Calcium electrogenesis in distal apical dendrites of layer 5 pyramidal cells at a critical frequency of back-propagating action potentials

Action potentials in juvenile and adult rat layer-5 neocortical pyramidal neurons can be initiated at both axonal and distal sites of the apical dendrite. However, little is known about the interaction between these two initiation sites. Here, we report that layer 5 pyramidal neurons are very sensitive to a critical frequency of back-propagating action potentials varying between 60 and 200 Hz in different neurons. Bursts of four to five back-propagating action potentials above the critical frequency elicited large regenerative potentials in the distal dendritic initiation zone. The critical frequency had a very narrow range (10–20 Hz), and the dendritic regenerative activity led to further depolarization at the soma. The dendritic frequency sensitivity was suppressed by blockers of voltage-gated calcium channels, and also by synaptically mediated inhibition. Calcium-fluorescence imaging revealed that the site of largest transient increase in intracellular calcium above the critical frequency was located 400–700 μm from the soma at the site for initiation of calcium action potentials. Thus, the distal dendritic initiation zone can interact with the axonal initiation zone, even when inputs to the neuron are restricted to regions close to the soma, if the output of the neuron exceeds a critical frequency.

A structural motif for the voltage-gated potassium channel pore.

Mutation studies have identified a region of the S5-S6 loop of voltage-gated K+ channels (P region) responsible for teraethylammonium (TEA) block and permeation/selectivity properties. We previously modeled a similar region of the Na+ channel as four beta-hairpins with the C strands from each of the domains forming the external vestibule and with charged residues at the beta-turns forming the selectivity filter. However, the K+ channel P region amino acid composition is much more hydrophobic in this area. Here we propose a structural motif for the K+ channel pore based on the following postulates (Kv2.1 numbering). (i) The external TEA binding site is formed by four Tyr-380 residues; P loop residues participating in the internal TEA binding site are four Met-371 and Thr-372 residues. (ii) P regions form extended hairpins with beta-turns in sequence ITMT. (iii) only C ends of hairpins form the inner walls of the pore. (iv) They are extended nonregular strands with backbone carbonyl oxygens of segment VGYGD facing the pore with the conformation BRLRL. (v) Juxtaposition of P loops of the four subunits forms the pore. Fitting the external and internal TEA sites to TEA molecules predicts an hourglass-like pore with the narrowest point (GYG) as wide as 5.5 A, suggesting that selectivity may be achieved by interactions of carbonyls with partially hydrated K+. Other potential cation binding sites also exist in the pore.

Characterization of a voltage-gated K+ channel beta subunit expressed in human heart.

Voltage-gated K+ channels are important modulators of the cardiac action potential. However, the correlation of endogenous myocyte currents with K+ channels cloned from human heart is complicated by the possibility that heterotetrameric alpha-subunit combinations and function-altering beta subunits exist in native tissue. Therefore, a variety of subunit interactions may generate cardiac K+ channel diversity. We report here the cloning of a voltage-gated K+ channel beta subunit, hKv beta 3, from adult human left ventricle that shows 84% and 74% amino acid sequence identity with the previously cloned rat Kv beta 1 and Kv beta 2 subunits, respectively. Together these three Kv beta subunits share > 82% identity in the carboxyl-terminal 329 aa and show low identity in the amino-terminal 79 aa. RNA analysis indicated that hKv beta 3 message is 2-fold more abundant in human ventricle than in atrium and is expressed in both healthy and diseased human hearts. Coinjection of hKv beta 3 with a human cardiac delayed rectifier, hKv1.5, in Xenopus oocytes increased inactivation, induced an 18-mV hyperpolarizing shift in the activation curve, and slowed deactivation (tau = 8.0 msec vs. 35.4 msec at -50 mV). hKv beta 3 was localized to human chromosome 3 by using a human/rodent cell hybrid mapping panel. These data confirm the presence of functionally important K+ channel beta subunits in human heart and indicate that beta-subunit composition must be accounted for when comparing cloned channels with endogenous cardiac currents.

