Neuron-glia cross talk revealed in reverberating networks by simultaneous extracellular recording of spikes and astrocytes' glutamate transporter and K+ currents.

Astrocytes uptake synaptically released glutamate with electrogenic transporters (GluT) and buffer the spike-dependent extracellular K+ excess with background K+ channels. We studied neuronal spikes and the slower astrocytic signals on reverberating neocortical cultures and organotypic slices from mouse brains. Spike trains and glial responses were simultaneously captured from individual sites of multielectrode arrays (MEA) by splitting the recorded traces into appropriate filters and reconstructing the original signal by deconvolution. GluT currents were identified by using dl-threo-ß-benzyloxyaspartate (TBOA). K+ currents were blocked by 30 µM Ba2+, suggesting a major contribution of inwardly rectifying K+ currents. Both types of current were tightly correlated with the spike rate, and their astrocytic origin was tested in primary cultures by blocking glial proliferation with cytosine ß-d-arabinofuranoside (AraC). The spike-related, time-locked inward and outward K+ currents in different regions of the astrocyte syncytium were consistent with the assumptions of the spatial K+ buffering model. In organotypic slices from ventral tegmental area and prefrontal cortex, the GluT current amplitudes exceeded those observed in primary cultures by several orders of magnitude, which allowed to directly measure transporter currents with a single electrode. Simultaneously measuring cell signals displaying widely different amplitudes and kinetics will help clarify the neuron-glia interplay and make it possible to follow the cross talk between different cell types in excitable as well as nonexcitable tissue.

Functional regeneration of the ex-vivo reconstructed mesocorticolimbic dopaminergic system.

CNS reparative-medicine therapeutic strategies need answers on the putative recapitulation of the basic rules leading to mammalian CNS development. To achieve this aim, we focus on the regeneration of functional connections in the mesocorticolimbic dopaminergic system. We used organotypic slice cocultures of ventral tegmental area/substantia nigra (VTA/SN) and prefrontal cortex (PFC) on a multielectrode array (MEA) platform to record spikes and local field potentials. The spontaneously growing synaptically based bidirectional bursting activity was followed from 2 to 28 days in vitro (DIV). A statistical analysis of excitatory and inhibitory neurons properties of the physiological firing activity demonstrated a remarkable, exponentially increasing maturation with a time constant of about 5-7 DIV. Immunohistochemistry demonstrated that the ratio of excitatory/inhibitory neurons (3:1) was in line with the functional results obtained. Exemplary pharmacology suggested that GABAA receptors were able to exert phasic and tonic inhibition typical of an adulthood network. Moreover, dopamine D2 receptor inactivation was equally inhibitory both on the spontaneous neuronal activity recorded by MEA and on patch-clamp electrophysiology in PFC pyramidal neurons. These results demonstrate that axon growth cones reach synaptic targets up to full functionality and that organotypic cocultures of the VTA/SN-PFC perfectly model their newly born dopaminergic, glutamatergic and GABAergic neuronal circuitries.

Target promiscuity and heterogeneous effects of tarantula venom peptides affecting Na+ and K+ ion channels.

Venom-derived peptide modulators of ion channel gating are regarded as essential tools for understanding the molecular motions that occur during the opening and closing of ion channels. In this study, we present the characterization of five spider toxins on 12 human voltage-gated ion channels, following observations about the target promiscuity of some spider toxins and the ongoing revision of their "canonical" gating-modifying mode of action. The peptides were purified de novo from the venom of Grammostola rosea tarantulas, and their sequences were confirmed by Edman degradation and mass spectrometry analysis. Their effects on seven tetrodotoxin-sensitive Na(+) channels, the three human ether-à-go-go (hERG)-related K(+) channels, and two human Shaker-related K(+) channels were extensively characterized by electrophysiological techniques. All the peptides inhibited ion conduction through all the Na(+) channels tested, although with distinctive patterns. The peptides also affected the three pharmaceutically relevant hERG isoforms differently. At higher concentrations, all peptides also modified the gating of the Na(+) channels by shifting the activation to more positive potentials, whereas more complex effects were recorded on hERG channels. No effects were evident on the two Shaker-related K(+) channels at concentrations well above the IC(50) value for the affected channels. Given the sequence diversity of the tested peptides, we propose that tarantula toxins should be considered both as multimode and target-promiscuous ion channel modulators; both features should not be ignored when extracting mechanistic interpretations about ion channel gating. Our observations could also aid in future structure-function studies and might help the development of novel ion channel-specific drugs.

