Channels In Hippocampal Neurons Research Articles

Glucocorticoids Rapidly Modulate CaV1.2-Mediated Calcium Signals through Kv2.1 Channel Clusters in Hippocampal Neurons.

The precise regulation of Ca2+ signals plays a crucial role in the physiological functions of neurons. Here, we investigated the rapid effect of glucocorticoids on Ca2+ signals in cultured hippocampal neurons from both female and male rats. In cultured hippocampal neurons, glucocorticoids inhibited the spontaneous somatic Ca2+ spikes generated by Kv2.1-organized Ca2+ microdomains. Furthermore, glucocorticoids rapidly reduced the cell surface expressions of Kv2.1 and CaV1.2 channels in hippocampal neurons. In HEK293 cells transfected with Kv2.1 alone, glucocorticoids significantly reduced the surface expression of Kv2.1 with little effect on K+ currents. In HEK293 cells transfected with CaV1.2 alone, glucocorticoids inhibited CaV1.2 currents but had no effect on the cell surface expression of CaV1.2. Notably, in the presence of wild-type Kv2.1, glucocorticoids caused a decrease in the surface expression of CaV1.2 channels in HEK293 cells. However, this effect was not observed in the presence of nonclustering Kv2.1S586A mutant channels. Live-cell imaging showed that glucocorticoids rapidly decreased Kv2.1 clusters on the plasma membrane. Correspondingly, Western blot results indicated a significant increase in the cytoplasmic level of Kv2.1, suggesting the endocytosis of Kv2.1 clusters. Glucocorticoids rapidly decreased the intracellular cAMP concentration and the phosphorylation level of PKA in hippocampal neurons. The PKA inhibitor H89 mimicked the effect of glucocorticoids on Kv2.1, while the PKA agonist forskolin abrogated the effect. In conclusion, glucocorticoids rapidly suppress CaV1.2-mediated Ca2+ signals in hippocampal neurons by promoting the endocytosis of Kv2.1 channel clusters through reducing PKA activity.

Benzo[a]pyrene represses synaptic vesicle exocytosis by inhibiting P/Q-type calcium channels in hippocampal neurons

Humans are exposed to the common carcinogen benzo[a]pyrene (BaP) by ingesting contaminated foods and water or inhaling polluted air. Given the enriched lipids and reduced antioxidative properties in the brain and the accumulation of BaP in the brain due to its high lipophilicity, the brain is susceptible to BaP-induced toxicity. Exposure to BaP leads to impairments in learning and memory, increased anxiety behavior, and neuronal death. It induces protein dysfunctions in neuronal compartments that play essential roles in neuronal activity or physiology. However, the neurotoxicity of BaP on presynaptic terminals, which is crucial to neurotransmission by releasing synaptic vesicles that contain neurotransmitters, has not yet been investigated. In the present study, we investigated the toxicity of BaP at presynaptic terminals in living hippocampal neurons. These neurons were sourced from transgenic mice pups (postnatal 1-day, a total of 12 pups, equal numbers for each sex) that endogenously express synaptic vesicle-fused pHluorin, which is a green fluorescent protein that enables monitoring of synaptic vesicle dynamics. We observed that BaP suppressed synaptic vesicle exocytosis by inhibiting presynaptic Ca2+ entry via P/Q-type Ca2+ channels. Together with molecular docking simulation, we speculate that BaP and metabolites may bind to the P/Q Ca2+ channels. These results suggest the toxic mechanism of BaP exposure-induced abnormal behavior that provides a basis to evaluate the risk assessment of BaP-induced neurotoxicity.

