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1.
Biosci Biotechnol Biochem ; 80(11): 2198-2207, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27399872

ABSTRACT

In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52-63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52 kDa globulin, 33 kDa glyoxalase I, and 14-16 kDa α-amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens.

2.
Mol Pharmacol ; 86(2): 159-67, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24898004

ABSTRACT

Voltage-gated sodium channels are critical determinants of nerve and muscle excitability. Although numerous toxins and small molecules target sodium channels, identifying the mechanisms of action is challenging. Here we used gating-pore currents selectively generated in each of the voltage-sensors from the four α-subunit domains (DI-DIV) to monitor the activity of individual voltage-sensors and to investigate the molecular determinants of sodium channel pharmacology. The tarantula toxin huwentoxin-IV (HWTX-IV), which inhibits sodium channel current, exclusively enhanced inward gating-pore currents through the DII voltage-sensor. By contrast, the tarantula toxin ProTx-II, which also inhibits sodium channel currents, altered the gating-pore currents in multiple voltage-sensors in a complex manner. Thus, whereas HWTX-IV inhibits central-pore currents by selectively trapping the DII voltage-sensor in the resting configuration, ProTx-II seems to inhibit central-pore currents by differentially altering the configuration of multiple voltage-sensors. The sea anemone toxin anthopleurin B, which impairs open-channel inactivation, exclusively enhanced inward gating-pore currents through the DIV voltage-sensor. This indicates that trapping the DIV voltage-sensor in the resting configuration selectively impairs open-channel inactivation. Furthermore, these data indicate that although activation of all four voltage-sensors is not required for central-pore current generation, activation of the DII voltage-sensor is crucial. Overall, our data demonstrate that gating-pore currents can determine the mechanism of action for sodium channel gating modifiers with high precision. We propose this approach could be adapted to identify the molecular mechanisms of action for gating modifiers of various voltage-gated ion channels.


Subject(s)
Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Sodium Channels/metabolism , Toxins, Biological/pharmacology , Cell Line , HEK293 Cells , Humans , Intercellular Signaling Peptides and Proteins , Peptides/pharmacology , Sodium Channel Blockers/pharmacology
3.
Biochemistry ; 52(30): 5103-5116, 2013 Jul 30.
Article in English | MEDLINE | ID: mdl-23837633

ABSTRACT

We have shown previously, using confocal imaging and transport assays, that the N-terminus of sodium-dependent vitamin C transporter 2 (SVCT2) can redirect apical SVCT1 to the basolateral membrane. Here, the SVCT model was used to further characterize the basolateral targeting peptide signal. Both the length (31 amino acids) and sequence accuracy of the N-terminus of SVCT2 were found to be important in basolateral targeting activity, suggesting a structural requirement. However, the N-terminal basolateral targeting sequence did not appear to act alone, based on analyses of heterologous chimeras. Although diverse N-terminal basolateral targeting signals from multipass membrane proteins can all redirect apical protein from the same gene family to the basolateral membrane, none of the N-terminal basolateral targeting signals can redirect the transmembrane and C-terminal regions from a different gene family. Instead, the presence of these heterologous N-terminal basolateral targeting signals affected the trafficking of otherwise apical protein, causing their accumulation in a stable tubulin-like non-actin structure. Nontargeting N-terminal sequences had no effect. Similar protein retention was observed previously and in this study when the C-terminus of apical or basolateral protein was mutated. These results suggest that the N- and C-termini interact, directly or indirectly, within each gene family for basolateral targeting. Circular dichroism and two-dimensional nuclear magnetic resonance analyses both found a lack of regular secondary structure in the conserved N-terminus of SVCT2, consistent with the presence of partner(s) in the targeting unit. Our finding, a departure from the prevailing single-peptide motif model, is consistent with the evolution of basolateral transporters from the corresponding apical genes. The interaction among the N-terminus, its partner(s), and the cellular basolateral targeting machinery needs to be further elucidated.