Purging of the neuroblastoma stem cell compartment and tumor regression on exposure to hypoxia or cytotoxic treatment.

We worked out an experimental protocol able to purge the stem cell compartment of the SH-SY5Y neuroblastoma clone. This protocol was based on the prolonged treatment of the wild-type cell population with either hypoxia or the antiblastic etoposide. Cell fate was monitored by immunocytochemical and electrophysiologic (patch-clamp) techniques. Both treatments produced the progressive disappearance of neuronal type (N) cells (which constitute the bulk of the tumor), leaving space for a special category of epithelial-like substrate-adherent cells (S(0)). The latter represent a minimal cell component of the untreated population and are endowed with immunocytochemical markers (p75, c-kit, and CD133) and the electrophysiologic "nude" profile, typical of the neural crest stem cells. S(0) cells displayed a highly clonogenic potency and a substantial plasticity, generating both the N component and an alternative subpopulation terminally committed to the fibromuscular lineage. Unlike the N component, this lineage was highly insensitive to the apoptotic activity of hypoxia and etoposide and developed only when the neuronal option was abolished. Under these conditions, the fibromuscular progeny of S(0) expanded and progressed up to the exhaustion of the staminal compartment and to the extinction of the tumor. When combined, hypoxia and etoposide cooperated in abolishing the N cell generation and promoting the conversion of the tumor described. This synergy might mirror a natural condition in the ischemic areas occurring in cancer. These results have relevant implications for the understanding of the documented tendency of neuroblastomas to regress from a malignant to a benign phenotype, either spontaneously or on antiblastic treatment.

Modulatory proteins can rescue a trafficking defective epileptogenic Nav1.1 Na+ channel mutant.

Familial epilepsies are often caused by mutations of voltage-gated Na+ channels, but correlation genotype-phenotype is not yet clear. In particular, the cause of phenotypic variability observed in some epileptic families is unclear. We studied Na(v)1.1 (SCN1A) Na+ channel alpha subunit M1841T mutation, identified in a family characterized by a particularly large phenotypic spectrum. The mutant is a loss of function because when expressed alone, the current was no greater than background. Function was restored by incubation at temperature <30 degrees C, showing that the mutant is trafficking defective, thus far the first case among neuronal Na+ channels. Importantly, also molecular interactions with modulatory proteins or drugs were able to rescue the mutant. Protein-protein interactions may modulate the effect of the mutation in vivo and thus phenotype; variability in their strength may be one of the causes of phenotypic variability in familial epilepsy. Interacting drugs may be used to rescue the mutant in vivo.

ERG voltage-gated K+ channels regulate excitability and discharge dynamics of the medial vestibular nucleus neurones.

The discharge properties of the medial vestibular nucleus neurones (MVNn) critically depend on the activity of several ion channel types. In this study we show, immunohistochemically, that the voltage-gated K(+) channels ERG1A, ERG1B, ERG2 and ERG3 are highly expressed within the vestibular nuclei of P10 and P60 mice. The role played by these channels in the spike-generating mechanisms of the MVNn and in temporal information processing was investigated electrophysiologically from mouse brain slices, in vitro, by analysing the spontaneous discharge and the response to square-, ramp- and sinusoid-like intracellular DC current injections in extracellular and whole-cell patch-clamp studies. We show that more than half of the recorded MVNn were responsive to ERG channel block (WAY-123,398, E4031), displaying an increase in spontaneous activity and discharge irregularity. The response to step and ramp current injection was also modified by ERG block showing a reduction of first spike latency, enhancement of discharge rate and reduction of the slow spike-frequency adaptation process. ERG channels influence the interspike slope without affecting the spike shape. Moreover, in response to sinusoid-like current, ERG channel block caused frequency-dependent gain enhancement and phase-lead shift. Taken together, the data demonstrate that ERG channels control the excitability of MVNn, their discharge regularity and probably their resonance properties.

