Identification, Characterization, and Modeling of a Bioinsecticide Protein Isolated from Scorpion Venom gland: A Three-Finger Protein.

Background: The majority of insecticides target sodium channels. The increasing emergence of resistance to the current insecticides has persuaded researchers to search for alternative compounds. Scorpion venom gland as a reservoir of peptides or proteins, which selectively target insect sodium channels. These proteins would be an appropriate source for finding new suitable anti-insect components. Methods: Transcriptome of venom gland of scorpion Mesobuthus eupeus was obtained by RNA extraction and complementary DNA library synthesis. The obtained transcriptome was blasted against protein databases to find insect toxins against sodium channel based on the statistically significant similarity in sequence. Physicochemical properties of the identified protein were calculated using bioinformatics software. The three-dimensional structure of this protein was determined using homology modeling, and the final structure was assessed by molecular dynamics simulation. Results: The sodium channel blocker found in the transcriptome of M. eupeus venom gland was submitted to the GenBank under the name of meuNa10, a stable hydrophilic protein consisting of 69 amino acids, with the molecular weight of 7721.77 g/mol and pI of 8.7. The tertiary structure of meuNa10 revealed a conserved LCN-type cysteine-stabilized alpha/beta domain stabilized by eight cysteine residues. The meuNa10 is a member of the 3FP superfamily consisting of three finger-like beta strands. Conclusion: This study identified meuNa10 as a small insect sodium channel-interacting protein with some physicochemical properties, including stability and water-solubility, which make it a good candidate for further in vivo and in vitro experiments in order to develop a new bioinsecticide.

Interactions of Sea Anemone Toxins with Insect Sodium Channel-Insights from Electrophysiology and Molecular Docking Studies.

Animal venoms are considered as a promising source of new drugs. Sea anemones release polypeptides that affect electrical activity of neurons of their prey. Voltage dependent sodium (Nav) channels are the common targets of Av1, Av2, and Av3 toxins from Anemonia viridis and CgNa from Condylactis gigantea. The toxins bind to the extracellular side of a channel and slow its fast inactivation, but molecular details of the binding modes are not known. Electrophysiological measurements on Periplaneta americana neuronal preparation revealed differences in potency of these toxins to increase nerve activity. Av1 and CgNa exhibit the strongest effects, while Av2 the weakest effect. Extensive molecular docking using a modern SMINA computer method revealed only partial overlap among the sets of toxins' and channel's amino acid residues responsible for the selectivity and binding modes. Docking positions support earlier supposition that the higher neuronal activity observed in electrophysiology should be attributed to hampering the fast inactivation gate by interactions of an anemone toxin with the voltage driven S4 helix from domain IV of cockroach Nav channel (NavPaS). Our modelling provides new data linking activity of toxins with their mode of binding in site 3 of NavPaS channel.

Modulation on tetrodotoxin-resistant sodium current of loureirin B in rat dorsal root ganglion neurons via cyclic AMP-dependent protein kinase A.

To search the modulation mechanism of loureirin B, a flavonoid is extracted from Dracaena cochinchinensis, on tetrodotoxin-resistant (TTX-R) sodium channel in dorsal root ganglion (DRG) neurons of rats. Experiments were carried out based on patch-clamp technique and molecular biological methods. We observed the time-dependent inhibition of loureirin B on TTX-R sodium currents in DRG neurons and found that neither occupancy theory nor rate theory could well explain the time-dependent inhibitory effect of loureirin B on TTX-R sodium currents. It suggested that a second messenger-mediated signaling pathway may be involved in the modulation mechanism. So the cyclin AMP (cAMP) level of the DRG neurons before and after incubation with loureirin B was tested by ELISA Kit. Results showed that loureirin B could increase the cAMP level and the increased cAMP was caused by the enhancement of adenylate cyclase (AC) induced by loureirin B. Immunolabelling experiments further confirmed that loureirin B can promote the production of PKA in DRG neurons. In the presence of the PKA inhibitor H-89, the inhibitory effect of loureirin B on TTX-R sodium currents was reversed. Forskolin, a tool in biochemistry to raise the levels of cAMP, also could reduce TTX-R sodium currents similar to that of loureirin B. These studies demonstrated that loureirin B can modulate the TTX-R sodium channel in DRG neurons via an AC/cAMP/PKA pathway involving the activation of AC and PKA, which also can be used to explain the other pharmacological effects of loureirin B.

