Epidemiology of arthropods envenomation in Brazil: a public health issue.

Brazil is located between the Equator and Tropic of Capricorn, which allows diverse climates, reliefs, and habitats for arthropods, which sting represents a risk to human health and a public health issue. This manuscript updates the epidemiological data of cases of human envenoming by spiders, scorpions, and insects with medical relevance in Brazil from 2010 to 2021. Epidemiological data were taken using the Brazilian Notifiable Diseases Information System. Statistics of non-parametric data used the Kruskal-Wallis followed by the Nemenyi test. On average, more than 145,000 envenomation and 145 deaths are recorded annually, and more than 60% of deaths are caused by scorpion bites. When the number of deaths was pondered by the number of cases with each arthropod, bees kill the most. Most stings cause mild symptoms and affect men of working age. The incidence decreases during the colder months, which is better noticeable in regions with well-defined seasons. The distribution is distinct among the regions: Southeast, Northeast, and South have the highest rate of bites. The growing number of cases of envenomation reported annually is a serious public health concern, especially involving scorpions, and highlights the importance of studying arthropod venom and improving the therapies.

Scorpion envenomation in Brazil: Current scenario and perspectives for containing an increasing health problem.

Opportunistic scorpion species can colonize urban environments, establishing high-density communities that enhance the chances of human accidents. This scenario has been taking place in Brazil, in which some Tityus species have taken city centers, causing an explosion in the number of scorpion envenoming cases. The characteristics of this scorpionism epidemic in Brazil is discussed in the present work. The number of Brazilian scorpion stings has surpassed 120,000 cases in 2017, and has been maintained above this number ever since, representing a more than 3-fold increase in 10 years, which was higher than the number of cases for most of the neglected tropical diseases in the country. The escalation in scorpionism cases is even higher in some regions of Brazil. Fortunately, the proportion of mild cases has also increased in the analyzed period, as well as the number of victims seeking for medical attention within the first hour after the accident. The species Tityus serrulatus, Tityus stigmurus, Tityus bahiensis, and Tityus obscurus are traditionally accountable for most of the scorpion accidents in different regions of Brazil, but other species deserve to be closely watched. Despite scorpionism being a notable health problem in Brazil, accident prevention and pest control regarding this venomous animal have not been properly addressed by the scientific community nor by policy makers. Therefore, this review also aims to point possible fields of research that could help to contain the aggravation of the current scorpionism landscape in Brazil.

Preliminary report on the hemagglutinating activity of the Scorpaena plumieri fish venom.

The scorpionfish Scorpaena plumieri is one of the most venomous fish species in the Brazilian coast. Amongst many biological activities, the S. plumieri fish venom (SpV) promotes hemagglutination. Although this activity appears to be associated to the presence of C-type lectins in the venom, it has not yet been chemically or functionally characterized. In the present work we sought to advance the characterization of the hemagglutinating activity associated to this venom. By fractionating SpV through saline precipitation followed by size exclusion chromatography we obtained two purified fractions - HF1 and HF3 - with Ca2+-dependent agglutinating activity against rabbit erythrocytes, which remained stable upon storage at 4 and -80oC. HF1 and HF3 were bacteriostatic against Gram-positive bacteria (Staphylococcus aureus), displaying minimum inhibitory concentration (MIC) of 50 and 200 µg/mL, respectively. In addition, a resazurin-based viability assay revealed that both fractions, at doses up to 370 µg/mL, were cytotoxic against tumor and non-tumor cell lines. Finally, a tendency towards edema formation could be detected when the fractions - particularly HF1 - were injected into mice footpads. We believe our data contribute to a better understanding of the biological properties of the so often neglected fish venoms.

Comparative venomic profiles of three spiders of the genus Phoneutria.

Background: Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.

Comparative venomic profiles of three spiders of the genus Phoneutria

Abstract Background: Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.