Four disulfide-bridged scorpion beta neurotoxin CssII: heterologous expression and proper folding in vitro.

The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and a FXa proteolytic cleavage region. This recombinant vector was transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The level of expression was 24.6 mg/l of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7538.6 Da as expected. Since native CssII (nCssII) is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1 Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na(v)1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.

Revisiting cangitoxin, a sea anemone peptide: purification and characterization of cangitoxins II and III from the venom of Bunodosoma cangicum.

Sodium channel toxins from sea anemones are employed as tools for dissecting the biophysical properties of inactivation in voltage-gated sodium channels. Cangitoxin (CGTX) is a peptide containing 48 amino acid residues and was formerly purified from Bunodosoma cangicum. Nevertheless, previous works reporting the isolation procedures for such peptide from B. cangicum secretions are controversial and may lead to incorrect information. In this paper, we report a simple and rapid procedure, consisting of two chromatographic steps, in order to obtain a CGTX analog directly from sea anemone venom. We also report a substitution of N16D in this peptide sample and the co-elution of an inseparable minor isoform presenting the R14H substitution. Peptides are named as CGTX-II and CGTX-III, and their effects over Nav1.1 channels in patch clamp experiments are demonstrated.

Human ether-a-go-go-related gene 1 channels are physically linked to beta1 integrins and modulate adhesion-dependent signaling.

Adhesive receptors of the integrin family are primarily involved in cell-extracellular matrix adhesion. Additionally, integrins trigger multiple signaling pathways that are involved in cell migration, proliferation, survival, and differentiation. We previously demonstrated that the activation of integrins containing the beta(1) subunit leads to a selective increase in potassium currents carried by the human ether-a-go-go-related gene (hERG) channels in neuroblastoma and leukemia cells; this current activation modulates adhesion-dependent differentiation in these cells. We hypothesized that the cross-talk between integrins and hERG channels could be traced back to the assembly of a macromolecular signaling complex comprising the two proteins. We tested this hypothesis in both SH-SY5Y neuroblastoma cells and in human embryonic kidney 293 cells stably transfected with hERG1 and, therefore, expressing only the full-length hERG1 protein on the plasma membrane. The beta(1) integrin and hERG1 coprecipitate in these cells and colocalize in both intracellular and surface membrane compartments. The two proteins also coprecipitate with caveolin-1, suggesting the localization of the complex in lipid rafts/caveolae. hERG1-transfected cells undergo an activation of hERG currents after beta(1) integrin-mediated adhesion to fibronectin; concomitant with this activation, the focal adhesion kinase associates with the hERG1 protein and becomes tyrosine phosphorylated. Using hERG1-specific inhibitors, we show that the tyrosine phosphorylation of focal adhesion kinase is strictly dependent on hERG channel activity. Similarly, the activity of the small GTPase Rac1 turned out to be dependent on hERG currents. On the whole, these data indicate that the hERG1 protein associates with beta(1) integrins and modulates adhesion receptor signaling.

Proteomic analysis of the venom and characterization of toxins specific for Na+ - and K+ -channels from the Colombian scorpion Tityus pachyurus.

The Colombian scorpion Tityus pachyurus is toxic to humans and is capable of producing fatal accidents, but nothing is known about its venom components. This communication reports the separation of at least 57 fractions from the venom by high performance liquid chromatography. From these, at least 104 distinct molecular weight compounds were identified by mass spectrometry analysis. The complete amino acid sequences of three peptides were determined and the partial sequences of three others were also identified. Electrophysiological experiments conducted with ion-channels expressed heterologously on Sf9 cells showed the presence of a potent Shaker B K(+)-channel blocker. This peptide (trivial name Tpa1) contains 23 amino acid residues closely packed by three disulfide bridges with a molecular mass of 2,457 atomic mass units. It is the third member of the sub-family 13, for which the systematic name is proposed to be alpha-KTx13.3. The mice assay showed clearly the presence of toxic peptides to mammals. One of them named Tpa2, containing 65 amino acid residues with molecular mass of 7,522.5 atomic mass units, is stabilized by four disulfide bridges. It was shown to modify the Na(+)-currents of F-11 and TE671 cells in culture, similar to the beta scorpion toxins. These results demonstrate the presence of toxic peptides in the venom of T. pachyurus and confirm that accidents with this species of scorpion should be considered an important human hazard in Colombia.