Discovery of a Novel Series of Tricyclic Oxadiazine 4a-Methyl Esters Based on Indoxacarb as Potential Sodium Channel Blocker/Modulator Insecticides.

Indoxacarb, a commercialized oxadiazine insecticide, nearly irreversibly blocks open/inactivated, but not resting sodium channels. The structure-activity relationships showed that the substituents at the position of the chiral atom in the oxadiazine ring are very important to the biological activity of oxadiazine insecticide. Here we synthesized a series of tricyclic oxadiazine 4a-methyl ester derivatives. The chiral atom in the oxadiazine ring has been epimerized and substituted with either pyrethric acid or cinnamic acid derivatives. Benzene ring in the tricyclic moiety was substituted with a chlorine, fluorine, or bromine atom, and nitrogen-linked benzene ring was substituted with a trifluoromethyl or trifluoromethoxy group. Toxicity of these compounds against Spodoptera litura F. was evaluated. Diastereoisomers of most toxic compounds J7 and J9 with pyrethric acid moiety were separated by flash column chromatography. The more polar diastereoisomers, J7-L-Rf and J9-L-Rf, and compounds J24 and J26 with cinnamic acid moiety exhibited highest insecticidal activities. We further used Monte Carlo energy minimizations to dock compound J7 and J24 in the NavMs-based homology model of the open cockroach sodium channel. In the low-energy binding modes, the compound interacted with residues in the inner pore and domain interfaces, which previously were proposed to contribute to receptors of pyrethroids and sodium channel blocker insecticides. Our results define compound J7 and J24 as a potentially useful optimized hit for the development of multiple sites sodium channel blocker or modulator.

Electrophysiological characterization of Tityus obscurus ß toxin 1 (To1) on Na+-channel isoforms.

To1, previously named Tc49b, is a peptide neurotoxin isolated from venom of the scorpion Tityus obscurus that is responsible for lethal human poisoning cases in the Brazilian Amazonian region. Previously, To1 was shown to be lethal to mice and to change Na+ permeation in cerebellum granular neurons from rat brain. In addition, To1 did not affect Shaker B K+ channels. Based on sequence similarities, To1 was described as a ß-toxin. In the present work, To1 was purified from T. obscurus venom and submitted to an electrophysiological characterization in human and invertebrate NaV channels. The analysis of the electrophysiological experiments reveal that To1 enhances the open probability at more negative potentials of human NaV 1.3 and 1.6, of the insect channel BgNaV1 and of arachnid VdNaV1 channel. In addition, To1 reduces the peak of Na+ currents in some of the NaVs tested. These results support the classification of the To1 as a ß-toxin. A structure and functional comparison to other ß-toxins that share sequence similarity to To1 is also presented.

Batrachotoxin acts as a stent to hold open homotetrameric prokaryotic voltage-gated sodium channels.