Comparative venomic profiles of three spiders of the genus Phoneutria

Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.(AU)

Preliminary report on the hemagglutinating activity of the Scorpaena plumieri fish venom

The scorpionfish Scorpaena plumieri is one of the most venomous fish species in the Brazilian coast. Amongst many biological activities, the S. plumieri fish venom (SpV) promotes hemagglutination. Although this activity appears to be associated to the presence of C-type lectins in the venom, it has not yet been chemically or functionally characterized. In the present work we sought to advance the characterization of the hemagglutinating activity associated to this venom. By fractionating SpV through saline precipitation followed by size exclusion chromatography we obtained two purified fractions - HF1 and HF3 - with Ca2+-dependent agglutinating activity against rabbit erythrocytes, which remained stable upon storage at 4 and -80oC. HF1 and HF3 were bacteriostatic against Gram-positive bacteria (Staphylococcus aureus), displaying minimum inhibitory concentration (MIC) of 50 and 200 μg/mL, respectively. In addition, a resazurin-based viability assay revealed that both fractions, at doses up to 370 μg/mL, were cytotoxic against tumor and non-tumor cell lines. Finally, a tendency towards edema formation could be detected when the fractions - particularly HF1 - were injected into mice footpads. We believe our data contribute to a better understanding of the biological properties of the so often neglected fish venoms.

Gene sequence analysis of toxins from the spider Phoneutria nigriventer revealed an intronless feature.

BACKGROUND: Phoneutria nigriventer spider venom contains several cysteine-rich peptide toxins that act on different ion channels. Despite extensive studies on its venom and description of cDNA sequences of several of its toxin precursors, the gene structure of these toxins remains unknown. METHODS: Genomic regions encoding the precursors of three previously characterized P. nigriventer toxins - PnTx1, PnTx2-5 and PnTx4(5-5) - were amplified by PCR using specific primers. PCR fragments were cloned and sequenced. Obtained sequences were compared with their corresponding cDNA sequences. RESULTS: The size of PCR fragments obtained and sequences corresponding to genomic regions encoding for the toxin precursors matched their cDNA sequences. CONCLUSIONS: Despite a few nucleotide substitutions in the genomic regions encoding for the toxin precursors when compared with cDNA sequences, the results of the present work indicate that P. nigriventer toxins do not contain introns in their genes sequences.

Gene sequence analysis of toxins from the spider Phoneutria nigriventer revealed an intronless feature

Phoneutria nigriventer spider venom contains several cysteine-rich peptide toxins that act on different ion channels. Despite extensive studies on its venom and description of cDNA sequences of several of its toxin precursors, the gene structure of these toxins remains unknown. Methods: Genomic regions encoding the precursors of three previously characterized P. nigriventer toxins - PnTx1, PnTx2-5 and PnTx4(5-5) - were amplified by PCR using specific primers. PCR fragments were cloned and sequenced. Obtained sequences were compared with their corresponding cDNA sequences. Results: The size of PCR fragments obtained and sequences corresponding to genomic regions encoding for the toxin precursors matched their cDNA sequences. Conclusions: Despite a few nucleotide substitutions in the genomic regions encoding for the toxin precursors when compared with cDNA sequences, the results of the present work indicate that P. nigriventer toxins do not contain introns in their genes sequences.(AU)

Differential effects of the recombinant toxin PnTx4(5-5) from the spider Phoneutria nigriventer on mammalian and insect sodium channels.

