R‐type Ca2+ channels contribute to fast synaptic excitation and action potentials in subsets of myenteric neurons in the guinea pig intestine

Background R‐type Ca²⁺ channels are expressed by myenteric neurons in the guinea pig ileum but the specific function of these channels is unknown. Methods In the present study, we used intracellular electrophysiological techniques to determine the function of R‐type Ca²⁺ channels in myenteric neurons in the acutely isolated longitudinal muscle‐myenteric plexus. We used immunohistochemical methods to localize the Ca_V2.3 subunit of the R‐type Ca²⁺ channel in myenteric neurons. We also studied the effects of the non‐selective Ca²⁺ channel antagonist, CdCl₂ (100 μmol L^?1), the R‐type Ca²⁺ channel blockers NiCl₂ (50 μmol L^?1) and SNX‐482 (0.1 μmol L^?1), and the N‐type Ca²⁺ channel blocker ω‐conotoxin GVIA (CTX 0.1 μmol L^?1) on action potentials and fast and slow excitatory postsynaptic potentials (fEPSPs and sEPSPs) in S and AH neurons in vitro. Key Results Ca_V2.3 co‐localized with calretinin and calbindin in myenteric neurons. NiCl₂ and SNX‐482 reduced the duration and amplitude of action potentials in AH but not S neurons. NiCl₂ inhibited the afterhyperpolarization in AH neurons. ω‐conotoxin GVIA, but not NiCl₂, blocked sEPSPs in AH neurons. NiCl₂ and SNX‐482 inhibited cholinergic, but not cholinergic/purinergic, fEPSPs in S neurons. Conclusions and Inferences These data show that R‐type Ca²⁺ channels contribute to action potentials, but not slow synaptic transmission, in AH neurons. R‐type Ca²⁺ channels contribute to release of acetylcholine as the mediator of fEPSPs in some S neurons. These data indicate that R‐type Ca²⁺ channels may be a target for drugs that selectively modulate activity of AH neurons or could alter fast synaptic excitation in specific pathways in the myenteric plexus.     

Developmental increase in D1‐like dopamine receptor‐mediated inhibition of glutamatergic transmission through P/Q‐type channel regulation in the basal forebrain of rats

Whole‐cell patch‐clamp recordings of non‐N‐methyl‐d ‐aspartate glutamatergic excitatory postsynaptic currents (EPSCs) were carried out from cholinergic neurons in slices of basal forebrain (BF) of developing rats aged 21–42 postnatal days to elucidate postnatal developmental change in Ca²⁺ channel subtypes involved in the transmission as well as that in dopamine D₁‐like receptor‐mediated presynaptic inhibition. The amplitude of EPSCs was inhibited by bath application of ω‐conotoxin GVIA (ω‐CgTX; 3 μm ) or ω‐agatoxin‐TK (ω‐Aga‐TK; 200 nm ) throughout the age range examined, suggesting that multiple types of Ca²⁺ channel are involved in the transmission. The EPSC fraction reduced by ω‐CgTX decreased with age, whereas that reduced by ω‐Aga‐TK increased. Inhibition of the EPSCs by a D₁‐like receptor agonist, SKF 81297 (SKF; 30 μm ) increased with age in parallel with the increase in ω‐Aga‐TK‐induced inhibition. An activator of the adenylyl cyclase (AC) pathway, forskolin (FK; 10 μm ) inhibited the EPSCs, and FK‐induced inhibition also increased with age in parallel with the increase in SKF‐induced inhibition. Throughout the age range examined, SKF showed no further inhibitory effect on the EPSCs after ω‐Aga‐TK‐ or FK‐induced effect had reached steady‐state. These findings suggest that D₁‐like receptor‐mediated presynaptic inhibition of glutamate release onto cholinergic BF neurons increases with age, and that the change is coupled with a developmental increase in the contribution of P/Q‐type Ca²⁺ channels as well as a developmental increase in AC pathway contribution.     