Structures of mu O-conotoxins from Conus marmoreus - Inhibitors of tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels in mammalian sensory neurons

The muO-conotoxins are an intriguing class of conotoxins targeting various voltage-dependent sodium channels and molluscan calcium channels. In the current study, we have shown MrVIA and MrVIB to be the first known peptidic inhibitors of the transient tetrodotoxin-resistant (TTX-R) Na+ current in rat dorsal root ganglion neurons, in addition to inhibiting tetrodotoxin-sensitive Na+ currents. Human TTX-R sodium channels are a therapeutic target for indications such as pain, highlighting the importance of the muO-conotoxins as potential leads for drug development. Furthermore, we have used NMR spectroscopy to provide the first structural information on this class of conotoxins. MrVIA and MrVIB are hydrophobic peptides that aggregate in aqueous solution but were solubilized in 50% acetonitrile/water. The three-dimensional structure of MrVIB consists of a small beta-sheet and a cystine knot arrangement of the three-disulfide bonds. It contains four backbone loops between successive cysteine residues that are exposed to the solvent to varying degrees. The largest of these, loop 2, is the most disordered part of the molecule, most likely due to flexibility in solution. This disorder is the most striking difference between the structures of MrVIB and the known delta- and omega-conotoxins, which along with the muO-conotoxins are members of the O superfamily. Loop 2 of omega-conotoxins has previously been shown to contain residues critical for binding to voltage-gated calcium channels, and it is interesting to speculate that the flexibility observed in MrVIB may accommodate binding to both sodium and molluscan calcium channels.

Ciguatoxins: Cyclic polyether modulators of voltage-gated Iion channel function

Ciguatoxins are cyclic polyether toxins, derived from marine dinoflagellates, which are responsible for the symptoms of ciguatera poisoning. Ingestion of tropical and subtropical fin fish contaminated by ciguatoxins results in an illness characterised by neurological, cardiovascular and gastrointestinal disorders. The pharmacology of ciguatoxins is characterised by their ability to cause persistent activation of voltage-gated sodium channels, to increase neuronal excitability and neurotransmitter release, to impair synaptic vesicle recycling, and to cause cell swelling. It is these effects, in combination with an action to block voltage-gated potassium channels at high doses, which are believed to underlie the complex of symptoms associated with ciguatera. This review examines the sources, structures and pharmacology of ciguatoxins. In particular, attention is placed on their cellular modes of actions to modulate voltage-gated ion channels and other Na+-dependent mechanisms in numerous cell types and to current approaches for detection and treatment of ciguatera.

Neuronal voltage-gated sodium channel subtypes: Key roles in inflammatory and neuropathic pain

Voltage-gated sodium channels (VGSCs) play an important role in neuronal excitability. Regulation of VGSC activity is a complex phenomenon that occurs at multiple levels in the cell, including transcriptional regulation, post-translational modification and membrane insertion and retrieval. Multiple VGSC subtypes exist that vary in their biophysical and pharmacological properties and tissue distribution. Any alteration of the VGSC subtype profile of a neuron or the mechanisms that regulate VGSC activity can cause significant changes in neuronal excitability. Inflammatory and neuropathic pain states are characterised by alterations in VGSC subtype composition and activity in sensory neurons. This review focuses on the VGSC subtypes involved in such pain states. (c) 2006 Elsevier Ltd. All rights reserved.