Targeting the Nav1.6:GSK3β Protein:Protein Interaction Complex to Mitigate Hippocampal Hyperexcitability in Early‐Stage Alzheimer’s Disease

AbstractBackgroundNav1.6 is the most densely expressed sodium channel isoform in the adult brain and is a primary determinant of action potential initiation and propagation due to its subcellular localization at the axon initial segment (AIS). Importantly, it has been shown that the firing frequency and Na+ currents of Nav 1.6 channels in hippocampal neurons are specifically increased by Aβ exposure in a concentration‐dependent manner. Nav1.6 is regulated through its interactions with key auxiliary proteins, notably Glycogen Synthase Kinase 3‐β (GSK3β), which has increased expression in the hippocampus of AD patients as well as peripheral circulating lymphocytes in both MCI and AD patients. We have shown that GSK3β regulates Nav1.6 channel activity through phosphorylation of and direct binding to the Nav1.6’s intracellular C‐terminal domain (CTD). Furthermore, genetic silencing of GSK3Ββ significantly decreased both Na+ currents and intrinsic firing compared to controls specifically through Nav1.6, while GSK3Ββ over‐expression or pharmacological disinhibition produced opposing phenotypes. Multiple lines of evidence indicate that hippocampal hyperactivity is present in patients with MCI and early‐stage AD and it is observed that reduction of hippocampal hyperactivity in MCI patients improves cognition and memory performance. We hypothesize that small‐molecule modulation of the Nav1.6:GSK3β PPI interface can counteract hippocampal hyperexcitability and could be used to treat early phases of AD, preventing the onset of cognitive decline.MethodHEK293 cells co‐transfected with WT or mutant Nav1.6 and GSK3β were utilized to interrogate the Nav1.6:GSK3β PPI and screen a series of 50 validated hit compounds using split‐luciferase complementation (LCA). Whole‐cell patch‐clamp electrophysiology was performed in heterologous cells and acute slice preparations to validate GSK3β‐mediated regulation of Nav1.6 activity and evaluate lead compound efficacy.ResultMutagenesis screening revealed putative interaction sites of the Nav1.6:GSK3β PPI. Counter‐screening of 50 validated hits revealed 5 compounds that inhibit Nav1.6:GSK3β complex assembly at low micromolar concentrations (Figure 1). Compound 5671063, identified from the counter‐screen, exhibits functional modulation of Nav1.6 activity in heterologous cells (Figure 2).ConclusionThe Nav1.6:GSK3β complex is a critical mediator of neuronal activity and represents a novel target for treatment of pathological hippocampal hyperexcitability associated with cognitive decline.

Increased Calcium Influx through L-Type Calcium Channels in Hippocampal Neurons with Exogenous Expression of Presenilin-1 ΔE9 Mutant.

Mutations in the PSEN1 gene encoding presenilin-1 (PS1) protein are the most common cause of familial Alzheimer's disease. One of these, deletion of exon 9, results in the production of shortened PS1 protein (PS1ΔE9). Neuronal hyperexcitability and hyperactivation of L-type calcium channels were observed in cellular and animal models of familial Alzheimer's disease. However, the effect of PS1ΔE9 on L-type calcium channels has not been studied before. We demonstrate enhanced calcium entry through L-type calcium channels in hippocampal mouse neurons with exogenous expression of PS1ΔE9. Additionally, we show that the same effect of the exogenous PS1ΔE9 can be observed in cells with predominant expression of L-type calcium channels subunit Cav1.2. Further research is required to unravel molecular mechanisms underlying hyperactivation L-type calcium channels caused by PS1ΔE9 expression.

Neuron Class and Target Variability in the Three-Dimensional Localization of SK2 Channels in Hippocampal Neurons as Detected by Immunogold FIB-SEM.

Small-conductance calcium-activated potassium (SK) channels are crucial for learning and memory. However, many aspects of their spatial organization in neurons are still unknown. In this study, we have taken a novel approach to answering these questions combining a pre-embedding immunogold labeling with an automated dual-beam electron microscope that integrates focused ion beam milling and scanning electron microscopy (FIB/SEM) to gather 3D map ultrastructural and biomolecular information simultaneously. Using this new approach, we evaluated the number and variability in the density of extrasynaptic SK2 channels in 3D reconstructions from six dendritic segments of excitatory neurons and six inhibitory neurons present in the stratum radiatum of the CA1 region of the mouse. SK2 immunoparticles were observed throughout the surface of hippocampal neurons, either scattered or clustered, as well as at intracellular sites. Quantitative volumetric evaluations revealed that the extrasynaptic SK2 channel density in spines was seven times higher than in dendritic shafts and thirty-five times higher than in interneurons. Spines showed a heterogeneous population of SK2 expression, some spines having a high SK2 content, others having a low content and others lacking SK2 channels. SK2 immunonegative spines were significantly smaller than those immunopositive. These results show that SK2 channel density differs between excitatory and inhibitory neurons and demonstrates a large variability in the density of SK2 channels in spines. Furthermore, we demonstrated that SK2 expression was associated with excitatory synapses, but not with inhibitory synapses in CA1 pyramidal cells. Consequently, regulation of excitability and synaptic plasticity by SK2 channels is expected to be neuron class- and target-specific. These data show that immunogold FIB/SEM represent a new powerful EM tool to correlate structure and function of ion channels with nanoscale resolution.