Activation of KCNN3/SK3/KCa2.3 channels attenuates enhanced calcium influx and inflammatory cytokine production in activated microglia

In neurons, small‐conductance calcium‐activated potassium (KCNN/SK/K_Ca2) channels maintain calcium homeostasis after N‐methyl‐D ‐aspartate (NMDA) receptor activation, thereby preventing excitotoxic neuronal death. So far, little is known about the function of KCNN/SK/K_Ca2 channels in non‐neuronal cells, such as microglial cells. In this study, we addressed the question whether KCNN/SK/K_Ca2 channels activation affected inflammatory responses of primary mouse microglial cells upon lipopolysaccharide (LPS) stimulation. We found that N‐cyclohexyl‐N‐[2‐(3,5‐dimethyl‐pyrazol‐1‐yl)‐6‐methyl‐4‐pyrimidinamine (CyPPA), a positive pharmacological activator of KCNN/SK/K_Ca2 channels, significantly reduced LPS‐stimulated activation of microglia in a concentration‐dependent manner. The general KCNN/SK/K_Ca2 channel blocker apamin reverted these effects of CyPPA on microglial proliferation. Since calcium plays a central role in microglial activation, we further addressed whether KCNN/SK/K_Ca2 channel activation affected the changes of intracellular calcium levels, [Ca²⁺]_i,, in microglial cells. Our data show that LPS‐induced elevation of [Ca²⁺]_i was attenuated following activation of KCNN2/3/K_Ca2.2/K_Ca2.3 channels by CyPPA. Furthermore, CyPPA reduced downstream events including tumor necrosis factor alpha and interleukin 6 cytokine production and nitric oxide release in activated microglia. Further, we applied specific peptide inhibitors of the KCNN/SK/K_Ca2 channel subtypes to identify which particular channel subtype mediated the observed anti‐inflammatory effects. Only inhibitory peptides targeting KCNN3/SK3/K_Ca2.3 channels, but not KCNN2/SK2/K_Ca2.2 channel inhibition, reversed the CyPPA‐effects on LPS‐induced microglial proliferation. These findings revealed that KCNN3/SK3/K_Ca2.3 channels can modulate the LPS‐induced inflammatory responses in microglial cells. Thus, KCNN3/SK3/K_Ca2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in the central nervous system. © 2012 Wiley Periodicals, Inc.     

N‐type calcium channels mediate a GABAB presynaptic modulation in the corticostriatal synapse in turtle's paleostriatum augmentatum

Spikes population evoked by a paired pulse protocol were used to assess the influence of GABA_A and GABA_B receptors agonists and antagonists on the synaptic potentials and in the S₂/S₁ ratio in a paired pulse (PP) protocol in the cortico‐paleostriatum augmentatum synapses of the turtle. GABA_A agonist, muscimol, decreased the amplitude of synaptic responses whereas the facilitation produced with the PP protocol did not change, suggesting a postsynaptic action for GABA_A receptors. GABA_B agonist, baclofen, enhanced paired pulse ratio indicating a presynaptic modulation through the GABA_B receptor. Selective antagonists for N‐ and P/Q‐type Ca²⁺‐channels also enhanced paired pulse ratio, suggesting that any of these channel types may be involved in neurotransmitter release. However, the strong paired pulse facilitation produced by baclofen was occluded by blocking the N‐type Ca²⁺ channels with ω‐conotoxin GVIA (1 μM), but not by the blockage of P/Q‐type Ca²⁺ channels with ω‐agatoxin TK (400 nM). These data suggest that N and P/Q channels participate in the neurotransmitter release, whereas only N‐type Ca²⁺ channels are involved in the presynaptic modulation of GABA_B in the corticostriatal synapse of the turtle. Synapse 63:855–862, 2009. © 2009 Wiley‐Liss, Inc.     