Renal- and calcium-dependent vascular effects of Polybia paulista wasp venom

In the present study, the effects of Polybia paulista venom (PPV) on renal and vascular tissues were investigated. Isolated kidneys perfused with PPV (1 and 3 mu g/mL) had increased perfusion pressure, renal vascular resistance, urinary flow, and glomerular filtration rate; and reduced sodium tubular transport. Histological evaluation demonstrated deposits of proteins in Bowman's space and tubular lumen, and focal areas of necrosis. The venom promoted a cytotoxic effect on Madin-Darby canine kidney (MDCK) cells. A significant increase in lactic dehydrogenase levels was observed in response to venom exposure. In isolated mesenteric vascular beds, pressure and vascular resistance augmented in a dose-dependent manner. PPV increased the contractility of aortic rings maintained under basal tension. This contractile response was inhibited when preparations were maintained in Ca2+-free medium. Likewise, verapamil, a voltage-gated calcium channel blocker, also inhibited the contractile response. In this study, phentolamine, a blocker of a-adrenergic receptor blocker, significantly reduced the contractile effect of PPV in the aortic ring. In conclusion, PPV produced nephrotoxicity, which suggests a direct effect on necrotic cellular death in renal tubule cells. The vascular contractile effect of PPV appears to involve calcium influx through voltage-gated calcium channels via adrenergic regulation.

Functional significance of the beta-hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a

omega -Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic specificity derives from its ability to antagonize insect, but not vertebrate, voltage-gated calcium channels. In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions. First, we constructed a Hairpinless mutant in which the C-terminal beta -hairpin, which is highly conserved in this family of neurotoxins, was excised without affecting the fold of the residual disulfide-rich core of the toxin. The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin. We subsequently developed a highly efficient system for production of recombinant toxin and then probed the hairpin for key functional residues using alanine-scanning mutagenesis followed by a second round of mutagenesis based on initial hits from the alanine scan. This revealed that two spatially proximal residues, Asn(27) and Arg(35), form a contiguous molecular surface that is essential for toxin activity. We propose that this surface of the beta -hairpin is a key site for interaction of the toxin with insect calcium channels.

Transmisión sináptica-canales de calcio y liberación de neurotransmisore

La comunicación neuronal en el sistema nervioso está mediada, en la gran mayoría de animales, por la transmisión sináptica química. Generalmente esta comunicación ocurre mediante la liberación de una sustancia transmisora en el terminal presináptico. Este transmisor sináptico se une a receptores postsinápticos y da lugar a una respuesta postsináptica en la célula blanco. La liberación del transmisor en la región presináptica, al parecer, es desencadenada por un aumento transitorio del calcio intracelular en el sitio de liberación. Este aumento se logra, principalmente, por la activación de canales de calcio dependientes de voltaje (VGCC), lo que da lugar a un ingreso de iones de calcio en el citosol presináptico, que desencadena la fusión de las vesículas sinápticas y la liberación del neurotransmisor. En este artículo se revisan las características moleculares y funcionales de los VGCC necesarias para la comprensión de alteraciones patológicas como las canalopatías y la transmisión sináptica anormal. Metodología: se consultaron las bases de datos Medline, Pubmed y los e-journals de la Biblioteca de la Universidad de Columbia, correspondientes a los años 1990 a 2004. Resultados: durante la última década se han logrado avances significativos en los aspectos moleculares y en la genética de los canales dependientes de voltaje. La integración de este conocimiento con la neurofisiología funcional y la neurología clínica apenas se está iniciando.

Signalling mechanisms affecting regulated neurotrophin secretion in hippocampal neurons