Endothelium-derived hydrogen sulfide acts as a hyperpolarizing factor and exerts neuroprotective effects via activation of large-conductance Ca2+ -activated K+ channels.

Endothelium-derived hyperpolarizing factor (EDHF) has been suggested as a therapeutic target for vascular protection against ischaemic brain injury. However, the molecular entity of EDHF and its action on neurons remains unclear. This study was undertaken to demonstrate whether the hydrogen sulfide (H2 S) acts as EDHF and exerts neuroprotective effect via large-conductance Ca2+ -activated K+ (BKCa /KCa 1.1) channels. The whole-cell patch-clamp technology was used to record the changes of BKCa currents in rat neurons induced by EDHF. The cerebral ischaemia/reperfusion model of mice and oxygen-glucose deprivation/reoxygenation (OGD/R) model of neurons were used to explore the neuroprotection of EDHF by activating BKCa channels in these neurons. Increases of BKCa currents and membrane hyperpolarization in hippocampal neurons induced by EDHF could be markedly inhibited by BKCa channel inhibitor iberiotoxin or endothelial H2 S synthase inhibitor propargylglycine. The H2 S donor, NaHS-induced BKCa current and membrane hyperpolarization in neurons were also inhibited by iberiotoxin, suggesting that H2 S acts as EDHF and activates the neuronal BKCa channels. Besides, we found that the protective effect of endothelium-derived H2 S against mice cerebral ischaemia/reperfusion injury was disrupted by iberiotoxin. Importantly, the inhibitory effect of NaHS or BKCa channel opener on OGD/R-induced neuron injury and the increment of intracellular Ca2+ level could be inhibited by iberiotoxin but enhanced by co-application with L-type but not T-type calcium channel inhibitor. Endothelium-derived H2 S acts as EDHF and exerts neuroprotective effects via activating the BKCa channels and then inhibiting the T-type calcium channels in hippocampal neurons.

Identifying mutation hotspots reveals pathogenetic mechanisms of KCNQ2 epileptic encephalopathy

Kv7 channels are enriched at the axonal plasma membrane where their voltage-dependent potassium currents suppress neuronal excitability. Mutations in Kv7.2 and Kv7.3 subunits cause epileptic encephalopathy (EE), yet the underlying pathogenetic mechanism is unclear. Here, we used novel statistical algorithms and structural modeling to identify EE mutation hotspots in key functional domains of Kv7.2 including voltage sensing S4, the pore loop and S6 in the pore domain, and intracellular calmodulin-binding helix B and helix B-C linker. Characterization of selected EE mutations from these hotspots revealed that L203P at S4 induces a large depolarizing shift in voltage dependence of Kv7.2 channels and L268F at the pore decreases their current densities. While L268F severely reduces expression of heteromeric channels in hippocampal neurons without affecting internalization, K552T and R553L mutations at distal helix B decrease calmodulin-binding and axonal enrichment. Importantly, L268F, K552T, and R553L mutations disrupt current potentiation by increasing phosphatidylinositol 4,5-bisphosphate (PIP2), and our molecular dynamics simulation suggests PIP2 interaction with these residues. Together, these findings demonstrate that each EE variant causes a unique combination of defects in Kv7 channel function and neuronal expression, and suggest a critical need for both prediction algorithms and experimental interrogations to understand pathophysiology of Kv7-associated EE.

