Emilio Salceda

The Sea Anemone Toxins BgII and BgIII Prolong the Inactivation Time Course of the Tetrodotoxin-Sensitive Sodium Current in Rat Dorsal Root Ganglion Neurons

We have characterized the effects of BgII and BgIII, two sea anemone peptides with almost identical sequences (they only differ by a single amino acid), on neuronal sodium currents using the whole-cell patch-clamp technique. Neurons of dorsal root ganglia of Wistar rats (P5-9) in primary culture (Leibovitz′s L15 medium; 37°C, 95% air/5% CO2) were used for this study (n = 154). These cells express two sodium current subtypes: tetrodotoxin-sensitive (TTX-S; K i = 0.3 nM) and tetrodotoxin-resistant (TTX-R;K i = 100 μM). Neither BgII nor BgIII had significant effects on TTX-R sodium current. Both BgII and BgIII produced a concentration-dependent slowing of the TTX-S sodium current inactivation (IC50 = 4.1 ± 1.2 and 11.9 ± 1.4 μM, respectively), with no significant effects on activation time course or current peak amplitude. For comparison, the concentration-dependent action of Anemonia sulcata toxin II (ATX-II), a well characterized anemone toxin, on the TTX-S current was also studied. ATX-II also produced a slowing of the TTX-S sodium current inactivation, with an IC50 value of 9.6 ± 1.2 μM indicating that BgII was 2.3 times more potent than ATX-II and 2.9 times more potent than BgIII in decreasing the inactivation time constant (τh) of the sodium current in dorsal root ganglion neurons. The action of BgIII was voltage-dependent, with significant effects at voltages below −10 mV. Our results suggest that BgII and BgIII affect voltage-gated sodium channels in a similar fashion to other sea anemone toxins and α-scorpion toxins.

A novel sea anemone peptide that inhibits acid-sensing ion channels

Sea anemones produce ion channels peptide toxins of pharmacological and biomedical interest. However, peptides acting on ligand-gated ion channels, including acid-sensing ion channel (ASIC) toxins, remain poorly explored. PhcrTx1 is the first compound characterized from the sea anemone Phymanthus crucifer, and it constitutes a novel ASIC inhibitor. This peptide was purified by gel filtration, ion-exchange and reversed-phase chromatography followed by biological evaluation on ion channels of isolated rat dorsal root ganglia (DRG) neurons using patch clamp techniques. PhcrTx1 partially inhibited ASIC currents (IC50∼100 nM), and also voltage-gated K(+) currents but the effects on the peak and on the steady state currents were lower than 20% in DRG neurons, at concentrations in the micromolar range. No significant effect was observed on Na(+) voltage-gated currents in DRG neurons. The N-terminal sequencing yielded 32 amino acid residues, with a molecular mass of 3477 Da by mass spectrometry. No sequence identity to other sea anemone peptides was found. Interestingly, the bioinformatic analysis of Cys-pattern and secondary structure arrangement suggested that this peptide presents an Inhibitor Cystine Knot (ICK) scaffold, which has been found in other venomous organisms such as spider, scorpions and cone snails. Our results show that PhcrTx1 represents the first member of a new structural group of sea anemones toxins acting on ASIC and, with much lower potency, on Kv channels. Moreover, this is the first report of an ICK peptide in cnidarians, suggesting that the occurrence of this motif in venomous animals is more ancient than expected.

CgNa, a type I toxin from the giant Caribbean sea anemone Condylactis gigantea shows structural similarities to both type I and II toxins, as well as distinctive structural and functional properties 1

CgNa (Condylactis gigantea neurotoxin) is a 47-amino-acid- residue toxin from the giant Caribbean sea anemone Condylactis gigantea. The structure of CgNa, which was solved by 1H-NMR spectroscopy, is somewhat atypical and displays significant homology with both type I and II anemone toxins. CgNa also displays a considerable number of exceptions to the canonical structural elements that are thought to be essential for the activity of this group of toxins. Furthermore, unique residues in CgNa define a characteristic structure with strong negatively charged surface patches. These patches disrupt a surface-exposed cluster of hydrophobic residues present in all anemone-derived toxins described to date. A thorough characterization by patch-clamp analysis using rat DRG (dorsal root ganglion) neurons indicated that CgNa preferentially binds to TTX-S (tetrodotoxin-sensitive) voltage-gated sodium channels in the resting state. This association increased the inactivation time constant and the rate of recovery from inactivation, inducing a significant shift in the steady state of inactivation curve to the left. The specific structural features of CgNa may explain its weaker inhibitory capacity when compared with the other type I and II anemone toxins.