Batrachotoxin (BTX), an alkaloid from skin secretions of dendrobatid frogs, causes paralysis and death by facilitating activation and inhibiting deactivation of eukaryotic voltage-gated sodium (Nav) channels, which underlie action potentials in nerve, muscle, and heart. A full understanding of the mechanism by which BTX modifies eukaryotic Nav gating awaits determination of high-resolution structures of functional toxin-channel complexes. Here, we investigate the action of BTX on the homotetrameric prokaryotic Nav channels NaChBac and NavSp1. By combining mutational analysis and whole-cell patch clamp with molecular and kinetic modeling, we show that BTX hinders deactivation and facilitates activation in a use-dependent fashion. Our molecular model shows the horseshoe-shaped BTX molecule bound within the open pore, forming hydrophobic H-bonds and cation-π contacts with the pore-lining helices, leaving space for partially dehydrated sodium ions to permeate through the hydrophilic inner surface of the horseshoe. We infer that bulky BTX, bound at the level of the gating-hinge residues, prevents the S6 rearrangements that are necessary for closure of the activation gate. Our results reveal general similarities to, and differences from, BTX actions on eukaryotic Nav channels, whose major subunit is a single polypeptide formed by four concatenated, homologous, nonidentical domains that form a pseudosymmetric pore. Our determination of the mechanism by which BTX activates homotetrameric voltage-gated channels reveals further similarities between eukaryotic and prokaryotic Nav channels and emphasizes the tractability of bacterial Nav channels as models of voltage-dependent ion channel gating. The results contribute toward a deeper, atomic-level understanding of use-dependent natural and synthetic Nav channel agonists and antagonists, despite their overlapping binding motifs on the channel proteins.

NMR Structure of µ-Conotoxin GIIIC: Leucine 18 Induces Local Repacking of the N-Terminus Resulting in Reduced NaV Channel Potency.

µ-Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of µ-conotoxin GIIIC was determined using 2D NMR spectroscopy and simulated annealing calculations. Despite high sequence similarity, GIIIC adopts a three-dimensional structure that differs from the previously observed conformation of µ-conotoxins GIIIA and GIIIB due to the presence of a bulky, non-polar leucine residue at position 18. The side chain of L18 is oriented towards the core of the molecule and consequently the N-terminus is re-modeled and located closer to L18. The functional characterization of GIIIC defines it as a canonical µ-conotoxin that displays substantial selectivity towards skeletal muscle sodium channels (NaV), albeit with ~2.5-fold lower potency than GIIIA. GIIIC exhibited a lower potency of inhibition of NaV1.4 channels, but the same NaV selectivity profile when compared to GIIIA. These observations suggest that single amino acid differences that significantly affect the structure of the peptide do in fact alter its functional properties. Our work highlights the importance of structural factors, beyond the disulfide pattern and electrostatic interactions, in the understanding of the functional properties of bioactive peptides. The latter thus needs to be considered when designing analogues for further applications.

Fibromyalgia and small fiber neuropathy: the plot thickens!

Several groups of investigators have described the presence of small fiber neuropathy in fibromyalgia patients. This writing discusses how this new finding could renovate fibromyalgia concept, diagnosis, and treatment. Predominant rheumatology thinking proposes fibromyalgia as a "centralized pain syndrome." An alternative hypothesis views fibromyalgia as a stress-related dysautonomia with neuropathic pain features. Dorsal root ganglia may be the key autonomic-nociceptive short-circuit sites. The recent recognition of small fiber neuropathy in a large subgroup of fibromyalgia patients reinforces the dysautonomia-neuropathic hypothesis and validates fibromyalgia pain. These new findings support fibromyalgia as a primarily neurological entity, nevertheless, rheumatologist will likely remain the best equipped specialist to diagnose fibromyalgia and differentiate it from other multi-symptomatic rheumatic syndromes. Skin biopsy and corneal confocal microscopy will probably become useful fibromyalgia diagnostic tests. Dorsal root ganglia sodium channel blockers are potential fibromyalgia analgesic medications. Subgroups of young girls with "autoimmune neuropathic fibromyalgia" may respond to immunoglobulin therapy. Multimodal intervention directed to regain autonomic nervous system resilience will likely remain the cornerstone for fibromyalgia therapy.

Novel NALCN biallelic truncating mutations in siblings with IHPRF1 syndrome.

