Differences in the binding sites of two site-3 sodium channel toxins

Site-3 toxins from scorpion and sea anemone bind to Na channels and selectively inhibit current decay. Anthopleurins A and B (ApA and ApB, respectively), toxins found in the venom of the sea anemone Anthopleura xanthogrammica, bind to closed states of mammalian skeletal and cardiac Na channels with differing affinities which arise from differences in first-order toxin/channel dissociation rate constants, koff. Using chimera comprising domain interchanges between channel isoforms, we examined the structural basis of this differential affinity. Toxin/channel association rates, kon, were similar for both toxins and both parental channels. Domain 4 determined koff for ApA, while ApB dissociated from all tested chimera in a cardiac-like manner. To probe this surprising difference between two such closely related toxins, we examined the interaction of chimeric channels with a form of ApB in which the two nonconserved basic residues, Arg-12 and Lys-49, were converted to the corresponding neutral amino acids from ApA. In the chimera comprising domain 1 from the cardiac muscle isoform and domains 2-4 from the skeletal muscle isoform, toxin dissociated at a rate intermediate between those of the parental channels. We conclude that the differential component of ApA binding is controlled by domain 4 and that some component of ApB binding is not shared by ApA. This additional component probably binds to an interface between channel domains and is partly mediated by toxin residues Arg-12 and Lys-49.     

Modulation of Na+ channel inactivation by the beta 1 subunit: a deletion analysis

Na+ currents recorded from Xenopus oocytes expressing the Na+ channel alpha subunit alone inactivate with two exponential components. The slow component predominates in monomeric channels, while co-expression with the beta 1 subunit favors the fast component. Macropatch recordings show that the relative rates of these components are much greater than previously estimated from two-electrode measurements (approximately 30-fold vs approximately 5-fold). A re-assessment of steady-state inactivation, h infinity (V), shows that there is no depolarized shift of the slow component, provided a sufficiently long prepulse duration and repetition interval are used to achieve steady-state entry and recovery from inactivation, respectively. Deletion mutagenesis of the beta 1 subunit was used to define which regions of the subunit are required to modulate inactivation kinetics. The carboxy tail, comprising the entire predicted intracellular domain, can be deleted without a loss of activity; whereas small deletions in the extracellular amino domain or the signal peptide totally disrupt function.     

Fast inactivation in Shaker K+ channels. Properties of ionic and gating currents

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as "charge immobilization" (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567-590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at -90 mV return potential changed from a single fast component to at least two components, the slower requiring approximately 200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at -120 and -90 mV. In contrast, at higher potentials (-70 and -50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of "parallel" inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH2-terminus region.     

Suicide inactivation of cytochrome P450 by midchain and terminal acetylenes. A mechanistic study of inactivation of a plant lauric acid omega-hydroxylase

Incubation of Vicia sativa microsomes, containing cytochrome P450-dependent lauric acid omega-hydroxylase (omega-LAH), with [1-(14)C]11-dodecynoic acid (11-DDYA) generates a major metabolite characterized as 1,12-dodecandioic acid. In addition to time- and concentration-dependent inactivation of lauric acid and 11-DDYA oxidation, irreversible binding of 11-DDYA (200 pmol of 11-DDYA bound/mg of microsomal protein) at a saturating concentration of 11-DDYA was observed. SDS-polyacrylamide gel electrophoresis analysis showed that 30% of the label was associated with several protein bands of about 53 kDa. The presence of beta-mercaptoethanol in the incubate reduces 1,12-dodecandioic acid formation and leads to a polar metabolite resulting from the interaction of oxidized 11-DDYA with the nucleophile. Although the alkylation of proteins was reduced, the lauric acid omega-hydroxylase activity was not restored, suggesting an active site-directed inactivation mechanism. Similar results were obtained when reconstituted mixtures of cytochrome P450 from family CYP4A from rabbit liver were incubated with 11-DDYA. In contrast, both 11- and 10-DDYA resulted in covalent labeling of the cytochrome P450 2B4 protein and irreversible inhibition of activity. These results demonstrate that acetylenic analogues of substrate are efficient mechanism-based inhibitors and that a correlation between the position of the acetylenic bond in the inhibitor and the regiochemistry of cytochromes P450 oxygenation is essential for enzyme inactivation.     