BgK anemone toxin inhibits outward K(+) currents in snail neurons.

We studied the effects of BgK toxin on outward K(+) currents in isolated neurons of the snail Helix aspersa, using the whole cell patch clamp technique. BgK partially and reversibly blocked K(+) currents in the 1 pM to 100 nM concentration range (n=53). The dose-response curve for BgK current inhibition had a maximum blocking effect at 100 nM. Our results indicate that BgK is a potent, apparently non-selective, K(+) channel blocker in molluscan neurons.

Antinociception produced by Thalassia testudinum extract BM-21 is mediated by the inhibition of acid sensing ionic channels by the phenolic compound thalassiolin B

BackgroundAcid-sensing ion channels (ASICs) have a significant role in the sensation of pain and constitute an important target for the search of new antinociceptive drugs. In this work we studied the antinociceptive properties of the BM-21 extract, obtained from the sea grass Thalassia testudinum, in chemical and thermal models of nociception in mice. The action of the BM-21 extract and the major phenolic component isolated from this extract, a sulphated flavone glycoside named thalassiolin B, was studied in the chemical nociception test and in the ASIC currents of the dorsal root ganglion (DRG) neurons obtained from Wistar rats.ResultsBehavioral antinociceptive experiments were made on male OF-1 mice. Single oral administration of BM-21 produced a significant inhibition of chemical nociception caused by acetic acid and formalin (specifically during its second phase), and increased the reaction time in the hot plate test. Thalassiolin B reduced the licking behavior during both the phasic and tonic phases in the formalin test. It was also found that BM-21 and thalassiolin B selectively inhibited the fast desensitizing (τ < 400 ms) ASIC currents in DRG neurons obtained from Wistar rats, with a nonsignificant action on ASIC currents with a slow desensitizing time-course. The action of thalassiolin B shows no pH or voltage dependence nor is it modified by steady-state ASIC desensitization or voltage. The high concentration of thalassiolin B in the extract may account for the antinociceptive action of BM-21.ConclusionsTo our knowledge, this is the first report of an ASIC-current inhibitor derived of a marine-plant extract, and in a phenolic compound. The antinociceptive effects of BM-21 and thalassiolin B may be partially because of this action on the ASICs. That the active components of the extract are able to cross the blood-brain barrier gives them an additional advantage for future uses as tools to study pain mechanisms with a potential therapeutic application.

Effects of ApC, a sea anemone toxin, on sodium currents of mammalian neurons

We have characterized the actions of ApC, a sea anemone polypeptide toxin isolated from Anthopleura elegantissima, on neuronal sodium currents (I(Na)) using current and voltage-clamp techniques. Neurons of the dorsal root ganglia of Wistar rats (P5-9) in primary culture were used for this study. These cells express tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) I(Na). In current-clamp experiments, application of ApC increased the average duration of the action potential. Under voltage-clamp conditions, the main effect of ApC was a concentration-dependent increase in the TTX-S I(Na) inactivation time course. No significant effects were observed on the activation time course or on the current peak-amplitude. ApC also produced a hyperpolarizing shift in the voltage at which 50% of the channels are inactivated and caused a significant decrease in the voltage dependence of Na+ channel inactivation. No effects were observed on TTX-R I(Na). Our results suggest that ApC slows the conformational changes required for fast inactivation of the mammalian Na+ channels in a form similar to other site-3 toxins, although with a greater potency than ATX-II, a highly homologous anemone toxin.