Multiple Ca2+ Channel‐Dependent Components in Growth Hormone Secretion from Rat Anterior Pituitary Somatotrophs

The involvement of L‐type Ca²⁺ channels in both ‘basal’ and ‘stimulated’ growth hormone (GH) secretion is well established; however, knowledge regarding the involvement of non‐L‐type Ca²⁺ channels is lacking. We investigated whether non‐L‐type Ca²⁺ channels regulate GH secretion from anterior pituitary (AP) cells. To this end, GH secretion was monitored from dissociated AP cells, which were incubated for 15 min with 2 mm K⁺ (‘basal’ secretion) or 60 mm K⁺ (‘stimulated’ secretion). The role of non‐L‐type Ca²⁺ influx was investigated using specific channel blockers, including ω‐agatoxin‐IVA, ω‐conotoxin GVIA or SNX‐482, to block P/Q‐, N‐ or R‐type Ca²⁺ channels, respectively. Our results demonstrate that P/Q‐, N‐ and R‐type Ca²⁺ channels contributed 21.2 ± 1.9%, 20.2 ± 7.6% and 11.4 ± 1.8%, respectively, to ‘basal’ GH secretion and 18.3 ± 1.0%, 24.4 ± 5.4% and 14.2 ± 4.8%, respectively, to ‘stimulated’ GH secretion. After treatment with a ‘cocktail’ that comprised the previously described non‐L‐type blockers, non‐L‐type Ca²⁺ channels contributed 50.9 ± 0.4% and 45.5 ± 2.0% to ‘basal’ and ‘stimulated’ GH secretion, respectively. Similarly, based on the effects of nifedipine (10 μM), L‐type Ca²⁺ channels contributed 34.2 ± 3.7% and 54.7 ± 4.1% to ‘basal’ and ‘stimulated’ GH secretion, respectively. Interestingly, the relative contributions of L‐type/non‐L‐type Ca²⁺ channels to ‘stimulated’ GH secretion were well correlated with the relative contributions of L‐type/non‐L‐type Ca²⁺ channels to voltage‐gated Ca²⁺ influx in AP cells. Finally, we demonstrated that compartmentalisation of Ca²⁺ channels is important for GH secretion. Lipid raft disruption (methyl‐β‐cyclodextrin, 10 mm ) abrogated the compartmentalisation of Ca²⁺ channels and substantially reduced ‘basal’ and ‘stimulated’ GH secretion by 43.2 ± 3.4% and 58.4 ± 4.0%, respectively. In summary, we have demonstrated that multiple Ca²⁺ channel‐dependent pathways regulate GH secretion. The proper function of these pathways depends on their compartmentalisation within AP cell membranes.     

Seletracetam (ucb 44212) inhibits high-voltage–activated Ca2+ currents and intracellular Ca2+ increase in rat cortical neurons in vitro

Purpose: We analyzed the effects of seletracetam (ucb 44212; SEL), a new antiepileptic drug candidate, in an in vitro model of epileptic activity. The activity of SEL was compared to the effects of levetiracetam (LEV; Keppra), in the same assays. Methods: Combined electrophysiologic and microfluorometric recordings were performed from layer V pyramidal neurons in rat cortical slices to study the effects of SEL on the paroxysmal depolarization shifts (PDSs), and the simultaneous elevations of intracellular Ca²⁺ concentration [Ca²⁺]_i. Moreover, the involvement of high‐voltage activated Ca²⁺ currents (HVACCs) was investigated by means of patch‐clamp recordings from acutely dissociated pyramidal neurons. Results: SEL significantly reduced both the duration of PDSs (IC₅₀ = 241.0 ± 21.7 nm ) as well as the number of action potentials per PDS (IC₅₀ = 82.7 ± 9.7 nm ). In addition, SEL largely decreased the [Ca²⁺]_i rise accompanying PDSs (up to 75% of control values, IC₅₀ = 345.0 ± 15.0 nm ). Furthermore, SEL significantly reduced HVACCs in pyramidal neurons. This effect was mimicked by ω‐conotoxin GVIA and, to a lesser extent, by ω‐conotoxin MVIIC, blockers of N‐ and Q‐type HVACC, respectively. The combination of these two toxins occluded the action of SEL, suggesting that N‐type Ca²⁺ channels, and partly Q‐type subtypes are preferentially targeted. Conclusions: These results demonstrate a powerful inhibitory effect of SEL on epileptiform events in vitro. SEL showed a higher potency than LEV. The effective limitation of [Ca²⁺]_i influx might be relevant for its antiepileptic efficacy and, more broadly, for pathologic processes involving neuronal [Ca²⁺]_i overload.     