Activation of Kir2.3 Channels by Tenidap Suppresses Epileptiform Burst Discharges in Cultured Hippocampal Neurons.

Tenidap, a selective human inwardly rectifying potassium (Kir) 2.3 channel opener, has been reported to have antiepileptic effect in the pilocarpine temporal lobe epilepsy rat model in our previous study. However, the effect of tenidap on neurons and its relationship with the epileptiform bursting charges in neuron is still required to be explored. In this study, cyclothiazide (CTZ) induced cultured hippocampal neuron epileptic model was used to study the antiepileptic effect of tenidap and the relationship between Kir2.3 channel and the neuronal epileptiform burst. Patch clamp recording showed that both acute (2h) and chronic (48h) CTZ pre-treatment all significantly induced robust epileptiform burst activities in cultured hippocampal neurons, and tenidap acutely application inhibited this highly synchronized abnormal activities. The effect of tenidap is likely due to increased activity of Kir2.3 channels, since tenidap significantly enhanced kir current recorded from those neurons. In addition, neurons overexpressing Kir2.3 channels, by transfection with Kir2.3 plasmid, showed a significant large increase of the Kir current, prevented CTZ treatment to induce epileptiform burst discharge. Our current study demonstrated that over activation of Kir2.3 channel in hippocampal neurons could positively interference with epileptiform burst activities, and tenidap, as a selective Kir2.3 channel opener, could be a potential candidate for seizure therapy.

Inhibitory effects of fucoidan on NMDA receptors and l-type Ca2+ channels regulating the Ca2+ responses in rat neurons

Context: Fucoidan, a sulphated polysaccharide extracted from brown algae [Fucus vesiculosus Linn. (Fucaceae)], has multiple biological activities.Objective: The effects of fucoidan on Ca2+ responses of rat neurons and its probable mechanisms with focus on glutamate receptors were examined.Materials and methods: The neurons isolated from the cortex and hippocampi of Wistar rats in postnatal day 1 were employed. The intracellular Ca2+ responses triggered by various stimuli were measured in vitro by Fura-2/AM. Fucoidan at 0.5 mg/mL or 1.5 mg/mL was applied for 3 min to determine its effects on Ca2+ responses. RT-PCR was used to determine the mRNA expression of neuron receptors treated with fucoidan at 0.5 mg/mL for 3 h.Results: The Ca2+ responses induced by NMDA were 100% suppressed by fucoidan, and those induced by Bay K8644 90% in the cortical neurons. However, fucoidan has no significant effect on the Ca2+ responses of cortical neurons induced by AMPA or quisqualate. Meanwhile, the Ca2+ responses of hippocampal neurons induced by glutamate, ACPD or adrenaline, showed only a slight decrease following fucoidan treatment. RT-PCR assays of cortical and hippocampal neurons showed that fucoidan treatment significantly decreased the mRNA expression of NMDA-NR1 receptor and the primer pair for l-type Ca2+ channels, PR1/PR2.Discussion and conclusions: Our data indicate that fucoidan suppresses the intracellular Ca2+ responses by selectively inhibiting NMDA receptors in cortical neurons and l-type Ca2+ channels in hippocampal neurons. A wide spectrum of fucoidan binding to cell membrane may be useful for designing a general purpose drug in future.

Inhibitory Signaling to Ion Channels in Hippocampal Neurons Is Differentially Regulated by Alternative Macromolecular Complexes of RGS7.