The toxin PnTx4(5-5) from the spider Phoneutria nigriventer is extremely toxic/lethal to insects but has no macroscopic behavioral effects observed in mice after intracerebral injection. Nevertheless, it was demonstrated that it inhibits the N-methyl-d-aspartate (NMDA) - subtype of glutamate receptors of cultured rat hippocampal neurons. PnTx4(5-5) has 63% identity to PnTx4(6-1), another insecticidal toxin from P. nigriventer, which can slow down the sodium current inactivation in insect central nervous system, but has no effect on Nav1.2 and Nav1.4 rat sodium channels. Here, we have cloned and heterologous expressed the toxin PnTx4(5-5) in Escherichia coli. The recombinant toxin rPnTx4(5-5) was tested on the sodium channel NavBg from the cockroach Blatella germanica and on mammalian sodium channels Nav1.2-1.6, all expressed in Xenopus leavis oocytes. We showed that the toxin has different affinity and mode of action on insect and mammalian sodium channels. The most remarkable effect was on NavBg, where rPnTx4(5-5) strongly slowed down channel inactivation (EC50 = 212.5 nM), and at 1 µM caused an increase on current peak amplitude of 105.2 ± 3.1%. Interestingly, the toxin also inhibited sodium current on all the mammalian channels tested, with the higher current inhibition on Nav1.3 (38.43 ± 8.04%, IC50 = 1.5 µM). Analysis of activation curves on Nav1.3 and Nav1.5 showed that the toxin shifts channel activation to more depolarized potentials, which can explain the sodium current inhibition. Furthermore, the toxin also slightly slowed down sodium inactivation on Nav1.3 and Nav1.6 channels. As far as we know, this is the first araneomorph toxin described which can shift the sodium channel activation to more depolarized potentials and also slows down channel inactivation.

PnPP-19, a Synthetic and Nontoxic Peptide Designed from a Phoneutria nigriventer Toxin, Potentiates Erectile Function via NO/cGMP.

PURPOSE: We designed a peptide, PnPP-19, comprising the potential active core of the Phoneutria nigriventer native toxin PnTx2-6. We investigated its role on erectile function, and its toxicity and immunogenicity. MATERIALS AND METHODS: Erectile function was evaluated by the intracavernous pressure-to-mean arterial pressure ratio during electrical field stimulation on rat pelvic ganglia. Cavernous strips were contracted with phenylephrine and relaxation was induced by electrical field stimulation with or without PnPP-19 (10(-8) M). Activity on sodium channels was evaluated by electrophysiological screening of transfected channels on Xenopus oocytes and dorsal root ganglion cells. Antibodies were detected by indirect enzyme-linked immunosorbent assay in mice previously treated with the peptide. Histopathological studies were performed with mouse organs treated with different doses of PnPP-19. RESULTS: PnPP-19 was able to potentiate erection at 4 and 8 Hz in vivo and ex vivo. It showed no toxicity and low immunogenicity in mice, and did not affect sodium channels or rat hearts. PnPP-19 increased cyclic guanosine monophosphate levels at 8 Hz. This effect was inhibited by L-NAME (10(-4) M). Erectile function was partially inhibited by 7-nitroindazole (10(-5) M), a selective inhibitor of neuronal nitric oxide synthase. CONCLUSIONS: PnPP-19 potentiates erection in vivo and ex vivo via the nitric oxide/cyclic guanosine monophosphate pathway. It does not affect sodium channels or rat hearts and shows no toxicity and low immunogenicity. These findings make it a promising candidate as a novel drug in the therapy of erectile dysfunction.

New insights on arthropod toxins that potentiate erectile function.

The use of natural substances for the treatment of diseases or injuries is an ancient practice of many cultures. According to folklore, natural aphrodisiacs may help to raise libido and increase desire. The supposed aphrodisiacs mainly include a plethora of preparations of plants, among other substances. However, the real boundary between myth and reality has not been established yet in most cases and such boundaries must be drawn by scientific methods. A growing interest of the scientific community has been focused on animal venoms, especially those from arthropods, i.e. spiders and scorpions, which cause priapism, a prolonged and painful erection. This review highlights the studies that have been performed with venoms and toxins from arthropods known to cause priapism, among other toxic symptoms, pointing out some pharmacological approaches for better understanding this effect. To date, the venom of some spiders, mainly Phoneutria nigriventer, and scorpions, such as the yellow South American scorpion Tityus serrulatus, among others, have been known to cause priapism. Since erectile dysfunction (ED) is a growing health problem in the world, more common in patients with vascular diseases as diabetes and hypertension, the use of animal venoms and toxins as pharmacological tools could not only shed light to the mechanisms involved in erectile function, but also represent a possible model for new drugs to treat ED. Unfortunately, attempts to correlate the structure of those priapism-related toxins were unfruitful. Such difficulties lie firstly on the poor data concerning purified priapism-related toxins, instead of whole venoms and/or semi-purified fractions, and secondly, on the scarce available primary sequences and structural data, mainly from spider toxins. It has been shown that all these toxins modify the sodium (Na(+)) channel activity, mostly slowing down its inactivation current. Improving the knowledge on the tertiary structure of these toxins could provide a key in the search of a new drug for ED treatment.