Infantile hypotonia with psychomotor retardation and characteristic facies-1 (IHPRF1) is a severe autosomal recessive neurologic disorder with onset at birth or in early infancy. It is caused by mutations in the NALCN gene that encodes a voltage-independent, cation channel permeable to NM, K+ and Ca2+ and forms a channel complex with UNCSO and UNC79. So far, only 4 homozygous mutations have been found in 11 cases belonging to 4 independent consanguineous families. We studied a Sardinian family with 2 siblings presenting dysmorphic facies, hypotonia, psychomotor retardation, epilepsy, absent speech, sleep disturbance, hyperkinetic movement disorder, cachexia and chronic constipation. Polymorphic generalized seizures started at 4 and 6 years, respectively. Anti-epileptic drugs (AEDs) therapy was efficient for female proband's epilepsy, but the male still has weekly seizures. Whole exome sequencing identified 2 novel truncating mutations in NALCN allowing to assess the clinical phenotype to IHPRF1. This is the fifth family reported worldwide, and these are the first European cases with IHPRF1 syndrome with biallelic truncating mutations of NALCN.

Distinct modulation of inactivation by a residue in the pore domain of voltage-gated Na+ channels: mechanistic insights from recent crystal structures.

Inactivation of voltage-gated Na+ channels (VGSC) is essential for the regulation of cellular excitability. The molecular rearrangement underlying inactivation is thought to involve the intracellular linker between domains III and IV serving as inactivation lid, the receptor for the lid (domain III S4-S5 linker) and the pore-lining S6 segements. To better understand the role of the domain IV S6 segment in inactivation we performed a cysteine scanning mutagenesis of this region in rNav 1.4 channels and screened the constructs for perturbations in the voltage-dependence of steady state inactivation. This screen was performed in the background of wild-type channels and in channels carrying the mutation K1237E, which profoundly alters both permeation and gating-properties. Of all tested constructs the mutation I1581C was unique in that the mutation-induced gating changes were strongly influenced by the mutational background. This suggests that I1581 is involved in specific short-range interactions during inactivation. In recently published crystal structures VGSCs the respective amino acids homologous to I1581 appear to control a bend of the S6 segment which is critical to the gating process. Furthermore, I1581 may be involved in the transmission of the movement of the DIII voltage-sensor to the domain IV S6 segment.

Forty Years of Sodium Channels: Structure, Function, Pharmacology, and Epilepsy.

Voltage-gated sodium channels initiate action potentials in brain neurons. In the 1970s, much was known about the function of sodium channels from measurements of ionic currents using the voltage clamp method, but there was no information about the sodium channel molecules themselves. As a postdoctoral fellow and staff scientist at the National Institutes of Health, I developed neurotoxins as molecular probes of sodium channels in cultured neuroblastoma cells. During those years, Bruce Ransom and I crossed paths as members of the laboratories of Marshall Nirenberg and Philip Nelson and shared insights about sodium channels in neuroblastoma cells from my work and electrical excitability and synaptic transmission in cultured spinal cord neurons from Bruce's pioneering electrophysiological studies. When I established my laboratory at the University of Washington in 1977, my colleagues and I used those neurotoxins to identify the protein subunits of sodium channels, purify them, and reconstitute their ion conductance activity in pure form. Subsequent studies identified the molecular basis for the main functions of sodium channels-voltage-dependent activation, rapid and selective ion conductance, and fast inactivation. Bruce Ransom and I re-connected in the 1990s, as ski buddies at the Winter Conference on Brain Research and as faculty colleagues at the University of Washington when Bruce became our founding Chair of Neurology and provided visionary leadership of that department. In the past decade my work on sodium channels has evolved into structural biology. Molecular modeling and X-ray crystallographic studies have given new views of sodium channel function at atomic resolution. Sodium channels are also the molecular targets for genetic diseases, including Dravet Syndrome, an intractable pediatric epilepsy disorder with major co-morbidities of cognitive deficit, autistic-like behaviors, and premature death that is caused by loss-of-function mutations in the brain sodium channel NaV1.1. Our work on a mouse genetic model of this disease has shown that its multi-faceted pathophysiology and co-morbidities derive from selective loss of electrical excitability and action potential firing in GABAergic inhibitory neurons, which disinhibits neural circuits throughout the brain and leads directly to the epilepsy, premature death and complex co-morbidities of this disease. It has been rewarding for me to use our developing knowledge of sodium channels to help understand the pathophysiology and to suggest potential therapeutic approaches for this devastating childhood disease.