Novel alpha-conotoxins from Conus spurius and the alpha-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors.

alpha-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [gamma15E]SrIB) were compared to those exerted by the alpha4/7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing alpha(1)beta(1)gammadelta, alpha(4)beta(2) and alpha(3)beta(4) nicotinic acetylcholine receptors. At high concentrations (10 microm), the peptides SrIA, SrIB and [gamma15E]SrIB showed weak blocking effects only on alpha(4)beta(2) and alpha(1)beta(1)gammadelta subtypes, but EI also strongly blocked alpha(3)beta(4) receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors if briefly (2-15 s) applied at concentrations several orders of magnitude lower (EC(50), 1.78 and 0.37 nm, respectively). These results suggest not only that the novel alpha-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin-receptor interaction, as well as models with which to design highly specific therapeutic drugs.

Toxins interacting with ether-à-go-go-related gene voltage-dependent potassium channels.

The critical role that ether-à-go-go-related gene (erg) K(+) channels play in mating in Caenorhabditis elegans, neuronal seizures in Drosophila and cardiac action potential repolarization in humans has been well documented. Three erg genes (erg1, erg2 and erg3) have been identified and characterized. A structurally diverse number of compounds block these channels, but do not display specificity among the different channel isoforms. In this review we describe the blocking properties of several peptides, purified from scorpion, sea anemone and spider venoms, which are selective for certain members of the ERG family of channels. These peptides do not behave as classical pore blockers and appear to modify the gating properties of the channel. Genomic studies predict the existence of many other novel peptides with the potential of being more selective for ERG channels than those discussed here.

Isolation and characterization of a novel toxin from the venom of the spider Grammostola rosea that blocks sodium channels.

This communication reports the chemical and physiological characterization of a novel peptide (GrTx1) isolated from the venom of the "rosean-tarantula"Grammostola rosea. This component was one among more than 15 distinct components separated from the soluble venom by high-performance liquid chromatography (HPLC). GrTx1 has 29 amino-acid residues, compactly folded by three disulfide bridges with a molecular weight of 3697 Da. Here we show that this peptide blocks Na(+) currents of neuroblastoma F-11 cells with an IC(50) of 2.8+/-0.1 microM, up to a maximum of about 85% at 10 microM. Moreover, the right-shift (+20.1+/-0.4 mV) of the fractional voltage-dependent conductance could be also compatible with a putative "gating-modifier" mechanism. No effects were seen on common K(+) channels, such as K(v)1.1 and 1.4, using concentrations of toxin up to 10 microM. Sequence analysis reveals that GrTx1 is closely related to other spider toxins reported to affect various distinct ion channel functions. A critical analysis of this study suggests the necessity to search for other potential receptor sites in order to establish the preferred specificity of these kind of peptides.

Crotamine inhibits preferentially fast-twitching muscles but is inactive on sodium channels.

Crotamine is a peptide toxin from the venom of the rattlesnake Crotalus durissus terrificus that induces a typical hind-limb paralysis of unknown nature. Hind limbs have a predominance of fast-twitching muscles that bear a higher density of sodium channels believed until now to be the primary target of crotamine. Hypothetically, this makes these muscles more sensitive to crotamine and would explain such hind-limb paralysis. To challenge this hypothesis, we performed concentration vs. response curves on fast (extensor digitorum longus (EDL)) and slow (soleus) muscles of adult male rats. Crotamine was tested on various human Na+ channel isoforms (Na(v)1.1-Na(v)1.6 alpha-subunits) expressed in HEK293 cells in patch-clamp experiments, as well as in acutely dissociated dorsal root ganglion (DRG) neurons. Also, the behavioral effects of crotamine intoxication were compared with those of a muscle-selective sodium channel antagonist mu-CgTx-GIIIA, and other sodium-acting toxins such as tetrodotoxin alpha- and beta-pompilidotoxins, sea anemone toxin BcIII, spider toxin Tx2-6. Results pointed out that EDL was more susceptible to crotamine than soleus under direct electrical stimulation. Surprisingly, electrophysiological experiments in human Na(v)1.1 to Na(v)1.6 Na+ channels failed to show any significant change in channel characteristics, in a clear contrast with former studies. DRG neurons did not respond to crotamine. The behavioral effects of the toxins were described in detail and showed remarkable differences. We conclude that, although differences in the physiology of fast and slow muscles may cause the typical crotamine syndrome, sodium channels are not the primary target of crotamine and therefore, the real mechanism of action of this toxin is still unknown.