Die Neurotrophine aus Säugetiere BDNF und NT-3 sind von Neuronen sekretierte Wachstumsfaktoren. Ferner sind Neurotrophine in verschiedene Formen der aktivitätsabhängigen synaptische Plastizität involviert. Obwohl die Ausschüttung von Neurotrophine aus Synapsen beschrieben worden ist, sind die intrazellulären Signalkaskaden, die die synaptische Ausschüttung von Neurotrophine regulieren, bei weitem nicht verstanden. Deswegen ist die Analyse der Sekretion von Neurotrophine auf subzellulärer Ebene erforderlich, um die genaue Rolle von präsynaptische und postsynaptische NT-Sekretion in der synaptischen Plastizität aufzudecken. In der vorliegenden Arbeit wurden die Kulturen von dissoziierten hippocampalen Neuronen aus Ratten mit grün fluoreszierenden Protein-markierten Konstrukten von BDNF und NT-3 transfiziert und Neurotrophine-enthaltenden Vesikeln durch die Colokalisierung mit dem cotransfizierten postsynaptischen Marker PSD-95-DsRed an glutamatergen Synapsen identifiziert. Depolarisationsinduzierte Sekretion von BDNF und NT-3 wurde per Direktaufnahme am Fluoreszenzmikroskop beobachtet. Die unvermittelte postsynaptische Depolarisation mit erhöhtem Kalium, in Gegenwart von Inhibitoren der synaptischen Transmission, erlaubte die Untersuchung der Signalwege, die am postsynaptischen Sekretionsprozess der Neurotrophinvesikel beteiligt sind. Es konnte gezeigt werden, dass die depolarisationsinduzierte postsynaptische Ausschüttung der Neurotrophine durch Calcium-Einstrom ausgelöst wird, entweder über L-Typ-spannungsabhängige Calcium-Kanäle oder über NMDA-Rezeptoren. Eine anschließende Freisetzung von Calcium aus intrazellulären Speichern über Ryanodin-Rezeptoren ist für den Sekretionsprozess erforderlich. Die postsynaptische Neurotrophinausschüttung wird durch KN-62 und KN-93 gehemmt, was auf eine unmittelbare Abhängigkeit von aktiver alpha-Calcium-Calmodulin-abhängige Proteinkinase II (CaMKII) hinweist. Der Inhibitor der cAMP/Proteinkinase A (PKA), Rp-cAMP-S, sowie der NO-Donor, SNP, minderten die Neurotrophinausschüttung. Hingegen blieben die Erhöhung des intrazellulären cAMP und der NO-Synthase-Inhibitor L-NMMA ohne Wirkung. Mit dem Trk-Inhibitor K252a konnte gezeigt werden, dass autokrine Neurotrophin-induzierte Neurotrophinausschüttung nicht an der synaptischen Freisetzung der Neurotrophine beiträgt und, dass BDNF seine eigene postsynaptische Sekretion nicht auslöst. Freisetzungsexperimente mit dem Fluoreszenz-Quencher Bromphenolblau konnten den Nachweis erbringen, dass asynchrone und anhaltende Fusionsporenöffnung von Neurotrophinvesikeln während der Sekretion stattfindet. Wegen der im Vergleich zum komplexen Sekretionsprozess schnellen Fusionsporenöffnung, scheint die Freisetzungsgeschwindigkeit von Neurotrophine durch ihre Diffusion aus dem Vesikel begrenzt. Zusammenfassend zeigen diese Ergebnisse eine starke Abhängigkeit der aktivitätsabhängigen postsynaptischen Neurotrophinausschüttung vom Calcium-Einstrom, von der Freisetzung von Calcium aus internen Speichern, von der Aktivierung der CaMKII und einem intakten Funktion der PKA, während der Trk-Signalweg, die Aktivierung von Natrium-Kanäle und NO-Signale nicht erforderlich sind.

Modulação da diferenciação neural de células troco embrionárias por transientes de cálcio intracelulares: papéis dos receptores purinérgicos e de canais de cálcio voltagem-dependentes

