β-Adrenergic Receptor Agonists Modulating the Slow Ca2+-activated K+ Current - IAHP in Hippocampd Neurons

Noradrenaline inhibits the Ca²⁺-activated K⁺ current I_AHP, which underlies the slow afterhyperpolarization and spike frequency adaptation in hippocampal and neocortical neurons. The resulting increase in excitability probably contributes to the state control of the forebrain during arousal and attention. The modulation of I_AHP by noradrenaline has previously been shown to be mediated by β₁ receptors, cyclic AMP and protein kinase A, but not by α receptors. We have now tested the possibility that a receptors also contribute to I_AHP modulation through interaction with β receptors, by the use of whole-cell recordings in CA1 pyramidal cells of rat hippocampal slices. The α-receptor agonist 6-fluoro-noradrenaline strongly potentiated the effect of isoproterenol on I_AHP. The synergistic effect of 6-fluoro-noradrenaline and soproterenol was blocked by the β-receptor antagonist timolol, but the receptor type mediating the effect of 6-fluoro-noradrenaline could not be unequivocally identified by using α-receptor antagonists. The effect of high concentrations of noradrenaline on I_AHP was only partly blocked by the β-receptor antagonist timolol, and was further reduced by blocking α receptors, again suggesting a contribution from α receptors. In contrast, the effect of low concentrations of noradrenaline seemed to be potentiated by the α-receptor antagonist phentolamine in 57% of the cells, suggesting concentration dependent antagonistic interaction between α and β receptors. Further tests indicated that the cross-talk between 6-fluoro-noradrenaline and isoproterenol occurs upstream from cyclic AMP production, and that protein kinase A serves as a final common path for the modulation of I_AHP by noradrenaline, and by the combination of 6-fluoro-noradrenaline and isoproterenol.     

Inhibitory gating modulation of small conductance Ca2+-activated K+ channels by the synthetic compound (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) reduces afterhyperpolarizing current in hippocampal CA1 neurons

SK channels are small conductance Ca(2+)-activated K(+) channels important for the control of neuronal excitability, the fine tuning of firing patterns, and the regulation of synaptic mechanisms. The classic SK channel pharmacology has largely focused on the peptide apamin, which acts extracellularly by a pore-blocking mechanism. 1-Ethyl-2-benzimidazolinone (1-EBIO) and 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) have been identified as positive gating modulators that increase the apparent Ca(2+) sensitivity of SK channels. In the present study, we describe inhibitory gating modulation as a novel principle for selective inhibition of SK channels. In whole-cell patch-clamp experiments, the compound (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) reversibly inhibited recombinant SK3-mediated currents (human SK3 and rat SK3) with potencies around 100 nM. However, in contrast to known pore blockers, NS8593 did not inhibit (125)I-apamin binding. Using excised patches, it was demonstrated that NS8593 decreased the Ca(2+) sensitivity by shifting the activation curve for Ca(2+) to the right, only slightly affecting the maximal Ca(2+)-activated SK current. NS8593 inhibited all the SK1-3 subtypes Ca(2+)-dependently (K(d) = 0.42, 0.60, and 0.73 microM, respectively, at 0.5 microM Ca(2+)), whereas the compound did not affect the Ca(2+)-activated K(+) channels of intermediate and large conductance (hIK and hBK channels, respectively). The site of action was accessible from both sides of the membrane, and the NS8593-mediated inhibition was prevented in the presence of a high concentration of the positive modulator NS309. NS8593 was further tested on mouse CA1 neurons in hippocampal slices and shown to inhibit the apaminand tubocurarine-sensitive SK-mediated afterhyperpolarizing current, at a concentration of 3 microM.     

An apamin-sensitive Ca2+-activated K+ current in hippocampal pyramidal neurons

In hippocampal and other cortical neurons, action potentials are followed by afterhyperpolarizations (AHPs) generated by the activation of small-conductance Ca2+-activated K+ channels (SK channels). By shaping the neuronal firing pattern, these AHPs contribute to the regulation of excitability and to the encoding function of neurons. Here we report that CA1 pyramidal neurons express an AHP current that is suppressed by apamin and is involved in the control of repetitive firing. This current presents distinct kinetic and pharmacological features, and it is modulated differently than the apamin-insensitive slow AHP current. Furthermore, our in situ hybridizations show that the apamin-sensitive SK subunits are expressed in CA1 pyramidal neurons, providing a potential molecular correlate to the apamin-sensitive AHP current. Altogether, these results clarify the discrepancy between the reported high density of apamin-binding sites in the CA1 region and the apparent lack of an apamin-sensitive current in CA1 pyramidal neurons, and they may explain the effects of this toxin on hippocampal synaptic plasticity and learning.     