Plant Polyphenols and Exendin-4 Prevent Hyperactivity and TNF-α Release in LPS-Treated In vitro Neuron/Astrocyte/Microglial Networks.

Increasing evidence supports a decisive role for neuroinflammation in the neurodegenerative process of several central nervous system (CNS) disorders. Microglia are essential mediators of neuroinflammation and can regulate a broad spectrum of cellular responses by releasing reactive oxygen intermediates, nitric oxide, proteases, excitatory amino acids, and cytokines. We have recently shown that also in ex-vivo cortical networks of neurons, astrocytes and microglia, an increased level of tumor necrosis factor-alpha (TNF-α) was detected a few hours after exposure to the bacterial endotoxin lipopolysaccharide (LPS). Simultaneously, an atypical "seizure-like" neuronal network activity was recorded by multi-electrode array (MEA) electrophysiology. These effects were prevented by minocycline, an established anti-inflammatory antibiotic. We show here that the same inhibitory effect against LPS-induced neuroinflammation is exerted also by natural plant compounds, polyphenols, such as curcumin (CU, curcuma longa), crocin (CR, saffron), and resveratrol (RE, grape), as well as by the glucagon like peptide-1 receptor (GLP-1R) agonist exendin-4 (EX-4). The drugs tested also caused per-se early transient (variable) changes of network activity. Since it has been reported that LPS-induced neuroinflammation causes rearrangements of glutamate transporters in astrocytes and microglia, we suggest that neural activity could be putatively increased by an imbalance of glial glutamate transporter activity, leading to prolonged synaptic glutamatergic dysregulation.

Blockade of Ca2+ -activated K+ channels by galantamine can also contribute to the potentiation of catecholamine secretion from chromaffin cells.

Galantamine is a drug in clinical use for the treatment of Alzheimer's disease, but its mechanism(s) of action remains controversial. Here we addressed the question whether galantamine could potentiate neurotransmitter release by inhibiting small conductance Ca2+ -activated K+ channels (KCa2). Galantamine potentiated catecholamine secretory responses induced by 10 s pulses of acetylcholine and high [K+]o applied to fast-superfused bovine adrenal chromaffin cell populations. Catecholamine release was significantly enhanced by galantamine although we did not find concentration dependence in the range 0.1-1 microM. The KCa2 channel blocker apamin (0.3 microM) occluded the potentiating effects of galantamine on acetylcholine-evoked secretion. Like apamin, galantamine also modified the firing of action potentials, but to a lesser extent. In addition, 1 microM galantamine reduced by 41% the KCa2 current without modifying the voltage-dependent Ca2+ currents. These results constitute the first direct evidence that galantamine can potentiate neurotransmitter release by blocking KCa2 channels, in addition to its already demonstrated capacity to mildly block acetylcholinesterase or potentiate allosterically nicotinic receptors.

herg1 gene and HERG1 protein are overexpressed in colorectal cancers and regulate cell invasion of tumor cells.

The acquisition of the capacity to invade surrounding tissues confers a more malignant phenotype to tumor cells and is necessary for the establishment of metastases. The understanding of the molecular mechanisms underlying cell invasion in human solid tumors such as colorectal cancers could provide not only more sensitive prognostic analyses but also novel molecular targets for cancer therapy. We report in this article that K(+) ion channels belonging to the HERG family are important determinants for the acquisition of an invasive phenotype in colorectal cancers. The herg1 gene and HERG1 protein are expressed in many colon cancer cell lines, and the activity of HERG channels modulates colon cancer cell invasiveness. Moreover, the amount of HERG1 protein expressed on the plasma membrane is directly related to the invasive phenotype of colon cancer cells. Finally, both the herg1 gene and HERG1 protein were expressed in a high percentage of primary human colorectal cancers, with the highest incidence occurring in metastatic cancers, whereas no expression could be detected either in normal colonic mucosa or in adenomas.