Receptores purinérgicos e canais de cálcio voltagem-dependentes estão envolvidos em diversos processos biológicos como na gastrulação, durante o desenvolvimento embrionário, e na diferenciação neural. Quando ativados, canais de cálcio voltagem-dependentes e receptores purinérgicos do tipo P2, ativados por nucleotídeos, desencadeiam transientes de cálcio intracelulares controlando diversos processos biológicos. Neste trabalho, nós estudamos a participação de canais de cálcio voltagem-dependentes e receptores do tipo P2 na geração de transientes de cálcio espontâneos e sua regulação na expressão de fatores de transcrição relacionados com a neurogênese utilizando como modelo células tronco (CTE) induzidas à diferenciação em células tronco neurais (NSC) com ácido retinóico. Descrevemos que CTE indiferenciadas podem ter a proliferação acelerada pela ativação de receptores P2X7, enquanto que a expressão e a atividade desse receptor precisam ser inibidas para o progresso da diferenciação em neuroblasto. Além disso, ao longo da diferenciação neural, por análise em tempo real dos níveis de cálcio intracelular livre identificamos 3 padrões de oscilações espontâneas de cálcio (onda, pico e unique), e mostramos que ondas e picos tiveram a frequência e amplitude aumentadas conforme o andamento da diferenciação. Células tratadas com o inibidor do receptor de inositol 1,4,5-trifosfato (IP3R), Xestospongin C, apresentaram picos mas não ondas, indicando que ondas dependem exclusivamente de cálcio oriundo do retículo endoplasmático pela ativação de IP3R. NSC de telencéfalo de embrião de camundongos transgênicos ou pré-diferenciadas de CTE tratadas com Bz-ATP, o agonista do receptor P2X7, e com 2SUTP, agonista de P2Y2 e P2Y4, aumentaram a frequência e a amplitude das oscilações espontâneas de cálcio do tipo pico. Dados, obtidos por microscopia de luminescência, da expressão em tempo real de gene repórter luciferase fusionado à Mash1 e Ngn2 revelou que a ativação dos receptores P2Y2/P2Y4 aumentou a expressão estável de Mash1 enquanto que ativação do receptor P2X7 levou ao aumento de Ngn2. Além disso, células na presença do quelante de cálcio extracelular (EGTA) ou do depletor dos estoques intracelulares de cálcio do retículo endoplasmático (thapsigargin) apresentaram redução na expressão de Mash1 e Ngn2, indicando que ambos são regulados pela sinalização de cálcio. A investigação dos canais de cálcio voltagem-dependentes demonstrou que o influxo de cálcio gerado por despolarização da membrana de NSC diferenciadas de CTE é decorrente da ativação de canais de cálcio voltagem-dependentes do tipo L. Além disso, esse influxo pode controlar o destino celular por estabilizar expressão de Mash1 e induzir a diferenciação neuronal por fosforilação e translocação do fator de transcrição CREB. Esses dados sugerem que os receptores P2X7, P2Y2, P2Y4 e canais de cálcio voltagem-dependentes do tipo L podem modular as oscilações espontâneas de cálcio durante a diferenciação neural e consequentemente alteram o padrão de expressão de Mash1 e Ngn2 favorecendo a decisão do destino celular neuronal.

Synthesis and biological evaluation of nonpeptide mimetics of co-conotoxin GVIA

A benzothiazole-derived compound (4a) designed to mimic the C-alpha-C-beta bond vectors and terminal functionalities of Lys2, TyrI3 and Arg17 in omega-conotoxin GVIA was synthesised, together with analogues (4b-d), which had each side-chain mimic systematically truncated or eliminated. The affinity of these compounds for rat brain N-type and P/Q-type voltage gated calcium channels (VGCCs) was determined. In terms of N-type channel affinity and selectivity, two of these compounds (4a and 4d) were found to be highly promising, first generation mimetics of omega-conotoxin. The fully functionalised mimetic (4a) showed low PM binding affinity to N-type VGCCs (IC50 = 1.9 muM) and greater than 20-fold selectivity for this channel sub-type over P/Q-type VGCCs, whereas the mimetic in which the guanidine-type side chain was truncated back to an amine (4d, IC50 = 4.1 muM) showed a greater than 25-fold selectivity for the N-type channel. (C) 2004 Elsevier Ltd. All rights reserved.