Identification of subunits of voltage‐gated calcium channels and actions of pregabalin on intrinsic primary afferent neurons in the guinea‐pig ileum

Background The intrinsic primary afferent neurons (IPANs) in the intestine are the first neurons of intrinsic reflexes. Action potential currents of IPANs flow partly through calcium channels, which could feasibly be targeted by pregabalin. The aim was to determine whether pregabalin‐sensitive α2δ1 subunits associate with calcium channels of IPANs and whether α2δ1 subunit ligands influence IPAN neuronal properties. Methods We used intracellular electrophysiological recording and in situ hybridisation to investigate calcium channel subunit expression in guinea‐pig enteric neurons. Key Results The α subunits of N (α1B) and R (α1E) type calcium channels, and the auxiliary α2δ1 subunit, were expressed by IPANs. This is the first discovery of the α2δ1 subunit in enteric neurons; we therefore investigated its functional role, by determining effects of the α2δ1 subunit ligand, pregabalin, that inhibits currents carried by channels incorporating this subunit. Pregabalin (10 μmol L^?1) reduced the action potential duration. The effect was not increased with increase in concentration to 100 μmol L^?1. If N channels were first blocked by ω‐conotoxin GVIA (0.5 μmol L^?1), pregabalin had no effect on the residual inward calcium current. Reduction of the calcium current by pregabalin substantially inhibited the after‐hyperpolarising potential (AHP) and increased neuron excitability. Conclusion & Inferences Intrinsic primary afferent neurons express functional N (α1B) channel‐forming subunits that are associated with α2δ1 modulatory subunits and are inhibited by pregabalin, plus functional R (α1E) channels that are not sensitive to binding of pregabalin to α2δ subunits. The positive effects of pregabalin in irritable bowel syndrome (IBS) patients might be partly mediated by its effect on enteric neurons.     

Role of TRPM2 cation channels in dorsal root ganglion of rats after experimental spinal cord injury

Introduction: We sought to determine the contribution of oxidative stress–dependent activation of TRPM2 and L‐type voltage‐gated Ca²⁺ channels (VGCC) in dorsal root ganglion (DRG) neurons of rats after spinal cord injury (SCI). Methods: The rats were divided into 4 groups: control; sham control; SCI; and SCI+nimodipine groups. The neurons of the SCI groups were also incubated with non‐specific TRPM2 channel blockers, 2‐aminoethoxydiphenylborate (2‐APB) and N‐(p‐amylcinnamoyl)anthranilic acid (ACA), before H₂O₂ stimulation. Results:The [Ca²⁺]_i concentrations were higher in the SCI group than in the control groups, although their concentrations were decreased by nimodipine and 2‐APB. The H₂O₂‐induced TRPM2 current densities in patch‐clamp experiments were decreased by ACA and 2‐APB incubation. In the nimodipine group, the TRPM2 channels of neurons were not activated by H₂O₂ or cumene hydroperoxide. Conclusions: Increased Ca²⁺ influx and currents in DRG neurons after spinal injury indicated TRPM2 and voltage‐gated Ca²⁺ channel activation. Muscle Nerve 48 : 945–950, 2013     

Nitric oxide as intracellular modulator: internal production of NO increases neuronal excitability via modulation of several ionic conductances