Comparative analyses by HPLC and the sodium channel and saxiphilin 3H-saxitoxin receptor assays for paralytic shellfish toxins in crustaceans and molluscs from tropical North West Australia

The increased frequency and distribution of red tides requires the development of high-throughput detection methods for paralytic shellfish toxins (PST). Community ethics also requires that there be a reduced reliance upon the standard mouse bioassay. A biomolecular assay such as the sodium channel 3H-saxitoxin binding assay can satisfy both of these requirements but may be compromised by cross-reactivity with the structurally unrelated tetrodotoxins (TTX). This study utilised the sodium channel assay but also an alternative 3H-saxitoxin binding assay based upon a saxiphilin isoform from the centipede Ethmostigmus rubripes to screen for PSTs. Saxiphilin is a novel transferrin which binds saxitoxin (STX) but differs from the sodium channel in not having any measurable affinity for TTX. A detailed analysis of toxin composition was achieved by high performance liquid chromatography (HPLC). Various crustaceans and molluscs accumulate PSTs and TTX, thus proving useful biomarkers for these toxins in their immediate environment and an ideal challenge to the detection and analysis of PSTs in this presumptive screening program. Also, there has been little investigation of PSTs in invertebrates from the Indian Ocean so this region was selected to extend our knowledge of the distribution of these toxins. 190 crabs and shellfish encompassing 31 species were collected from reefs along the North-West Australian coast and tested for PSTs and TTX by sodium channel and saxiphilin bioassays as well as HPLC. PSTs were detected in 18 species of crabs and shellfish of the 31 species tested. Eight of these species have not been previously described as toxic, these being the crabs Euzanthus exsculptus, Lophozozymus octodentatus, Metopograpsus frontalis, Pilumnus pulcher, Platypodia pseudogranulosa and Portunus pelagicus, and the molluscs Tectus fenestratus and Trochus hanleyanus. By HPLC, only one or both of STX and decarbamoyl-STX was detected in any extract. Some extracts markedly inhibited 3H-saxitoxin binding by the sodium channel but not by saxiphilin. The close agreement between toxin quantification by the PST specific methods of HPLC and the saxiphilin bioassay is indicative that the additional toxicity detected by the sodium channel assay is TTX.     

Plausibility and recovery from garden paths: An eye-tracking study.

Three eye-tracking experiments investigated plausibility effects on recovery from misanalysis in sentence comprehension. On the initially favored analysis, a noun phrase served as the object of the preceding verb. On the ultimately correct analysis, it served as the subject of a main clause in Experiments 1 and 3 and of a complement clause in Experiment 2. If the object analysis was implausible, disruption occurred during processing of the noun phrase. If it was plausible, disruption occurred after disambiguation. In Experiment 3, discourse context affected plausibility of the initial analysis and subsequent reanalysis. The authors argue that readers performed substantial semantic processing on the initial analysis and committed strongly when it was plausible. Experiment 3 showed that these effects were not due to selectional restrictions or word co-occurrences and that the interpretation of the target sentence was not computed in isolation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Psychotherapy and recovery from schizophrenia: A review of potential applications and need for future study.

Recovery from schizophrenia has been conceptualized to involve not only symptom remission of symptoms and achievement of psychosocial milestones but also subjective changes in how persons appraise their lives and the extent to which they experience themselves as meaningful agents in the world. In this article we review the potential of individual psychotherapy to address these more subjective aspects of recovery. Literature on the effectiveness of psychotherapy for persons with schizophrenia is discussed, and two different paths by which psychotherapy might modify self-experience are described. First, we detail how psychotherapy could be conceptualized and tailored to help persons with schizophrenia to construct richer and fuller narrative accounts of their lives, including their strengths, challenges, losses, and hopes. Second, we explore how psychotherapy could target the capacity for metacognition or thinking about thinking, assisting persons with psychosis to become able to think about themselves and others in a generally more complex and flexible manner. The needs for future research are discussed along with a commentary on how current evidence- and skill-based treatments may contain key elements that could be considered psychotherapeutic. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Irreversible inactivation of Caldariomyces fumago chloroperoxidase by hydrogen peroxide. A kinetic study in chloride and bromide system

The rate constants for the H2O2-induced irreversible inactivation (kinact) of chloroperoxidase from Caldariomyces fumago evaluated from the analysis of complete kinetic curves of chlorination or bromination of monochlorodimedon were found to follow the rate law kinact = k[H2O2]/(K + [H2O2]) with k = 0.009 > or = 0.002 and 0.0095 > or = 0.010 s-1 and K = (13 > or = 4) x 10(-3) and (9 > or = 2) x 10(-3) M in the presence of 0.01 M chloride and bromide, respectively, at pH 2.75 and 25 degrees C. The data show that chloroperoxidase investigated is more than by a factor of 10 less resistant toward hydrogen peroxide compared to horseradish peroxidase. The possible reason for it and the biotechnological implications are briefly discussed.     