The neuromodulatory effects of GABA on pyramidal neurons are mediated by GABAB receptors (GABABRs) that signal via a conserved G-protein-coupled pathway. Two prominent effectors regulated by GABABRs include G-protein inwardly rectifying K+ (GIRK) and P/Q/N type voltage-gated Ca2+ (CaV2) ion channels that control excitability and synaptic output of these neurons, respectively. Regulator of G-protein signaling 7 (RGS7) has been shown to control GABAB effects, yet the specificity of its impacts on effector channels and underlying molecular mechanisms is poorly understood. In this study, we show that hippocampal RGS7 forms two distinct complexes with alternative subunit configuration bound to either membrane protein R7BP (RGS7 binding protein) or orphan receptor GPR158. Quantitative biochemical experiments show that both complexes account for targeting nearly the entire pool of RGS7 to the plasma membrane. We analyzed the effect of genetic elimination in mice of both sexes and overexpression of various components of RGS7 complex by patch-clamp electrophysiology in cultured neurons and brain slices. We report that RGS7 prominently regulates GABABR signaling to CaV2, in addition to its known involvement in modulating GIRK. Strikingly, only complexes containing R7BP, but not GPR158, accelerated the kinetics of both GIRK and CaV2 modulation by GABABRs. In contrast, GPR158 overexpression exerted the opposite effect and inhibited RGS7-assisted temporal modulation of GIRK and CaV2 by GABA. Collectively, our data reveal mechanisms by which distinctly composed macromolecular complexes modulate the activity of key ion channels that mediate the inhibitory effects of GABA on hippocampal CA1 pyramidal neurons.SIGNIFICANCE STATEMENT This study identifies the contributions of distinct macromolecular complexes containing a major G-protein regulator to controlling key ion channel function in hippocampal neurons with implications for understanding molecular mechanisms underlying synaptic plasticity, learning, and memory.

T-type calcium channels functionally interact with spectrin (\u03b1/\u03b2) and ankyrin B

This study describes the functional interaction between the Cav3.1 and Cav3.2 T-type calcium channels and cytoskeletal spectrin (α/β) and ankyrin B proteins. The interactions were identified utilizing a proteomic approach to identify proteins that interact with a conserved negatively charged cytosolic region present in the carboxy-terminus of T-type calcium channels. Deletion of this stretch of amino acids decreased binding of Cav3.1 and Cav3.2 calcium channels to spectrin (α/β) and ankyrin B and notably also reduced T-type whole cell current densities in expression systems. Furthermore, fluorescence recovery after photobleaching analysis of mutant channels lacking the proximal C-terminus region revealed reduced recovery of both Cav3.1 and Cav3.2 mutant channels in hippocampal neurons. Knockdown of spectrin α and ankyrin B decreased the density of endogenous Cav3.2 in hippocampal neurons. These findings reveal spectrin (α/β) / ankyrin B cytoskeletal and signaling proteins as key regulators of T-type calcium channels expressed in the nervous system.

Multiple molecular mechanisms form a positive feedback loop driving amyloid \u03b242 peptide-induced neurotoxicity via activation of the TRPM2 channel in hippocampal neurons

Emerging evidence supports an important role for the ROS-sensitive TRPM2 channel in mediating age-related cognitive impairment in Alzheimer’s disease (AD), particularly neurotoxicity resulting from generation of excessive neurotoxic Aβ peptides. Here we examined the elusive mechanisms by which Aβ42 activates the TRPM2 channel to induce neurotoxicity in mouse hippocampal neurons. Aβ42-induced neurotoxicity was ablated by genetic knockout (TRPM2-KO) and attenuated by inhibition of the TRPM2 channel activity or activation through PARP-1. Aβ42-induced neurotoxicity was also inhibited by treatment with TPEN used as a Zn2+-specific chelator. Cell imaging revealed that Aβ42-induced lysosomal dysfunction, cytosolic Zn2+ increase, mitochondrial Zn2+ accumulation, loss of mitochondrial function, and mitochondrial generation of ROS. These effects were suppressed by TRPM2-KO, inhibition of TRPM2 or PARP-1, or treatment with TPEN. Bafilomycin-induced lysosomal dysfunction also resulted in TRPM2-dependent cytosolic Zn2+ increase, mitochondrial Zn2+ accumulation, and mitochondrial generation of ROS, supporting that lysosomal dysfunction and accompanying Zn2+ release trigger mitochondrial Zn2+ accumulation and generation of ROS. Aβ42-induced effects on lysosomal and mitochondrial functions besides neurotoxicity were also suppressed by inhibition of PKC and NOX. Furthermore, Aβ42-induced neurotoxicity was prevented by inhibition of MEK/ERK. Therefore, our study reveals multiple molecular mechanisms, including PKC/NOX-mediated generation of ROS, activation of MEK/ERK and PARP-1, lysosomal dysfunction and Zn2+ release, mitochondrial Zn2+ accumulation, loss of mitochondrial function, and mitochondrial generation of ROS, are critically engaged in forming a positive feedback loop that drives Aβ42-induced activation of the TRPM2 channel and neurotoxicity in hippocampal neurons. These findings shed novel and mechanistic insights into AD pathogenesis.