Nitric oxide (NO) has been shown to regulate neuronal excitability in the nervous system, but little is known as to whether NO, which is synthesized in certain neurons, also serves functional roles within NO‐producing neurons themselves. We investigated this possibility by using a nitric oxide synthase (NOS)‐expressing neuron, and studied the role of intrinsic NO production on neuronal firing properties in single‐cell culture. B5 neurons of the pond snail Helisoma trivolvis fire spontaneous action potentials (APs), but once the intrinsic activity of NOS was inhibited, neurons became hyperpolarized and were unable to fire evoked APs. These striking long‐term effects could be attributed to intrinsic NO acting on three types of conductances, a persistent sodium current (I_NaP), voltage‐gated Ca currents (I_Ca) and small‐conductance calcium‐activated potassium (SK) channels. We show that NOS inhibitors 7‐nitroindazole and S‐methyl‐l ‐thiocitrulline resulted in a decrease in I_NaP, and that their hyperpolarizing and inhibiting effects on spontaneous spiking were mimicked by the inhibitor of I_NaP, riluzole. Moreover, inhibition of NOS, soluble guanylate cyclase (sGC) or protein kinase G (PKG) attenuated I_Ca, and blocked spontaneous and depolarization‐induced spiking, suggesting that intrinsic NO controlled I_Ca via the sGC/PKG pathway. The SK channel inhibitor apamin partially prevented the hyperpolarization observed after inhibition of NOS, suggesting a downregulation of SK channels by intrinsic NO. Taken together, we describe a novel mechanism by which neurons utilize their self‐produced NO as an intrinsic modulator of neuronal excitability. In B5 neurons, intrinsic NO production is necessary to maintain spontaneous tonic and evoked spiking activity.     

Microglial K+ channel expression in young adult and aged mice

The K⁺ channel expression pattern of microglia strongly depends on the cells' microenvironment and has been recognized as a sensitive marker of the cells' functional state. While numerous studies have been performed on microglia in vitro, our knowledge about microglial K⁺ channels and their regulation in vivo is limited. Here, we have investigated K⁺ currents of microglia in striatum, neocortex and entorhinal cortex of young adult and aged mice. Although almost all microglial cells exhibited inward rectifier K⁺ currents upon membrane hyperpolarization, their mean current density was significantly enhanced in aged mice compared with that determined in young adult mice. Some microglial cells additionally exhibited outward rectifier K⁺ currents in response to depolarizing voltage pulses. In aged mice, microglial outward rectifier K⁺ current density was significantly larger than in young adult mice due to the increased number of aged microglial cells expressing these channels. Aged dystrophic microglia exhibited outward rectifier K⁺ currents more frequently than aged ramified microglia. The majority of microglial cells expressed functional BK‐type, but not IK‐ or SK‐type, Ca²⁺‐activated K⁺ channels, while no differences were found in their expression levels between microglia of young adult and aged mice. Neither microglial K⁺ channel pattern nor K⁺ channel expression levels differed markedly between the three brain regions investigated. It is concluded that age‐related changes in microglial phenotype are accompanied by changes in the expression of microglial voltage‐activated, but not Ca²⁺‐activated, K⁺ channels. GLIA 2015;63:664–672     

Possible involvement of transient receptor potential ankyrin 1 in Ca2+ signaling via T‐type Ca2+ channel in mouse sensory neurons

T‐type Ca²⁺ channels and TRPA1 are expressed in sensory neurons and both are associated with pain transmission, but their functional interaction is unclear. Here we demonstrate that pharmacological evidence of the functional relation between T‐type Ca²⁺ channels and TRPA1 in mouse sensory neurons. Low concentration of KCl at 15 mM (15K) evoked increases of intracellular Ca²⁺ concentration ([Ca²⁺]_i), which were suppressed by selective T‐type Ca²⁺ channel blockers. RT‐PCR showed that mouse sensory neurons expressed all subtypes of T‐type Ca²⁺ channel. The magnitude of 15K‐induced [Ca²⁺]_i increase was significantly larger in neurons sensitive to allylisothiocyanate (AITC, a TRPA1 agonist) than in those insensitive to it, and in TRPA1^?/? mouse sensory neurons. TRPA1 blockers diminished the [Ca²⁺]_i responses to 15K in neurons sensitive to AITC, but failed to inhibit 40 mM KCl‐induced [Ca²⁺]_i increases even in AITC‐sensitive neurons. TRPV1 blockers did not inhibit the 15K‐induced [Ca²⁺]_i increase regardless of the sensitivity to capsaicin. [Ca²⁺]_i responses to TRPA1 agonist were enhanced by co‐application with 15K. These pharmacological data suggest the possibility of functional interaction between T‐type Ca²⁺ channels and TRPA1 in sensory neurons. Since TRPA1 channel is activated by intracellular Ca²⁺, we hypothesize that Ca²⁺ entered via T‐type Ca²⁺ channel activation may further stimulate TRPA1, resulting in an enhancement of nociceptive signaling. Thus, T‐type Ca²⁺ channel may be a potential target for TRPA1‐related pain.     

