Sodium Channel Myotonia Due to Novel Mutations in Domain I of Nav1.4.

Sodium channel myotonia is a form of muscle channelopathy due to mutations that affect the Nav1.4 channel. We describe seven families with a series of symptoms ranging from asymptomatic to clearly myotonic signs that have in common two novel mutations, p.Ile215Thr and p.Gly241Val, in the first domain of the Nav1.4 channel. The families described have been clinically and genetically evaluated. p.Ile215Thr and p.Gly241Val lie, respectively, on extracellular and intracellular loops of the first domain of the Nav1.4 channel. We assessed that the p.Ile215Thr mutation can be related to a founder effect in people from Southern Italy. Electrophysiological evaluation of the channel function showed that the voltage dependence of the activation for both the mutant channels was significantly shifted toward hyperpolarized potentials (Ile215Thr: -28.6 ± 1.5 mV and Gly241Val: -30.2 ± 1.3 mV vs. WT: -18.5 ± 1.3 mV). The slow inactivation was also significantly affected, whereas fast inactivation showed a different behavior in the two mutants. We characterized two novel mutations of the SCN4A gene expanding the knowledge about genetics of mild forms of myotonia, and we present, to our knowledge, the first homozygous patient with sodium channel myotonia.

Myotonia congenita: novel mutations in CLCN1 gene and functional characterizations in Italian patients.

Myotonia congenita is an autosomal dominantly or recessively inherited muscle disorder causing impaired muscle relaxation and variable degrees of permanent muscle weakness, abnormal currents linked to the chloride channel gene (CLCN1) encoding the chloride channel on skeletal muscle membrane. We describe 12 novel mutations: c.1606G>C (p.Val536Leu), c.2533G>A (p.Gly845Ser), c.2434C>T (p.Gln812X), c.1499T>G (p.E500X), c.1012C>T (p.Arg338X), c.2403+1G>A, c.2840T>A (p.Val947Glu), c.1598C>T (p.Thr533Ile), c.1110delC, c.590T>A (p.Ile197Arg), c.2276insA Fs800X, c.490T>C (p.Trp164Arg) in 22 unrelated Italian patients. To further understand the functional outcome of selected missense mutations (p.Trp164Arg, p.Ile197Arg and p.Gly845Ser, and the previously reported p.Gly190Ser) we characterized the biophysical properties of mutant ion channels in tsA cell model. In the physiological range of muscle membrane potential, all the tested mutations, except p.Gly845Ser, reduced the open probability, increased the fast and slow components of deactivation and affected pore properties. This suggests a decrease in macroscopic chloride currents impairing membrane potential repolarization and causing hyperexcitability in muscle membranes. Detailed clinical features are given of the 8 patients characterized by cell electrophysiology. These data expand the spectrum of CLCN1 mutations and may contribute to genotype-phenotype correlations. Furthermore, we provide insights into the fine protein structure of ClC-1 and its physiological role in the maintenance of membrane resting potential.

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.

Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms.

As voltage-gated Na(+) channels are responsible for the conduction of electrical impulses in most excitable tissues in the majority of animals (except nematodes), they have become important targets for the toxins of venomous animals, from sea anemones to molluscs, scorpions, spiders and even fishes. During their evolution, different animals have developed a set of cysteine-rich peptides capable of binding different extracellular sites of this channel protein. A fundamental question concerning the mechanism of action of these toxins is whether they act at a common receptor site in Na(+) channels when exerting their different pharmacological effects, or at distinct receptor sites in different Na(v) channels subtypes whose particular properties lead to these pharmacological differences. The alpha-subunits of voltage-gated Na(+) channels (Na(v)1.x) have been divided into at least nine subtypes on the basis of amino acid sequences. Sea anemones have been extensively studied from the toxinological point of view for more than 40 years. There are about 40 sea anemone type 1 peptides known to be active on Na(v)1.x channels and all are 46-49 amino acid residues long, with three disulfide bonds and their molecular weights range between 3000 and 5000 Da. About 12 years ago a general model of Na(v)1.2-toxin interaction, developed for the alpha-scorpion toxins, was shown to fit also to action of sea anemone toxin such as ATX-II. According to this model these peptides are specifically acting on the type 3 site known to be between segments 3 and 4 in domain IV of the Na(+) channel protein. This region is indeed responsible for the normal Na(+) currents fast inactivation that is potently slowed by these toxins. This fundamental "gain-of-function" mechanism is responsible for the strong increase in the action potential duration. They constitute a class of tools by means of which physiologists and pharmacologists can study the structure/function relationships of channel proteins. As most of the structural and electrophysiological studies were performed on type 1 sea anemone sodium channel toxins, we will present a comprehensive and updated review on the current understanding of the physiological actions of these Na channel modifiers.