Somatodendritic surface expression of epitope-tagged and KChIP binding-deficient Kv4.2 channels in hippocampal neurons.

Kv4.2 channels mediate a subthreshold-activating somatodendritic A-type current (ISA) in hippocampal neurons. We examined the role of accessory Kv channel interacting protein (KChIP) binding in somatodendritic surface expression and activity-dependent decrease in the availability of Kv4.2 channels. For this purpose we transfected cultured hippocampal neurons with cDNA coding for Kv4.2 wild-type (wt) or KChIP binding-deficient Kv4.2 mutants. All channels were equipped with an externally accessible hemagglutinin (HA)-tag and an EGFP-tag, which was attached to the C-terminal end. Combined analyses of EGFP self-fluorescence, surface HA immunostaining and patch-clamp recordings demonstrated similar dendritic trafficking and functional surface expression for Kv4.2[wt]HA,EGFP and the KChIP binding-deficient Kv4.2[A14K]HA,EGFP. Coexpression of exogenous KChIP2 augmented the surface expression of Kv4.2[wt]HA,EGFP but not Kv4.2[A14K]HA,EGFP. Notably, activity-dependent decrease in availability was more pronounced in Kv4.2[wt]HA,EGFP + KChIP2 coexpressing than in Kv4.2[A14K]HA,EGFP + KChIP2 coexpressing neurons. Our results do not support the notion that accessory KChIP binding is a prerequisite for dendritic trafficking and functional surface expression of Kv4.2 channels, however, accessory KChIP binding may play a potential role in Kv4.2 modulation during intrinsic plasticity processes.

Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of CaV1.2 calcium channels in hippocampal neurons

L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of CaV1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of CaV1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of CaV1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of CaV1.2 at signaling complexes.

Gating mechanisms of TRPC1 channel in hippocampal neurons.

Gating mechanisms of TRPC1 channel in hippocampal neurons.

Cav 1.3 channels play a crucial role in the formation of paroxysmal depolarization shifts in cultured hippocampal neurons.

An increase of neuronal Cav 1.3 L-type calcium channels (LTCCs) has been observed in various animal models of epilepsy. However, LTCC inhibitors failed in clinical trials of epileptic treatment. There is compelling evidence that paroxysmal depolarization shifts (PDSs) involve Ca2+ influx through LTCCs. PDSs represent a hallmark of epileptiform activity. In recent years, a probable epileptogenic role for PDSs has been proposed. However, the implication of the two neuronal LTCC isoforms, Cav 1.2 and Cav 1.3, in PDSs remained unknown. Moreover, Ca2+ -dependent nonspecific cation (CAN) channels have also been suspected to contribute to PDSs. Nevertheless, direct experimental support of an important role of CAN channel activation in PDS formation is still lacking. Primary neuronal networks derived from dissociated hippocampal neurons were generated from mice expressing a dihydropyridine-insensitive Cav 1.2 mutant (Cav 1.2DHP-/- mice) or from Cav 1.3-/- knockout mice. To investigate the role of Cav 1.2 and Cav 1.3, perforated patch-clamp recordings were made of epileptiform activity, which was elicited using either bicuculline or caffeine. LTCC activity was modulated using the dihydropyridines Bay K 8644 (agonist) and isradipine (antagonist). Distinct PDS could be elicited upon LTCC potentiation in Cav 1.2DHP-/- neurons but not in Cav 1.3-/- neurons. In contrast, when bicuculline led to long-lasting, seizure-like discharge events rather than PDS, these were prolonged in Cav 1.3-/- neurons but not in Cav 1.2DHP-/- neurons. Because only the Cav 1.2 isoform is functionally coupled to CAN channels in primary hippocampal networks, PDS formation does not require CAN channel activity. Our data suggest that the LTCC requirement of PDS relates primarily to Cav 1.3 channels rather than to Cav 1.2 channels and CAN channels in hippocampal neurons. Hence, Cav 1.3 may represent a new therapeutic target for suppression of PDS development. The proposed epileptogenic role of PDSs may allow for a prophylactic rather than the unsuccessful seizure suppressing application of LTCC inhibitors.