Serotonin 5‐HT1B receptor‐mediated calcium influx‐independent presynaptic inhibition of GABA release onto rat basal forebrain cholinergic neurons

Modulatory roles of serotonin (5‐HT) in GABAergic transmission onto basal forebrain cholinergic neurons were investigated, using whole‐cell patch‐clamp technique in the rat brain slices. GABA_A receptor‐mediated inhibitory postsynaptic currents (IPSCs) were evoked by focal stimulation. Bath application of 5‐HT (0.1–300 μm ) reversibly suppressed the amplitude of evoked IPSCs in a concentration‐dependent manner. Application of a 5‐HT_1B receptor agonist, CP93129, also suppressed the evoked IPSCs, whereas a 5‐HT_1A receptor agonist, 8‐OH‐DPAT had little effect on the evoked IPSCs amplitude. In the presence of NAS‐181, a 5‐HT_1B receptor antagonist, 5‐HT‐induced suppression of evoked IPSCs was antagonised, whereas NAN‐190, a 5‐HT_1A receptor antagonist did not antagonise the 5‐HT‐induced suppression of evoked IPSCs. Bath application of 5‐HT reduced the frequency of spontaneous miniature IPSCs without changing their amplitude distribution. The effect of 5‐HT on miniature IPSCs remained unchanged when extracellular Ca²⁺ was replaced by Mg²⁺. The paired‐pulse ratio was increased by CP93129. In the presence of ω‐CgTX, the N‐type Ca²⁺ channel blocker, ω‐Aga‐TK, the P/Q‐type Ca²⁺ channel blocker, or SNX‐482, the R‐type Ca²⁺ channel blocker, 5‐HT could still inhibit the evoked IPSCs. 4‐AP, a K⁺ channel blocker, enhanced the evoked IPSCs, and CP93129 had no longer inhibitory effect in the presence of 4‐AP. CP93129 increased the number of action potentials elicited by depolarising current pulses. These results suggest that activation of presynaptic 5‐HT_1B receptors on the terminals of GABAergic afferents to basal forebrain cholinergic neurons inhibits GABA release in Ca²⁺ influx‐independent manner by modulation of K⁺ channels, leading to enhancement of neuronal activities.     

Neuronal Synchronization and Ionic Mechanisms for Propagation of Excitation in the Functional Network of Immortalized GT1–7 Neurons: Optical Imaging with a Voltage-Sensitive Dye