Molecular basis of decreased Kir4.1 function in SeSAME/EAST syndrome.

SeSAME/EAST syndrome is a channelopathy consisting of a hypokalemic, hypomagnesemic, metabolic alkalosis associated with seizures, sensorineural deafness, ataxia, and developmental abnormalities. This disease links to autosomal recessive mutations in KCNJ10, which encodes the Kir4.1 potassium channel, but the functional consequences of these mutations are not well understood. In Xenopus oocytes, all of the disease-associated mutant channels (R65P, R65P/R199X, G77R, C140R, T164I, and A167V/R297C) had decreased K(+) current (0 to 23% of wild-type levels). Immunofluorescence demonstrated decreased surface expression of G77R, C140R, and A167V expressed in HEK293 cells. When we coexpressed mutant and wild-type subunits to mimic the heterozygous state, R199X, C140R, and G77R currents decreased to 55, 40, and 20% of wild-type levels, respectively, suggesting that carriers of these mutations may present with an abnormal phenotype. Because Kir4.1 subunits can form heteromeric channels with Kir5.1, we coexpressed the aforementioned mutants with Kir5.1 and found that currents were reduced at least as much as observed when we expressed mutants alone. Reduction of pH(i) from approximately 7.4 to 6.8 significantly decreased currents of all mutants except R199X but did not affect wild-type channels. In conclusion, perturbed pH gating may underlie the loss of channel function for the disease-associated mutant Kir4.1 channels and may have important physiologic consequences.

Small molecule disruption of G beta gamma signaling inhibits the progression of heart failure.

RATIONALE: Excess signaling through cardiac Gbetagamma subunits is an important component of heart failure (HF) pathophysiology. They recruit elevated levels of cytosolic G protein-coupled receptor kinase (GRK)2 to agonist-stimulated beta-adrenergic receptors (beta-ARs) in HF, leading to chronic beta-AR desensitization and downregulation; these events are all hallmarks of HF. Previous data suggested that inhibiting Gbetagamma signaling and its interaction with GRK2 could be of therapeutic value in HF. OBJECTIVE: We sought to investigate small molecule Gbetagamma inhibition in HF. METHODS AND RESULTS: We recently described novel small molecule Gbetagamma inhibitors that selectively block Gbetagamma-binding interactions, including M119 and its highly related analog, gallein. These compounds blocked interaction of Gbetagamma and GRK2 in vitro and in HL60 cells. Here, we show they reduced beta-AR-mediated membrane recruitment of GRK2 in isolated adult mouse cardiomyocytes. Furthermore, M119 enhanced both adenylyl cyclase activity and cardiomyocyte contractility in response to beta-AR agonist. To evaluate their cardiac-specific effects in vivo, we initially used an acute pharmacological HF model (30 mg/kg per day isoproterenol, 7 days). Concurrent daily injections prevented HF and partially normalized cardiac morphology and GRK2 expression in this acute HF model. To investigate possible efficacy in halting progression of preexisting HF, calsequestrin cardiac transgenic mice (CSQ) with extant HF received daily injections for 28 days. The compound alone halted HF progression and partially normalized heart size, morphology, and cardiac expression of HF marker genes (GRK2, atrial natriuretic factor, and beta-myosin heavy chain). CONCLUSIONS: These data suggest a promising therapeutic role for small molecule inhibition of pathological Gbetagamma signaling in the treatment of HF.