Matching molecules to function: neuronal Ca-activated K channels and afterhyperpolarizations

Potassium channels regulate the membrane excitability of neurons, play a major role in shaping action potentials, determining firing patterns and regulating neurotransmitter release, and thus significantly contribute to neuronal signal encoding and integration. This review focuses on the molecular and cellular basis for the specific function of small-conductance calcium-activated potassium channels (SK channels) in the nervous system. SK channels are activated by an intracellular increase of free calcium during action potentials. They mediate currents that modulate the firing frequency of neurons.

Three SK channel subunits have been cloned and form channels, which are voltage-insensitive, activated by submicromolar intracellular calcium concentrations, and are blocked, with different affinities, by a number of toxins and organic compounds. Different neurons in the central and peripheral nervous system express distinct subsets of SK channel subunits. Recent progress has been made in relating cloned SK channels to their native counterparts. These findings argue in favour of regulatory mechanisms conferring to native SK channels with specific subunit compositions distinct and specific functional profiles in different neurons.     

Protein kinase A-independent modulation of ion channels in the brain by cyclic AMP.

Ion channels underlying the electrical activity of neurons can be regulated by neurotransmitters via two basic mechanisms: ligand binding and covalent modification. Whereas neurotransmitters often act by binding directly to ion channels, the intracellular messenger cyclic AMP is thought usually to act indirectly, by activating protein kinase A, which in turn can phosphorylate channel proteins. Here we show that cyclic AMP, and transmitters acting via cyclic AMP, can act in a protein kinase A-independent manner in the brain. In hippocampal pyramidal cells, cyclic AMP and norepinephrine were found to cause a depolarization by enhancing the hyperpolarization-activated mixed cation current, IQ (also called Ih). This effect persisted even after protein kinase A activity was blocked, thus strongly suggesting a kinase-independent action of cyclic AMP. The modulation of this current by ascending monoaminergic fibers from the brainstem is likely to be a widespread mechanism, participating in the state control of the brain during arousal and attention.     

Pituitary adenylate cyclase‐activating polypeptide (PACAP) inhibits the slow afterhyperpolarizing current sIAHP in CA1 pyramidal neurons by activating multiple signaling pathways

The slow afterhyperpolarizing current (sI_AHP) is a calcium‐dependent potassium current that underlies the late phase of spike frequency adaptation in hippocampal and neocortical neurons. sI_AHP is a well‐known target of modulation by several neurotransmitters acting via the cyclic AMP (cAMP) and protein kinase A (PKA)‐dependent pathway. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP) and its receptors are present in the hippocampal formation. In this study we have investigated the effect of PACAP on the sI_AHP and the signal transduction pathway used to modulate intrinsic excitability of hippocampal pyramidal neurons. We show that PACAP inhibits the sI_AHP, resulting in a decrease of spike frequency adaptation, in rat CA1 pyramidal cells. The suppression of sI_AHP by PACAP is mediated by PAC₁ and VPAC₁ receptors. Inhibition of PKA reduced the effect of PACAP on sI_AHP, suggesting that PACAP exerts part of its inhibitory effect on sI_AHP by increasing cAMP and activating PKA. The suppression of sI_AHP by PACAP was also strongly hindered by the inhibition of p38 MAP kinase (p38 MAPK). Concomitant inhibition of PKA and p38 MAPK indicates that these two kinases act in a sequential manner in the same pathway leading to the suppression of sI_AHP. Conversely, protein kinase C is not part of the signal transduction pathway used by PACAP to inhibit sI_AHP in CA1 neurons. Our results show that PACAP enhances the excitability of CA1 pyramidal neurons by inhibiting the sI_AHP through the activation of multiple signaling pathways, most prominently cAMP/PKA and p38 MAPK. Our findings disclose a novel modulatory action of p38 MAPK on intrinsic excitability and the sI_AHP, underscoring the role of this current as a neuromodulatory hub regulated by multiple protein kinases in cortical neurons. © 2013 The Authors. Hippocampus Published by Wiley Periodicals, Inc.     

BK potassium channels control transmitter release at CA3–CA3 synapses in the rat hippocampus

Augmentation is a component of short-term synaptic plasticity with a gradual onset and duration in seconds. To investigate this component at the corticogeniculate synapse, whole cell patch-clamp recordings were obtained from principal cells in a slice preparation of the rat dorsal lateral geniculate nucleus. Trains with 10 stimuli at 25 Hz evoked excitatory postsynaptic currents (EPSCs) that grew in amplitude, primarily from facilitation. Such trains also induced augmentation that decayed exponentially with a time constant τ= 4.6 ± 2.6 s (mean ± standard deviation). When the trains were repeated at 1–10 s intervals, augmentation markedly increased the size of the first EPSCs, leaving late EPSCs unaffected. The magnitude of augmentation was dependent on the number of pulses, pulse rate and intervals between trains. Augmented EPSCs changed proportionally to basal EPSC amplitudes following alterations in extracellular calcium ion concentration. The results indicate that augmentation is determined by residual calcium remaining in the presynaptic terminal after repetitive spikes, competing with fast facilitation. We propose that augmentation serves to maintain a high synaptic strength in the corticogeniculate positive feedback system during attentive visual exploration.