Immortalized gonadotropin releasing hormone (GnRH) neurons (GT1 cell line) in culture release GnRH in a pulsatile manner, suggesting that GT1 cells form a functional neuronal network. Optical imaging techniques and a voltage-sensitive fluorescent dye (RH795) were used to study the mechanism of neuronal synchronization and intercellular communication in cultured GT1–7 cells (one of the subclones of the GT1 cell line). The majority (79%) of GT1–7 cells in contact with one another revealed synchronized fluctuations in spontaneous neuronal activity. When a cell in contact with other cells was electrically stimulated, the evoked excitation was propagated to neighbouring cells. The ionic mechanisms involved in the propagation of electrical signals between interconnected GT1–7 cells were investigated using various blockers of Na⁺, Ca²⁺ and K⁺ channels. The propagation of stimulus-evoked excitation was prevented by the voltage-dependent Na⁺ channel blocker tetrodotoxin. It was also prevented by the voltage-dependent Ca²⁺ channel blockers, Ni⁺ (nonselective), nimodipine (L-type) and flunarizine (T-type>L-type), but not apparently affected by &ohgr;-agatoxin IVA (P- and Q-type) and &ohgr;-conotoxin MVIIA (N-type). The propagation was not influenced by the K⁺ channel blockers, quinine, tetraethylammonium and Ba²⁺, but in some cases, it was enhanced by 4-aminopyridine (4-AP) and prevented by apamin. These results suggest that voltage-dependent Na⁺ channels and L- and T-type Ca²⁺ channels are involved in the propagation of electrical signals in the GT1–7 neuronal network. Ionic mechanisms, through 4-AP- or apamin-sensitive K⁺ channels, also seem to be involved in the regulation of signal propagation. These mechanisms may underlie the functioning of the neuronal network formed by immortalized GnRH neurons.     

Block of P-type Ca channels in freshly dissociated rat cerebellar Purkinje neurons by diltiazem and verapamil

We investigated the effects of organic Ca²⁺ channel blockers, diltiazem and verapamil, on the high voltage-activated P-type Ca²⁺ channels in freshly isolated rat Purkinje neurons. Both diltiazem and verapamil blocked P-type Ca²⁺ channel current without any change in the current-voltage relation. The block was concentration-dependent. In the presence of these agents, the inactivation curve was shifted to hyperpolarizing potentials. The characteristics of block of P-type Ca²⁺ channels by diltiazem and verapamil are similar to that of L-type Ca²⁺ channels. These results indicate that both benzothiazepine and phenylalkylamine react with P-type Ca²⁺ channels and suggest that some structural features common to which operate in both L-type and P-type Ca²⁺ channels may be involved in drug binding to these channels.     

Calcium influx through N- and P/Q-type channels activate apamin-sensitive calcium-dependent potassium channels generating the late afterhyperpolarization in lamprey spinal neurons

Lamprey spinal neurons exhibit a fast afterhyperpolarization and a late afterhyperpolarization (AHP) which is due to the activation of apamin-sensitive SK Ca²⁺-dependent K⁺ channels (K_Ca) activated by calcium influx through voltage-dependent channels during the action potential ( 1 , Neuroreport, 3, 943–945). In this study we have investigated which calcium channel subtypes are responsible for the activation of the K_Ca channels underlying the AHP. The effects of applying specific calcium channel blockers and agonists were analysed with regard to their effects on the AHP. Blockade of N-type calcium channels by ω-conotoxin GVIA resulted in a significant decrease in the amplitude of the AHP by 76.2 ± 14.9% (mean ± SD). Application of the P/Q-type calcium channel blocker ω-agatoxin IVA reduced the amplitude of the AHP by 20.3 ± 10.4%. The amplitude of the AHP was unchanged during application of the L-type calcium channel antagonist nimodipine or the agonist (±)-BAY K 8644, as was the compound afterhyperpolarization after a train of 10 spikes at 100 Hz. The effects of calcium channel blockers were also tested on the spike frequency adaptation during a train of action potentials induced by a 100–200 ms depolarizing pulse. The N- and P/Q-type calcium channel antagonists decreased the spike frequency adaptation, whereas blockade of L-type channels had no effect. Thus in lamprey spinal cord motor- and interneurons, apamin-sensitive K_Ca channels underlying the AHP are activated primarily by calcium entering through N-type channels, and to a lesser extent through P/Q-type channels.     