Identification of a posttranslational mechanism for the regulation of hERG1 K+ channel expression and hERG1 current density in tumor cells.

A common feature of tumor cells is the aberrant expression of ion channels on their plasma membrane. The molecular mechanisms regulating ion channel expression in cancer cells are still poorly known. K(+) channels that belong to the human ether-a-go-go-related gene 1 (herg1) family are frequently misexpressed in cancer cells compared to their healthy counterparts. We describe here a posttranslational mechanism for the regulation of hERG1 channel surface expression in cancer cells. This mechanism is based on the activity of hERG1 isoforms containing the USO exon. These isoforms (i) are frequently overexpressed in human cancers, (ii) are retained in the endoplasmic reticulum, and (iii) form heterotetramers with different proteins of the hERG family. (iv) The USO-containing heterotetramers are retained intracellularly and undergo ubiquitin-dependent degradation. This process results in decreased hERG1 current (I(hERG1)) density. We detailed such a mechanism in heterologous systems and confirmed its functioning in tumor cells that endogenously express hERG1 proteins. The silencing of USO-containing hERG1 isoforms induces a higher I(hERG1) density in tumors, an effect that apparently regulates neurite outgrowth in neuroblastoma cells and apoptosis in leukemia cells.

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.

Binding specificity of sea anemone toxins to Nav 1.1-1.6 sodium channels: unexpected contributions from differences in the IV/S3-S4 outer loop.

Sea anemones are an important source of various biologically active peptides, and it is known that ATX-II from Anemonia sulcata slows sodium current inactivation. Using six different sodium channel genes (from Nav1.1 to Nav1.6), we investigated the differential selectivity of the toxins AFT-II (purified from Anthopleura fuscoviridis) and Bc-III (purified from Bunodosoma caissarum) and compared their effects with those recorded in the presence of ATX-II. Interestingly, ATX-II and AFT-II differ by only one amino acid (L36A) and Bc-III has 70% similarity. The three toxins induced a low voltage-activated persistent component primarily in the Nav1.3 and Nav1.6 channels. An analysis showed that the 18 dose-response curves only partially fit the hypothesized binding of Lys-37 (sea anemone toxin Anthopleurin B) to the Asp (or Glu) residue of the extracellular IV/S3-S4 loop in cardiac (or nervous) Na+ channels, thus suggesting the substantial contribution of some nearby amino acids that are different in the various channels. As these channels are atypically expressed in mammalian tissues, the data not only suggest that the toxicity is highly dependent on the channel type but also that these toxins and their various physiological effects should be considered prototype models for the design of new and specific pharmacological tools.

Depolarization differentially affects the secretory and migratory properties of two cell lines of immortalized luteinizing hormone-releasing hormone (LHRH) neurons.

In this report we studied and compared the biochemical and the electrophysiological characteristics of two cell lines (GT1-7 and GN11) of immortalized mouse LHRH-expressing neurons and the correlation with their maturational stage and migratory activity. In fact, previous results indicated that GN11, but not GT1-7, cells exhibit an elevated motility in vitro. The results show that the two cell lines differ in terms of immunoreactivity for tyrosine hydroxylase and nestin as well as of production and release of 3,4-dihydroxyphenylalanine (DOPA) and of intracellular distribution and release of the LHRH. Patch-clamp recordings in GN11 cells, reveal the presence of a single inward rectifier K+ current indicative of an immature neuronal phenotype (neither firing nor electrical activity). In contrast, as known from previous studies, GT1-7 cells show the characteristics of mature LHRH neurons with a high electrical activity characterized by spontaneous firing and excitatory postsynaptic potentials. K+-induced depolarization induces in GT1-7 cells, but not in GN11 cells, a strong increase in the release of LHRH in the culture medium. However, depolarization of GN11 cells significantly decreases their chemomigratory response. In conclusion, these results indicate that GT1-7 and GN11 cells show different biochemical and electrophysiological characteristics and are representative of mature and immature LHRH neurons, respectively. The early stage of maturation of GN11 cells, as well as the low electrical activity detected in these cells, appears to correlate with their migratory activity in vitro.

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.