Evaluating the potential of a loop-extended scorpion toxin-like peptide as a protein scaffold.

Grafting of exogenous bioactive sites or functional motifs onto structurally stable scaffolds to gain new functions represents an important research direction in protein engineering. Some engineered proteins have been developed into therapeutic drugs. MeuNaTxα-3 (abbreviated as MT-3) is a newly characterized scorpion sodium channel toxin-like peptide isolated from the venom of the scorpion Mesobuthus eupeus, which contains a rigid scaffold highly similar to classical scorpion sodium channel toxins and an extension of eight amino acids in its J-loop region. This extended loop constitutes a flexible region extruded from the scaffold and could be substituted by exogenous functional sequences. In this study, we experimentally evaluated the scaffold potential of MT-3 through grafting two small antimicrobial motifs to replace residues within the loop. Functional assays showed that the two engineered molecules exhibited elevated antimicrobial potency, as compared with the unmodified scaffold, without structural disruption, providing experimental evidence in favor of MT-3 as a promising scaffold in protein engineering.

Structure of the C-terminal domain of the prokaryotic sodium channel orthologue NsvBa.

Crystallographic and electrophysiological studies have recently provided insight into the structure, function, and drug binding of prokaryotic sodium channels. These channels exhibit significant sequence identities, especially in their transmembrane regions, with human voltage-gated sodium channels. However, rather than being single polypeptides with four homologous domains, they are tetramers of single domain polypeptides, with a C-terminal domain (CTD) composed of an inter-subunit four helix coiled coil. The structures of the CTDs differ between orthologues. In NavBh and NavMs, the C-termini form a disordered region adjacent to the final transmembrane helix, followed by a coiled-coil region, as demonstrated by synchrotron radiation circular dichroism (SRCD) and double electron-electron resonance electron paramagnetic resonance spectroscopic measurements. In contrast, in the crystal structure of the NavAe orthologue, the entire C-terminus is comprised of a helical region followed by a coiled coil. In this study, we have examined the CTD of the NsvBa from Bacillus alcalophilus, which unlike other orthologues is predicted by different methods to have different types of structures: either a disordered region adjacent to the transmembrane region, followed by a helical coiled coil, or a fully helical CTD. To discriminate between the two possible structures, we have used SRCD spectroscopy to experimentally determine the secondary structure of the C-terminus of this orthologue and used the results as the basis for modeling the open and closed conformations of the channel.

Molecular basis of ion permeability in a voltage-gated sodium channel.

Voltage-gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na(+) ≈ Li(+) â‰« K(+), Ca(2+), Mg(2+)) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.

Structural Basis for the Inhibition of Voltage-gated Sodium Channels by Conotoxin µO§-GVIIJ.

Cone snail toxins are well known blockers of voltage-gated sodium channels, a property that is of broad interest in biology and therapeutically in treating neuropathic pain and neurological disorders. Although most conotoxin channel blockers function by direct binding to a channel and disrupting its normal ion movement, conotoxin µO§-GVIIJ channel blocking is unique, using both favorable binding interactions with the channel and a direct tether via an intermolecular disulfide bond. Disulfide exchange is possible because conotoxin µO§-GVIIJ contains anS-cysteinylated Cys-24 residue that is capable of exchanging with a free cysteine thiol on the channel surface. Here, we present the solution structure of an analog of µO§-GVIIJ (GVIIJ[C24S]) and the results of structure-activity studies with synthetic µO§-GVIIJ variants. GVIIJ[C24S] adopts an inhibitor cystine knot structure, with two antiparallel ß-strands stabilized by three disulfide bridges. The loop region linking the ß-strands (loop 4) presents residue 24 in a configuration where it could bind to the proposed free cysteine of the channel (Cys-910, rat NaV1.2 numbering; at site 8). The structure-activity study shows that three residues (Lys-12, Arg-14, and Tyr-16) located in loop 2 and spatially close to residue 24 were also important for functional activity. We propose that the interaction of µO§-GVIIJ with the channel depends on not only disulfide tethering via Cys-24 to a free cysteine at site 8 on the channel but also the participation of key residues of µO§-GVIIJ on a distinct surface of the peptide.