Electrophysiological characterization of a novel small peptide from the venom of Conus californicus that targets voltage-gated neuronal Ca2+ channels

Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. californicus has been shown to be distantly related to all Conus species, but showing unusual biological features. We report a novel peptide isolated from C. californicus with a significant inhibitory action over neuronal voltage-gated calcium channels. The new toxin is formed by 13-amino acid residues with two disulfide bonds, whose sequence (NCPAGCRSQGCCM) is strikingly different from regular ω-conotoxins. In the HPLC purification procedure, the venom fraction eluted in the first 10-15 min produced a significant decrease (54% ± 3%) of the Ca(2+) current in Xenopus laevis oocytes transfected with purified rat-brain mRNA. A specific peptide obtained from the elution at 13 min decreased the Ca(2+) current in the adult rat dorsal-root ganglion neurons in a primary culture by 34% ± 2%. The cysteine pattern of this peptide corresponds to the framework XVI described for the M-superfamily of conopeptides and is unprecedented among Conus peptides acting on Ca(2+) channels.

α-Dendrotoxin inhibits the ASIC current in dorsal root ganglion neurons from rat

Dendrotoxins are a group of peptide toxins purified from the venom of several mamba snakes. α-Dendrotoxin (α-DTx, from the Eastern green mamba Dendroaspis angusticeps) is a well-known blocker of voltage-gated K(+) channels and specifically of K(v)1.1, K(v)1.2 and K(v)1.6. In this work we show that α-DTx inhibited the ASIC currents in DRG neurons (IC50=0.8 μM) when continuously perfused during 25 s (including a 5 s pulse to pH 6.1), but not when co-applied with the pH drop. Additionally, we show that α-DTx abolished a transient component of the outward current that, in some experiments, appeared immediately after the end of the acid pulse. Our data indicate that α-DTx inhibits ASICs in the high nM range while some Kv are inhibited in the low nM range. The α-DTx selectivity and its potential interaction with ASICs should be taken in consideration when DTx is used in the high nM range.

Microcomputer program for automated action potential waveform analysis.

A program for action potential waveform analysis based on a PC compatible computer is described. Single or averaged action potentials are analyzed by obtaining its first derivative and using criteria which allow automatic measurement of several action potential components, including: depolarization rate, repolarization rate, amplitude, duration, resting membrane potential and afterhyperpolarization amplitude and slope. Data can be imported from pClamp (Axon Instruments) and exported to other software such as Excel, Sigmaplot and MatLab for example.

Functional diversity of secreted cestode Kunitz proteins: Inhibition of serine peptidases and blockade of cation channels

We previously reported a multigene family of monodomain Kunitz proteins from Echinococcus granulosus (EgKU-1-EgKU-8), and provided evidence that some EgKUs are secreted by larval worms to the host interface. In addition, functional studies and homology modeling suggested that, similar to monodomain Kunitz families present in animal venoms, the E. granulosus family could include peptidase inhibitors as well as channel blockers. Using enzyme kinetics and whole-cell patch-clamp, we now demonstrate that the EgKUs are indeed functionally diverse. In fact, most of them behaved as high affinity inhibitors of either chymotrypsin (EgKU-2-EgKU-3) or trypsin (EgKU-5-EgKU-8). In contrast, the close paralogs EgKU-1 and EgKU-4 blocked voltage-dependent potassium channels (Kv); and also pH-dependent sodium channels (ASICs), while showing null (EgKU-1) or marginal (EgKU-4) peptidase inhibitory activity. We also confirmed the presence of EgKUs in secretions from other parasite stages, notably from adult worms and metacestodes. Interestingly, data from genome projects reveal that at least eight additional monodomain Kunitz proteins are encoded in the genome; that particular EgKUs are up-regulated in various stages; and that analogous Kunitz families exist in other medically important cestodes, but not in trematodes. Members of this expanded family of secreted cestode proteins thus have the potential to block, through high affinity interactions, the function of host counterparts (either peptidases or cation channels) and contribute to the establishment and persistence of infection. From a more general perspective, our results confirm that multigene families of Kunitz inhibitors from parasite secretions and animal venoms display a similar functional diversity and thus, that host-parasite co-evolution may also drive the emergence of a new function associated with the Kunitz scaffold.