The role of Ca2+‐dependent K+‐ channels at the rat corticostriatal synapses revealed by paired pulse stimulation

Potassium channels play an important role in modulating synaptic activity both at presynaptic and postsynaptic levels. We have shown before that presynaptically located K_V and K_IR channels modulate the strength of corticostriatal synapses in rat brain, but the role of other types of potassium channels at these synapses remains largely unknown. Here, we show that calcium‐dependent potassium channels BK‐type but not SK‐type channels are located presynaptically in corticostriatal synapses. We stimulated cortical neurons in rat brain slices and recorded postsynaptic excitatory potentials (EPSP) in medium spiny neurons (MSN) in dorsal neostriatum. By using a paired pulse protocol, we induced synaptic facilitation before applying either BK‐ or SK‐specific toxins. Thus, we found that blockage of BK_Ca with iberiotoxin (10 nM) reduces synaptic facilitation and increases the amplitude of the EPSP, while exposure to SK‐blocker apamin (100 nM) has no effect. Additionally, we induced train action potentials on striatal MSN by current injection before and after the exposure to K_Ca toxins. We found that the action potential becomes broader when the MSN is exposed to iberiotoxin, although it has no impact on frequency. In contrast, exposure to apamin results in loss of afterhyperpolarization phase and an increase of spike frequency. Therefore, we concluded that postsynaptic SK channels are involved in afterhyperpolarization and modulation of spike frequency while the BK channels are involved on the late repolarization phase of the action potential. Altogether, our results show that calcium‐dependent potassium channels modulate both input towards and output from the striatum.     

SK2 channels are neuroprotective for ischemia-induced neuronal cell death

In mouse hippocampal CA1 pyramidal neurons, the activity of synaptic small-conductance Ca²⁺-activated K⁺ channels type 2 (SK2 channels) provides a negative feedback on N-methyl--aspartate receptors (NMDARs), reestablishing Mg²⁺ block that reduces Ca²⁺ influx. The well-established role of NMDARs in ischemia-induced excitotoxicity led us to test the neuroprotective effect of modulating SK2 channel activity following cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Administration of the SK channel positive modulator, 1-ethyl-benzimidazolinone (1-EBIO), significantly reduced CA1 neuron cell death and improved CA/CPR-induced cognitive outcome. Electrophysiological recordings showed that CA/CPR-induced ischemia caused delayed and sustained reduction of synaptic SK channel activity, and immunoelectron microscopy showed that this is associated with internalization of synaptic SK2 channels, which was prevented by 1-EBIO treatment. These results suggest that increasing SK2 channel activity, or preventing ischemia-induced loss of synaptic SK2 channels, are promising and novel approaches to neuroprotection following cerebral ischemia.     

Calcium electrogenesis in neocortical pyramidal neurons in vivo

Much of what is known about Ca²⁺ electrogenesis in neocortical cells has been derived from in vitro studies. Since Ca²⁺ currents are controlled by various modulators, comparing these findings to in vivo data is essential. Here, we analysed tetrodotoxin (TTX)-resistant, presumably Ca²⁺-mediated potentials in intracellularly recorded neocortical neurons in vivo. TTX was applied locally to block Na⁺ channels. Its effectiveness was demonstrated by the elimination of fast spikes and orthodromic responses. In response to depolarizing current pulses bringing the membrane potential beyond ≈–33 mV, 71% of neurons generated high-threshold Ca²⁺ spikes averaging 17 mV. This is in contrast with in vitro findings, where high-threshold spikes could only be elicited following the blockade of K⁺ conductances. Consistent with this, neurons dialysed with K⁺ channel blockers in vivo generated high-threshold spikes that had a lower threshold (≈–40 mV) and, with intracellular Cs⁺, a larger amplitude, indicating the presence of K⁺ currents opposing the activation of Ca²⁺ channels. Only 15% of cortical cells displayed low-threshold Ca²⁺ spikes. To compare high-threshold Ca²⁺ spikes evoked by synaptic stimuli or current injection, another group of cortical neurons was dialysed with QX-314 and Cs⁺, in the absence of extracellular TTX. Synaptic stimuli applied on a background of membrane depolarization elicited presumed Ca²⁺ spikes whose amplitude varied in a stepwise fashion. Thus, although there are numerous similarities between in vivo and in vitro data, some significant differences were found, which suggest that the high-voltage activated Ca²⁺ currents and/or the K⁺ conductances that oppose them are subjected to different modulatory influences in vivo than in vitro.