An improved sensitive assay for the detection of PSP toxins with neuroblastoma cell-based impedance biosensor.

Paralytic shellfish poisoning (PSP) toxins are well-known sodium channel-blocking marine toxins, which block the conduction of nerve impulses and lead to a series of neurological disorders symptoms. However, PSP toxins can inhibit the cytotoxicity effect of compounds (e.g., ouabain and veratridine). Under the treatment of ouabain and veratridine, neuroblastoma cell will swell and die gradually, since veratridine causes the persistent inflow of Na(+) and ouabain inhibits the activity of Na(+)/K(+)-ATPases. Therefore, PSP toxins with antagonism effect can raise the chance of cell survival by blocking inflow of Na(+). Based on the antagonism effect of PSP toxins, we designed an improved cell-based assay to detect PSP toxins using a neuroblastoma cell-based impedance biosensor. The results demonstrated that this biosensor showed high sensitivity and good specificity for saxitoxins detection. The detection limit of this biosensor was as low as 0.03 ng/ml, which was lower than previous reported cell-based assays and mouse bioassays. With the improvement of biosensor performance, the neuroblastoma cell-based impedance biosensor has great potential to be a universal PSP screening method.

Microwave assisted synthesis and docking study of N-(2-oxo-2-(4-oxo-2-substituted thiazolidin-3ylamino)ethyl)benzamide derivatives as anticonvulsant agents.

Herewith, we report the design and synthesis of a series of N-(2-oxo-2((4-oxo-2-substituted thiazolidin-3yl)amino)ethyl) benzamide derivatives 7(a-j) under microwave irradiation, based on four component pharmacophoric model to get structural prerequisite indispensable for anticonvulsant activity. The synthesized derivatives were investigated in maximal electroshock seizure (MES), subcutaneous pentylenetetrazole (sc-PTZ) induced seizure and neurotoxicity screening. All the test compounds were administered at a dose of 30, 100 and 300 mg/kg body weight at the time interval of 0.5 h and 4 h. The compounds were also evaluated for behavioral activity and toxicity study. The compound 7 h was found to be most active in MES model. The anticonvulsant screening data shows that 65% of the compounds were found active against MES model when compared to 35% sc-PTZ model. The computational parameter such as docking study, logP determination and ADME prediction were performed to exploit the results.

Ion selectivity strategies of sodium channel selectivity filters.