Contribution of rare damaging variants in Autism Spectrum Disorder

Autism Spectrum Disorder (ASD) is a heterogeneous and highly heritable neurodevelopmental disorder with a complex genetic architecture, consisting of a combination of common low-risk and more penetrant rare variants. This PhD project aimed to explore the contribution of rare variants in ASD susceptibility through NGS approaches in a cohort of 106 ASD families including 125 ASD individuals. Firstly, I explored the contribution of inherited rare variants towards the ASD phenotype in a girl with a maternally inherited pathogenic NRXN1 deletion. Whole exome sequencing of the trio family identified an increased burden of deleterious variants in the proband that could modulate the CNV penetrance and determine the disease development. In the second part of the project, I investigated the role of rare variants emerging from whole genome sequencing in ASD aetiology. To properly manage and analyse sequencing data, a robust and efficient variant filtering and prioritization pipeline was developed, and by its application a stringent set of rare recessive-acting and ultra-rare variants was obtained. As a first follow-up, I performed a preliminary analysis on de novo variants, identifying the most likely deleterious variants and highlighting candidate genes for further analyses. In the third part of the project, considering the well-established involvement of calcium signalling in the molecular bases of ASD, I investigated the role of rare variants in voltage-gated calcium channels genes, that mainly regulate intracellular calcium concentration, and whose alterations have been correlated with enhanced ASD risk. Specifically, I functionally tested the effect of rare damaging variants identified in CACNA1H, showing that CACNA1H variation may be involved in ASD development by additively combining with other high risk variants. This project highlights the challenges in the analysis and interpretation of variants from NGS analysis in ASD, and underlines the importance of a comprehensive assessment of the genomic landscape of ASD individuals.

Effects of polyunsaturated fatty acids on voltage-gated K+ and Na+ channels in rat olfactory receptor neurons

Although the polyunsaturated fatty acids arachidonic acid (AA) and docosahexaenoic acid (DHA) are enriched in the olfactory mucosa, their possible contribution to olfactory transduction has not been investigated. This study characterized their effects on voltage-gated K+ and Na+ channels of rat olfactory receptor neurons. Physiological (3-10 mum) concentrations of AA and DHA potently and irreversibly inhibited the voltage-gated K+ current in a voltage-independent manner. In addition, both compounds significantly reduced the inhibitory potency of the odorants acetophenone and amyl acetate at these channels. By comparison, the steady-state effects of both AA and DHA on the voltage-gated Na+ channel were relatively weak, with half-maximal inhibition requiring approximate to 35 mum of either compound. However, a surprising finding was that the initial application of 3 mum AA to a naive neuron caused a strong but transient inhibition of the Na+ current. The channels became almost completely resistant to this inhibition within 1 min, and a 2-min wash in control solution was insufficient to restore the strong inhibitory effect. These observations suggest that polyunsaturated fatty acids have the potential to strongly influence the coding of odorant information by olfactory receptor neurons.

Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant beta(1) subunits.

Voltage-dependent calcium channel (Ca(v)) pores are modulated by cytosolic beta subunits. Four beta-subunit genes and their splice variants offer a wide structural array for tissue- or disease-specific biophysical gating phenotypes. For instance, the length of the N terminus of beta(2) subunits has major effects on activation and inactivation rates. We tested whether a similar mechanism principally operates in a beta(1) subunit. Wild-type beta(1a) subunit (N terminus length 60 aa) and its newly generated N-terminal deletion mutants (51, 27 and 18 aa) were examined within recombinant L-type calcium channel complexes (Ca(v)1.2 and alpha(2)delta2) in HEK293 cells at the whole-cell and single-channel level. Whole-cell currents were enhanced by co-transfection of the full-length beta(1a) subunit and by all truncated constructs. Voltage dependence of steady-state activation and inactivation did not depend on N terminus length, but inactivation rate was diminished by N terminus truncation. This was confirmed at the single-channel level, using ensemble average currents. Additionally, gating properties were estimated by Markov modeling. In confirmation of the descriptive analysis, inactivation rate, but none of the other transition rates, was reduced by shortening of the beta(1a) subunit N terminus. Our study shows that the length-dependent mechanism of modulating inactivation kinetics of beta(2) calcium channel subunits can be confirmed and extended to the beta(1) calcium channel subunit.