Heparinase II from Flavobacterium heparinum. Role of cysteine in enzymatic activity as probed by chemical modification and site- directed mutagenesis

Heparinase II (no EC number) is one of three lyases isolated from Flavobacterium heparinum that degrade heparin-like complex polysaccharides. Heparinase II is unique among the heparinases in that it has broad substrate requirements and possesses the ability to degrade both heparin and heparan sulfate-like regions of glycosaminoglycans. This study set out to investigate the role of cysteines in heparinase II activity. Through a series of chemical modification experiments, it was found that one of the three cysteines in heparinase II is surface-accessible and possesses unusual chemical reactivity toward cysteine-specific chemical modifying reagents. Substrate protection experiments suggest that this surface-accessible cysteine is proximate to the active site, since addition of substrate shields the cysteine from modifying reagents. The cysteine, present in an ionic environment, was mapped by radiolabeling with N-[3H]ethylmaleimide and identified as cysteine 348. Site-directed mutagenesis of cysteine 348 to an alanine resulted in loss of activity toward heparin but not heparan sulfate, indicating that cysteine 348 is required for heparinase II activity toward heparin but is not essential for the breakdown of heparan sulfate. Furthermore, we show in this study that cysteine 164 and cysteine 189 are functionally unimportant for heparinase II.     

Augmentation of the cardiac natriuretic peptides by beta-receptor antagonism: evidence from a population-based study

OBJECTIVES: The present retrospective analysis of data derived from a population-based study examined the relationship between intake of beta-receptor antagonists and plasma concentrations of the cardiac natriuretic peptides and their second messenger. BACKGROUND: Beta-receptor antagonists are widely used for treatment of cardiovascular disease. In addition to direct effects on heart rate and cardiac contractility, recent evidence suggests that beta-receptor antagonists may also modulate the cross talk between the sympathetic nervous system and the cardiac natriuretic peptide system. METHODS: Plasma concentrations of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and their second messenger cyclic guanosine monophosphate (cGMP) were assessed in addition to anthropometric, hemodynamic and echocardiographic parameters in a population-based sample (n = 672), of which 80 subjects used beta-receptor antagonists. RESULTS: Compared to subjects without medication, subjects receiving beta-receptor antagonists were characterized by substantially elevated ANP, BNP and cGMP plasma concentrations (plus 32%, 89% and 18%, respectively, p < 0.01 each). Analysis of subgroups revealed that this effect was highly consistent and present even in the absence of hypertension, left atrial enlargement, left ventricular hypertrophy or left ventricular dysfunction. The most prominent increase was observed in a subgroup with increased left ventricular mass index. By multivariate analysis, a statistically significant and independent association between beta-receptor antagonism and ANP, BNP and cGMP concentrations was confirmed. Such an association could not be demonstrated for other antihypertensive agents such as angiotensin-converting enzyme inhibitors or diuretics. CONCLUSIONS: Beta-receptor antagonists appear to augment plasma ANP, BNP and cGMP concentrations. The current observation suggests an important contribution of the cardiac natriuretic peptide system to the therapeutic mechanism of beta-receptor antagonists.     

A study of cumulative effects and recovery from anaesthesia with intermittent doses of althesin. A comparison with methohexitone

The cumulative effects and post-anaesthetic recovery of Althesin were studied by comparing the drug with methohexitone in a series of 60 patients undergoing surgery for varicose veins. Anaesthesia was maintained with each anaesthetic agent in 30 patients by administration of intermittent doses of the respective drugs in accordance with the surgical stimulus. When repeat doses were required at intervals of 2 to 5 minutes, the fall off in requirements was observed with both anaesthetics, more distinctly with Althesin than with methohexitone. No significant difference could be observed in the time required for immediate awakening after the two drugs. However, the majority of the patients anaesthetized with Althesin displayed a peculiar lack of mental clarity for a short period after recovering consciousness. The recovery from anaesthesia was studied by various tests and observations. Recovery after Althesin appeared to proceed slightly faster than after methohexitone. When Althesin was required in high total dosage (exceeding 150 mu1/kg), the immediate awakening was associated with emotional upset and confusion. Frequency of nausea and vomiting after anaesthesia was considerably higher in the Althesin group than in the methohexitone group. These symptoms might be toxic due to the excessive dosage given. Using the induction time as a basis for calculation of the potency ratio, Althesin and methohexiton were found to have the ratio of 1:33 (expressed in mu1/kg : mg/kg).     