PKA and PKC partially rescue long QT type 1 phenotype by restoring channel-PIP2 interactions.

Long-QT syndrome causes torsade de pointes arrhythmia, ventricular fibrillation, and sudden death. The most commonly inherited form of long-QT syndrome, LQT1, is due to mutations on the potassium channel gene KCNQ1, which forms one of the main repolarizing cardiac K(+) channels, IKs. IKs has been shown to be regulated by both beta-adrenergic receptors, via protein kinase A (PKA), and by Gq protein coupled receptors (GqPCR), via protein kinase C (PKC) and phosphatidylinositol 4,5-bisphosphate (PIP(2)). These regulatory pathways were shown to crosstalk, with PKA phosphorylation increasing the apparent affinity of IKs to PIP(2). Here we study the effects of LQT1 mutations in putative PIP(2)-KCNQ1 interaction sites on regulation of IKs by PKA and GqPCR. The effect of the LQT1 mutations on IKs regulation was tested for mutations in conserved, positively charged amino acids, located in four distinct cytoplamic domains of the KCNQ1 subunit: R174C (S2-S3), R243C (S4-S5), R366Q (proximal c-terminus) and R555C (distal c-terminus). Mutations in the c-terminus of IKs (both proximal and distal) enhanced channel sensitivity to changes in membrane PIP(2) levels, consistent with a decrease in apparent channel-PIP(2) affinity. These mutant channels were more sensitive to inhibition caused by receptor mediated PIP(2)-depletion and more sensitive to stimulation of PIP(2) production, by overexpression of phosphatidylinositol-4-phosphate-5-kinase (PI5-kinase). In addition, c-terminus mutants showed a potentiated regulation by PKA. On the other hand, for the two cytoplasmic-loop mutations, an impaired activation by PKA was observed. The effects of the mutations on PKC stimulation of the channel paralleled the effects on PKA stimulation, suggesting that both regulatory inputs are similarly affected by the mutations. We tested whether PKC-mediated activation of IKs, similarly to the PKA-mediated activation, can regulate the channel response to PIP(2). After PKC activation, channel was less sensitive to changes in membrane PIP(2) levels, consistent with an increase in apparent channel-PIP(2) affinity. PKC-activated channel was less sensitive to inhibition caused by block of synthesis of PIP(2) by the lipid kinase inhibitor wortmannin and less sensitive to stimulation of PIP(2) production. Our data indicates that stimulation by PKA and PKC can partially rescue LQT1 mutant channels with weakened response to PIP(2) by strengthening channel interactions with PIP(2).

PKC activation and PIP(2) depletion underlie biphasic regulation of IKs by Gq-coupled receptors.

KCNQ1 is co-assembled with KCNE1 subunits in the heart to form the cardiac delayed rectifier K(+) current (IKs), which is one of the main currents responsible for myocyte repolarization. The most commonly inherited form of cardiac arrhythmias, long-QT syndrome type 1 (LQT1), is due to mutations on KCNQ1. Gq-coupled receptors (GqPCRs) are known to mediate positive inotropism in human ventricular myocardium. The mechanism of IKs current modulation by GqPCRs remains incompletely understood. Here we studied the molecular mechanisms underlying Gq regulation of the IKs channel. Heterologously expressed IKs (human KCNQ1/KCNE1 subunits) was measured in Xenopus oocytes, expressed together with GqPCRs. Our data from several GqPCRs shows that IKs is regulated in a biphasic manner, showing both an activation and an inhibition phase. Receptor-mediated inhibition phase was irreversible when recycling of agonist-sensitive pools of phosphatidylinositol-4,5-bisphosphate (PIP2) was blocked by the lipid kinase inhibitor wortmannin. In addition, stimulation of PIP(2) production, by overexpression of phosphatidylinositol-4-phosphate-5-kinase (PIP5-kinase), decreased receptor-mediated inhibition. The receptor-mediated activation phase was inhibited by the PKC inhibitor calphostin C and by a mutation in a putative PKC phosphorylation site in the KCNE1 subunit. Our results indicate that the depletion of membrane PIP(2) underlies receptor-mediated inhibition of IKs and that phosphorylation by PKC of the KCNE1 subunit underlies the GqPCR-mediated channel activation.