Role of TRPC1 ion channel in hippocampal neurons

Event Abstract Back to Event Role of TRPC1 ion channel in hippocampal neurons Sophie Lepannetier1*, Olivier Schakman1, Francois Seghers1 and Philippe Gailly1 1 Université catholique de Louvain, Institute of Neuroscience, Belgium Transient receptor potential canonical 1 (TRPC1) is a non selective cation channel. It is involved in stored-operated Ca2+ entry in cooperation with Orai channel and activated by STIM1, a sensor of Ca2+ contents in the endoplamic reticulum. However, several pieces of evidence suggest that TRPC1 can be activated independently of store depletion in response to agonists. Using LacZ reporter mouse, we found that TRPC1 is abundantly expressed in neurons from CA1-CA3 regions and to a lesser extent from the dentate gyrus region of the hippocampus. We compared cellular culture of hippocampal neurons from Trpc1-/- mice to wild type mice. Interestingly, stimulation with dihydroxyphenylglycine (DHPG), an agonist of group I metabotropic glutamate receptors (consisting of mGluR1 and mGluR5) induced a store-independent entry of Ca2+ that was very much reduced in hippocampal neurons from Trpc1-/- mice. Studying brains slices, we observed that LTP in Schaffer collaterals-CA1 synpases was diminished in Trpc1-/- mice when induced with a mild stimulus (theta burst consisting of 4 stimuli at 100Hz repeated at 5 Hz during 1s). Cognition tests such as Modified Y maze and contextual fear conditioning, realized on 4-8 months old mice revealed a deficit memory. Moreover, we noted that the specific hippocampal overexpression of the immediate early gene zif268 related to new environment learning was blunted in Trpc1-/- mice. We conclude that TRPC1 channels are activated by mGluR5 stimulation and play a role in neuronal plasticity. Keywords: TRPC1, mGluR, Hippocampus, Neuronal Plasticity, Memory Conference: 12th National Congress of the Belgian Society for Neuroscience, Gent, Belgium, 22 May - 22 May, 2017. Presentation Type: Poster Presentation Topic: Neural Excitability, Synapses, and Glia: Cellular Mechanisms Citation: Lepannetier S, Schakman O, Seghers F and Gailly P (2019). Role of TRPC1 ion channel in hippocampal neurons. Front. Neurosci. Conference Abstract: 12th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2017.94.00031 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 Apr 2017; Published Online: 25 Jan 2019. * Correspondence: Mrs. Sophie Lepannetier, Université catholique de Louvain, Institute of Neuroscience, Brussels, 1150, Belgium, slepannetier@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sophie Lepannetier Olivier Schakman Francois Seghers Philippe Gailly Google Sophie Lepannetier Olivier Schakman Francois Seghers Philippe Gailly Google Scholar Sophie Lepannetier Olivier Schakman Francois Seghers Philippe Gailly PubMed Sophie Lepannetier Olivier Schakman Francois Seghers Philippe Gailly Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Determinants of frequency-dependent regulation of Kv1.2-containing potassium channels