Ion transport across leech integument. I. Electrogenic Na+ transport and current fluctuation analysis of the apical Na+ channel

BACKGROUND: The development of functional diversity through gene duplication and subsequent divergent evolution can give rise to proteins that have little or no sequence similarity, but retain similar topologies. RESULTS: The crystal structures of nerve growth factor, transforming growth factor-beta 2 and platelet-derived growth factor-BB show that all three are based on a cystine-knot plus beta-strands topology. There is very little sequence identity between the three proteins and the relationship between the structures had not been deduced from sequence comparisons. Each growth factor is usually active as a dimer; each exists as a dimer in the crystal, but the relative orientations of the protomers are different in each case. CONCLUSION: The structural motif of disulphide bonds and hydrogen-bonded beta-strands unexpectedly found in these three growth factors acts as a stable framework for elaboration of loops of low sequence similarity that contain the specificity for receptor interaction.     

Inactivation of rabbit muscle glycogen synthase by glycogen synthase kinase-3. Dominant role of the phosphorylation of Ser-640 (site-3a)

Rabbit skeletal muscle glycogen synthase, a rate-limiting enzyme for glycogen biosynthesis, is regulated by multisite phosphorylation. The protein kinase glycogen synthase kinase 3 (GSK-3) phosphorylates 4 Ser residues (Ser-640, Ser-644, Ser-648, and Ser-652; also known as sites 3a, 3b, 3c, and 4, respectively) at the COOH terminus of the subunit. Phosphorylation of these sites by GSK-3 is sequential, from COOH- to NH2-terminal, and is wholly dependent on prior phosphorylation by casein kinase II at Ser-656 (site 5). Expression in Escherichia coli was used to generate mutant forms of glycogen synthase, S640A, S644A, and S648A, in which site 3a, site 3b, or site 3c was changed to Ala, respectively. The purified enzymes had -/+ glucose-6-P activity ratios in the range of 0.8-0.9. Phosphorylation by casein kinase II and GSK-3 gave results consistent with the model of obligate sequential action of GSK-3. Phosphorylation at site 5, sites 4 + 5, or sites 3c + 4 + 5 had no measurable effect on activity. When sites 3b + 3c + 4 + 5 were phosphorylated, modest inactivation resulted. Additional phosphorylation at site 3a, however, was potently inactivating, reducing the -/+ glucose-6-P activity ratio to 0.1 and increasing the glucose-6-P concentration needed for half-maximal activation by an order of magnitude. Introduction of each additional phosphate, in the order site 4, 3c, 3b, and 3a, caused an incremental reduction in the mobility of the subunit when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results of this study demonstrate that GSK-3 phosphorylation of site 3a (Ser-640), and to a lesser extent, site 3b, correlates with inactivation of glycogen synthase by GSK-3. Evidence is also presented for an allosteric mechanism of inactivation whereby modification of one subunit influences the activity state of adjacent subunits.     

Polyamine spider toxins and mammalian N-methyl-D-aspartate receptors. Structural basis for channel blocking and binding of argiotoxin636

Recombinant N-methyl-D-aspartate receptors composed of NR1/NR2A subunits were expressed in Xenopus oocytes to analyse the voltage-dependent and use-dependent channel blocking activity of argiotoxin636. Functional assays demonstrate that the toxin competes with other open channel blockers such as Mg2+ and MK-801. Direct binding or competition assays using radiolabeled ligands and isolated rat brain membranes, in contrast, reveal no specific binding or yield binding constants which differ by orders of magnitude from the IC50 values of the functional assays. One explanation is that argiotoxin636 does not bind with high affinity to the inhibitory site in the N-methyl-D-aspartate-receptor channel under in vitro conditions when membranes are depolarised. The structure of argiotoxin636 was investigated by NMR spectroscopy. In solution the positively charged argiotoxin636 acquires an extended conformation and its dimensions might allow permeation deep into the channel. In the absence of direct structural information on the channel protein, the detailed analysis of blockade in conjunction with structural information, as provided here, may be of aid in the deduction of structural features of glutamate-receptor channel ion pores.     

Mutations causing neurodegeneration in Caenorhabditis elegans drastically alter the pH sensitivity and inactivation of the mammalian H+-gated Na+ channel MDEG1

The mammalian degenerin MDEG1 belongs to the nematode degenerin/epithelial Na+ channel superfamily. It is constitutively activated by the same mutations that cause gain-of-function of the Caenorhabditis elegans degenerins and neurodegeneration. ASIC and DRASIC, which were recently cloned, are structural homologues of MDEG1 and behave as H+-gated cation channels. MDEG1 is also a H+-activated Na+ channel, but it differs from ASIC in its lower pH sensitivity and slower kinetics. In addition to the generation of a constitutive current, mutations in MDEG1 also alter the properties of the H+-gated current. Replacement of Gly-430 in MDEG1 by bulkier amino acids, such as Val, Phe, or Thr, drastically increases the H+ sensitivity of the channel (half-maximal pH (pHm) approximately 4.4 for MDEG1, pHm approximately 6.7 for the different mutants). Furthermore, these replacements completely suppress the inactivation observed with the wild-type channel and increase the sensitivity of the H+-gated channel to blockade by amiloride by a factor of 10 without modification of its conductance and ionic selectivity. These results as well as those obtained with other mutants clearly indicate that the region surrounding Gly-430, situated just before the second transmembrane segment, is essential for pH sensitivity and gating.     