Stochastic modeling of spontaneous bursting activity to simulate neural responses of in-vitro networks on multielectrode arrays.

The study of neuronal bursting activity, observed in cell-culture, is physiologically important because is correlated with synaptic transmission, plasticity, and information processing. However, besides strong ethical issues related to the use of animal models, there are many limitations due to experimental setup and neural signaling acquisition. In this study, we propose a stochastic modeling approach to simulate neural dynamics observed in networks of neocortical neurons from an ex vivo normal mouse. Specifically, we devised a stochastic version of the Izhichevich's model of cortical neurons, and simulated a network of excitatory and inhibitory neurons also accounting for cell signaling delays. No specific learning rules were used throughout the simulation time. Results show spontaneous bursting activity while mimicking balanced and hyper-excitable networks through modulation of the inhibitory synaptic weights. Furthermore, we validate our findings comparing the simulated cumulative probability functions of the firing spike histograms with the ones obtained in cultured networks of dissociated cortical neurons from ex vivo mice.

Isolation of a long-lasting eag-related gene-type K+ current in MMQ lactotrophs and its accommodating role during slow firing and prolactin release.

Native rat lactotrophs express thyrotrophin-releasing hormone-dependent K+ currents consisting of fast and slow deactivating components that are both sensitive to the class III anti-arrhythmic drugs that block the eag-related gene (ERG) K+ current (I(ERG)). Here we describe in MMQ prolactin-releasing pituitary cells the isolation of the slowly deactivating long-lasting component (I(ERGS)), which, unlike the fast component (I(ERGF)), is insensitive to verapamil 2 microm but sensitive to a novel scorpion toxin (ErgTx-2) that hardly affects I(ERGF). The time constants of I(ERGS) activation, deactivation, and recovery from inactivation are more than one order of magnitude greater than in I(ERGF), and the voltage-dependent inactivation is left-shifted by approximately 25 mV. The very slow MMQ firing frequency (approximately 0.2 Hz) investigated in perforated patch is increased approximately four times by anti-arrhythmic agents, by ErgTx-2, and by the abrupt I(ERGS) deactivation. Prolactin secretion in the presence of anti-arrhythmics is three- to fourfold higher in comparison with controls. We provide evidence from I(ERGS) and I(ERGF) simulations in a firing model cell to indicate that only I(ERGS) has an accommodating role during the experimentally observed very slow firing. Thus, we suggest that I(ERGS) potently modulates both firing and prolactin release in lactotroph cells.

Expression pattern of the ether-a-go-go-related (ERG) family proteins in the adult mouse central nervous system: evidence for coassembly of different subunits.

Voltage-dependent K+ channels are the main determinants in controlling cellular excitability within the central nervous system. Among voltage-dependent K+ channels, the ERG subfamily is deeply involved in the control of cellular excitability, both in mammals and in invertebrates. ERG channels are encoded by different genes: the erg1 gene, which can generate two alternative transcripts (erg1a and erg1b), erg2 and erg3. The aim of the present study was to determine the expression pattern and cellular localization of ERG proteins (ERG1, ERG2, and ERG3) in the mouse CNS, differentiating, for the first time, the ERG1A and ERG1B isoforms. To this purpose, novel specific antibodies were raised against the various channel proteins and their specificity and immunoreactivity tested. It emerged that: 1) all the erg genes were indeed translated in neuronal tissue; 2) ERG proteins distribution in the mouse CNS often overlapped, and only in specific areas each ERG protein showed a distinct pattern of expression; and 3) ERG proteins were generally expressed in neuronal soma, but dendritic and/or white matter labeling could be detected in specific areas. The finding that ERG proteins often have an overlapping expression suggests that neuronal ERG currents could be determined, at least in part, by heterotetrameric ERG channels. This suggestion is demonstrated to occur for ERG1A/ERG1B by showing that the two isoforms coassemble in mouse brain.