ABSTRACTVoltage-gated potassium channels are important regulators of electrical excitation in many tissues, with Kv1.2 standing out as an essential contributor in the CNS. Genetic deletion of Kv1.2 invariably leads to early lethality in mice. In humans, mutations affecting Kv1.2 function are linked to epileptic encephalopathy and movement disorders. We have demonstrated that Kv1.2 is subject to a unique regulatory mechanism in which repetitive stimulation leads to dramatic potentiation of current. In this study, we explore the properties and molecular determinants of this use-dependent potentiation/activation. First, we examine how alterations in duty cycle (depolarization and repolarization/recovery times) affect the onset and extent of use-dependent activation. Also, we use trains of repetitive depolarizations to test the effects of a variety of Thr252 (S2-S3 linker) mutations on use-dependent activation. Substitutions of Thr with some sterically similar amino acids (Ser, Val, and Met, but not Cys) retain use-dependent activation, while bulky or charged amino acid substitutions eliminate use-dependence. Introduction of Thr at the equivalent position in other Kv1 channels (1.1, 1.3, 1.4), was not sufficient to transfer the phenotype. We hypothesize that use-dependent activation of Kv1.2 channels is mediated by an extrinsic regulator that binds preferentially to the channel closed state, with Thr252 being necessary but not sufficient for this interaction to alter channel function. These findings extend the conclusions of our recent demonstration of use-dependent activation of Kv1.2-containing channels in hippocampal neurons, by adding new details about the molecular mechanism underlying this effect.

Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations, but Mediate Identical Transport Functions

ClC-3 is a member of the CLC family of anion channels and transporters, for which multiple functional properties and subcellular localizations have been reported. Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3. We identified three alternatively spliced ClC-3 isoforms, ClC-3a, ClC-3b, and ClC-3c, in mouse brain, with ClC-3c being the predominant splice variant. Whereas ClC-3a and ClC-3b are present in late endosomes/lysosomes, ClC-3c is targeted to recycling endosomes via a novel N-terminal isoleucine-proline (IP) motif. Surface membrane insertion of a fraction of ClC-3c transporters permitted electrophysiological characterization of this splice variant through whole-cell patch clamping on transfected mammalian cells. In contrast, neutralization of the N-terminal dileucine-like motifs was required for functional analysis of ClC-3a and ClC-3b. Heterologous expression of ClC-3a or ClC-3b carrying mutations in N-terminal dileucine motifs as well as WTClC-3c in HEK293T cells resulted in outwardly rectifying Cl(-) currents with significant capacitive current components. We conclude that alternative splicing of Clcn3 results in proteins with different subcellular localizations, but leaves the transport function of the proteins unaffected.

Transient Receptor Potential Vanilloid 4-Induced Modulation of Voltage-Gated Sodium Channels in Hippocampal Neurons.

Transient receptor potential vanilloid 4 (TRPV4) is reported to control the resting membrane potential and increase excitability in many types of cells. Voltage-gated sodium channels (VGSCs) play an important role in initiating action potentials in neurons. However, whether VGSCs can be modulated by the activation of TRPV4 in hippocampal pyramidal neurons remains unknown. In this study, we tested the effect of TRPV4 agonists (GSK1016790A and 4α-PDD) on voltage-gated sodium current (I Na) in hippocampal CA1 pyramidal neurons and the protein levels of α/β-subunit of VGSCs in the hippocampus of mice subjected to intracerebroventricular (icv.) injection of GSK1016790A (GSK-injected mice). Herein, we report that I Na was inhibited by acute application of GSK1016790A or 4α-PDD. In the presence of TRPV4 agonists, the voltage-dependent inactivation curve shifted to the hyperpolarization, whereas the voltage-dependent activation curve remained unchanged. The TRPV4 agonist-induced inhibition of I Na was blocked by the TRPV4 antagonist or tetrodotoxin. Moreover, blocking protein kinase A (PKA) markedly attenuated the GSK1016790A-induced inhibition of I Na, whereas antagonism of protein kinase C or p38 mitogen-activated protein kinase did not change GSK1016790A action. Finally, the protein levels of Nav1.1, Nav1.2, and Nav1.6 in the hippocampus increased in GSK-injected mice, whereas those of Nav1.3 and Navβ1 remained nearly unchanged. We conclude that I Na is inhibited by the acute activation of TRPV4 through PKA signaling pathway in hippocampal pyramidal neurons, but protein expression of α-subunit of VGSCs is increased by sustained TRPV4 activation, which may compensate for the acute inhibition of I Na and provide a possibility for hyper-excitability upon sustained TRPV4 activation.