CONSPECTUS: Sodium ion channels selectively transport Na(+) cations across the cell membrane. These integral parts of the cell machinery are implicated in regulating the cardiac, skeletal and smooth muscle contraction, nerve impulses, salt and water homeostasis, as well as pain and taste perception. Their malfunction often results in various channelopathies of the heart, brain, skeletal muscles, and lung; thus, sodium channels are key drug targets for various disorders including cardiac arrhythmias, heart attack, stroke, migraine, epilepsy, pain, cancer, and autoimmune disorders. The ability of sodium channels to discriminate the native Na(+) among other competing ions in the surrounding fluids is crucial for proper cellular functions. The selectivity filter (SF), the narrowest part of the channel's open pore, lined with amino acid residues that specifically interact with the permeating ion, plays a major role in determining Na(+) selectivity. Different sodium channels have different SFs, which vary in the symmetry, number, charge, arrangement, and chemical type of the metal-ligating groups and pore size: epithelial/degenerin/acid-sensing ion channels have generally trimeric SFs lined with three conserved neutral serines and/or backbone carbonyls; eukaryotic sodium channels have EKEE, EEKE, DKEA, and DEKA SFs with an invariant positively charged lysine from the second or third domain; and bacterial voltage-gated sodium (Nav) channels exhibit symmetrical EEEE SFs, reminiscent of eukaryotic voltage-gated calcium channels. How do these different sodium channel SFs achieve high selectivity for Na(+) over its key rivals, K(+) and Ca(2+)? What factors govern the metal competition in these SFs and which of these factors are exploited to achieve Na(+) selectivity in the different sodium channel SFs? The free energies for replacing K(+) or Ca(2+) bound inside different model SFs with Na(+), evaluated by a combination of density functional theory and continuum dielectric calculations, have shed light on these questions. The SFs of epithelial and eukaryotic Nav channels select Na(+) by providing an optimal number and ligating strength of metal ligands as well as a rigid pore whose size fits the cognate Na(+) ideally. On the other hand, the SFs of bacterial Nav channels select Na(+), as the protein matrix attenuates ion-protein interactions relative to ion-solvent interactions by enlarging the pore and allowing water to enter, so the ion interacts indirectly with the conserved glutamates via bridging water molecules. This shows how these various SFs have adapted to the specific physicochemical properties of the native ion, using different strategies to select Na(+) among its contenders.

Hapalindoles from the cyanobacterium fischerella: potential sodium channel modulators.

Hapalindoles make up a large group of bioactive metabolites of the cyanobacterial order Stigonematales. 12-epi-Hapalindole E isonitrile, 12-epi-hapalindole C isonitrile, 12-epi-hapalindole J isonitrile, and hapalindole L from Fischerella are acutely toxic for insect larvae; however, the biochemical targets responsible for the biological activities of hapalindoles are not understood. We describe here the electron impact mass spectra of these four hapalindole congeners; their structures were confirmed by nuclear magnetic resonance spectroscopy. In combination with the presented mass spectra of (15)N-labeled species and their retention times on a gas chromatography capillary column, a rapid and reliable determination should be possible in future research. The bioactivity of these hapalindoles was tested on mammalian cells focusing on their effects in the BE(2)-M17 excitable human neuroblastoma cell line. The fluorescent dye Alamar Blue was applied to monitor cytotoxicity, fura-2 to evaluate changes in the cytosolic calcium concentrations, and bis-oxonol to detect effects on membrane potential. Data showed that the hapalindoles did not affect cell viability of the neuroblastoma cells, even when they were incubated for 72 h. Neither depolarization nor initiation of calcium influx was observed in the cells upon hapalindole treatment. However, the data provide evidence that hapalindoles are sodium channel-modulating neurotoxins. They inhibited veratridine-induced depolarization in a manner similar to that of neosaxitoxin. Our data suggest hapalindoles should be added to the growing number of neurotoxic secondary metabolites, such as saxitoxins and anatoxins, already known in freshwater cyanobacteria. As stable congeners, hapalindoles may be a risk in freshwater ecosystems or agricultural water usage and should therefore be considered in water quality assessment.

Volumetric deformation of live cells induced by pressure-activated cross-membrane ion transport.

In this work, we developed a method that allows precise control over changes in the size of a cell via hydrostatic pressure changes in the medium. Specifically, we show that a sudden increase, or reduction, in the surrounding pressure, in the physiologically relevant range, triggers cross-membrane fluxes of sodium and potassium ions in leukemia cell lines K562 and HL60, resulting in reversible volumetric deformation with a characteristic time of around 30 min. Interestingly, healthy leukocytes do not respond to pressure shocks, suggesting that the cancer cells may have evolved the ability to adapt to pressure changes in their microenvironment. A model is also proposed to explain the observed cell deformation, which highlights how the apparent viscoelastic response of cells is governed by the microscopic cross-membrane transport.