Structure and activity analysis of two spider toxins that alter sodium channel inactivation kinetics.

In this work, Phoneutria nigriventer toxins PnTx2-5 and PnTx2-6 were shown to markedly delay the fast inactivation kinetics of neuronal-type sodium channels. Furthermore, our data show that they have significant differences in their interaction with the channel. PnTx2-6 has an affinity 6 times higher than that of PnTx2-5, and its effects are not reversible within 10-15 min of washing. PnTx2-6 partially (59%) competes with the scorpion alpha-toxin AaHII, but not with the scorpion beta-toxin CssIV, thus suggesting a mode of action similar to that of site 3 toxins. However, PnTx2-6 is not removed by strong depolarizing pulses, as in the known site 3 toxins. We have also established the correct PnTx2-5 amino acid sequence and confirmed the sequence of PnTx2-6, in both cases establishing that the cysteines are in their oxidized form. A structural model of each toxin is proposed. They show structures with poor alpha-helix content. The model is supported by experimental and theoretical tests. A likely binding region on PnTx2-5 and PnTx2-6 is proposed on the basis of their different affinities and sequence differences.

Protein kinase A modulates PLC-dependent regulation and PIP2-sensitivity of K+ channels.

Neurotransmitter and hormone regulation of cellular function can result from a concomitant stimulation of different signaling pathways. Signaling cascades are strongly regulated during disease and are often targeted by commonly used drugs. Crosstalk of different signaling pathways can have profound effects on the regulation of cell excitability. Members of all the three main structural families of potassium channels: inward-rectifiers, voltage-gated and 2-P domain, have been shown to be regulated by direct phosphorylation and Gq-coupled receptor activation. Here we test members of each of the three families, Kir3.1/Kir3.4, KCNQ1/KCNE1 and TREK-1 channels, all of which have been shown to be regulated directly by phosphatidylinositol bisphosphate (PIP2). The three channels are inhibited by activation of Gq-coupled receptors and are differentially regulated by protein kinase A (PKA). We show that Gq-coupled receptor regulation can be physiologically modulated directly through specific channel phosphorylation sites. Our results suggest that PKA phosphorylation of these channels affects Gq-coupled receptor inhibition through modulation of the channel sensitivity to PIP2.

Electrophysiological characterization and molecular identification of the Phoneutria nigriventer peptide toxin PnTx2-6.

A cDNA with 403 nucleotides encoding the precursor of the toxin PnTx2-6 was cloned and sequenced. Subsequent analysis revealed that the precursor begins with a signal peptide and a glutamate-rich propeptide. The succeeding peptide confirmed the reported sequence of PnTx2-6. The purified toxin exerted complex effects on Na(+) current of frog skeletal muscle. There was a marked decrease of the inactivation kinetics, and a shift to hyperpolarizing potentials of both the Na(+) conductance and the steady-state inactivation voltage dependences, along with a reduction of the current amplitude. The concentration dependence of the modified current suggests a K(D) of 0.8 microM for the toxin-channel complex.

Use of a purified and functional recombinant calcium-channel beta4 subunit in surface-plasmon resonance studies.

Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Ca(v) and at least two auxiliary subunits alpha(2)delta and beta. The beta subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native beta subunit. Here, we report the purification of a recombinant calcium-channel beta(4) subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Ca(v)-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the beta(4) subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-beta(4) subunit for the full-length Ca(v)2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Ca(v)/beta interaction and (ii) crystallographic studies of the calcium-channel beta(4) subunit.