K+ channel inactivation mediated by the concerted action of the cytoplasmic N- and C-terminal domains

Mice lacking thymic function of the GTPase Rho show severe defects in fetal and adult thymopoiesis. Rho thymi are deficient in CD44+ CD25+ pro-T cells and CD44- CD25+ early pre-T cells because Rho function is required for survival but not G1/S phase cell cycle progression in these populations. The selective apoptosis defect in Rho prothymocytes can be rescued by expression of a bcl-2 transgene. A second function for Rho is seen in CD44- CD25- late pre-T cells: Rho regulates cell cycle progression but not survival of this population. These studies show that the critical processes of proliferation and survival are independently regulated during thymopoiesis and establish two different functions for Rho in the development of early thymic progenitors.     

Work, recovery activities, and individual well-being: A diary study.

This study extends previous research on respite from work and addresses the question of how individuals use their leisure time to recover from work. It is hypothesized that time spent on work-related and household activities has a negative effect on well-being, whereas low-effort, social, and physical activities are assumed to have a positive effect. One hundred Dutch teachers completed a diary on leisure time activities and situational well-being for 5 days, and work situation variables were assessed with a questionnaire. Multilevel analyses in which preleisure well-being and work situation variables were entered as control variables supported 4 of the 5 hypotheses. Moreover, a lagged effect of high time pressure on poor situational well-being was found. The study showed that leisure time activities and a low-stress work situation contribute independently to an individual's well-being. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Parallel recovery in a bilingual aphasic: A neurolinguistic and fMRI study.

In bilingual aphasics, the neural correlates of rehabilitation benefits and their generalization across languages are still scarcely understood. The authors present the case of a highly proficient bilingual woman (Flemish, L1/Italian, L2) with chronic aphasia who, in the presence of the same pattern of impairment in both languages, showed parallel recovery in both languages after long-term rehabilitation therapy in L2. The authors postulated that this recovery was due to the engagement of the same neural substrates. To confirm this the authors used an event-related functional magnetic resonance imaging (fMRI) paradigm to explore cortical activation during an overt picture naming task, performed in both Flemish and Italian once before and once after 2 weeks of training in L2. Behaviorally, the patient showed complete recovery of both languages. The fMRI results indicated that the same cerebral regions were recruited for both languages before and after training. Increasing activations were observed perilesionally and in homologous contralesional areas. Our data, in agreement with previous results of fMRI studies in healthy bilinguals, indicate a promising direction for future research on the neural mechanisms associated with recovery in bilingual aphasics. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule

The marine guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX), have played crucial roles in the study of voltage-gated Na+ channels. Because they have similar actions, sizes, and functional groups, they have been thought to associate with the channel in the same manner, and early mutational studies supported this idea. Recent experiments by. Biophys. J. 67:2305-2315) have suggested that the toxins bind differently to the isoform-specific domain I Phe/Tyr/Cys location. In the adult skeletal muscle Na+ channel isoform (microliter), we compared the effects on both TTX and STX affinities of mutations in eight positions known to influence toxin binding. The results permitted the assignment of energies contributed by each amino acid to the binding reaction. For neutralizing mutations of Asp400, Glu755, and Lys1237, all thought to be part of the selectivity filter of the channel, the loss of binding energy was identical for the two toxins. However, the loss of binding energy was quite different for vestibule residues considered to be more superficial. Specifically, STX affinity was reduced much more by neutralizations of Glu758 and Asp1532. On the other hand, mutation of Tyr401 to Cys reduced TTX binding energy twice as much as it reduced STX binding energy. Kinetic analysis suggested that all outer vestibule residues tested interacted with both toxins early in the binding reaction (consistent with larger changes in the binding than unbinding rates) before the transition state and formation of the final bound complex. We propose a revised model of TTX and STX binding in the Na+ channel outer vestibule in which the toxins have similar interactions at the selectivity filter, TTX has a stronger interaction with Tyr401, and STX interacts more strongly with the more extracellular residues.