Subject(s)
Cell Membrane/metabolism , Models, Biological , Protein Sorting Signals , Sodium-Coupled Vitamin C Transporters/metabolism , Amino Acid Sequence , Animals , Cell Polarity , Conserved Sequence , Dogs , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Madin Darby Canine Kidney Cells , Mutant Chimeric Proteins/chemistry , Mutant Chimeric Proteins/metabolism , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Conformation , Protein Interaction Domains and Motifs , Protein Transport , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sodium-Coupled Vitamin C Transporters/chemistry , Sodium-Coupled Vitamin C Transporters/genetics
4.
Mol Pharmacol ; 78(6): 1124-34, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20855463

ABSTRACT

The voltage-gated sodium channel Na(v)1.7 plays a crucial role in pain, and drugs that inhibit hNa(v)1.7 may have tremendous therapeutic potential. ProTx-II and huwentoxin-IV (HWTX-IV), cystine knot peptides from tarantula venoms, preferentially block hNa(v)1.7. Understanding the interactions of these toxins with sodium channels could aid the development of novel pain therapeutics. Whereas both ProTx-II and HWTX-IV have been proposed to preferentially block hNa(v)1.7 activation by trapping the domain II voltage-sensor in the resting configuration, we show that specific residues in the voltage-sensor paddle of domain II play substantially different roles in determining the affinities of these toxins to hNa(v)1.7. The mutation E818C increases ProTx-II's and HWTX-IV's IC(50) for block of hNa(v)1.7 currents by 4- and 400-fold, respectively. In contrast, the mutation F813G decreases ProTx-II affinity by 9-fold but has no effect on HWTX-IV affinity. It is noteworthy that we also show that ProTx-II, but not HWTX-IV, preferentially interacts with hNa(v)1.7 to impede fast inactivation by trapping the domain IV voltage-sensor in the resting configuration. Mutations E1589Q and T1590K in domain IV each decreased ProTx-II's IC(50) for impairment of fast inactivation by ~6-fold. In contrast mutations D1586A and F1592A in domain-IV increased ProTx-II's IC(50) for impairment of fast inactivation by ~4-fold. Our results show that whereas ProTx-II and HWTX-IV binding determinants on domain-II may overlap, domain II plays a much more crucial role for HWTX-IV, and contrary to what has been proposed to be a guiding principle of sodium channel pharmacology, molecules do not have to exclusively target the domain IV voltage-sensor to influence sodium channel inactivation.


Subject(s)
Sodium Channel Blockers/metabolism , Sodium Channels/metabolism , Spider Venoms/metabolism , Amino Acid Sequence , Animals , Female , HEK293 Cells , Humans , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Molecular Sequence Data , NAV1.7 Voltage-Gated Sodium Channel , Sodium Channel Blockers/pharmacology , Spider Venoms/pharmacology , Spiders
5.
Toxicon ; 56(4): 624-36, 2010 Sep 15.
Article in English | MEDLINE | ID: mdl-20600227

ABSTRACT

Toxins have been used extensively to probe the gating mechanisms of voltage-gated ion channels. Relatively few such tools are available to study the low-voltage activated T-type Ca channels, which underlie thalamic neuron firing and affect sleep, resistance to seizures, and weight gain. Here we show that ProTxII, a peptide toxin recently isolated from the venom of the tarantula spider Thrixopelma pruriens, dose-dependently inhibited Ca(V)3.1 causing a decrease in current (81.6% +/- 3.1% at -30 mV in 5 microM toxin) and a positive shift in the voltage range of activation (+34.5 mV +/- 4.4 mV). Toxin-modified currents were slower to activate and faster to deactivate and they displayed a longer lag in the onset of current, i.e. the Cole-Moore shift, consistent with the inhibition of gating transitions along the activation pathway, particularly the final opening transition. Single-channel current amplitude and total gating charge were unaffected by toxin, ruling out a change in ion flux or channel dropout as mechanisms for the decrease in macroscopic conductance. A positive shift in the voltage range of gating charge movement (+30.6 mV +/- 2.6 mV shift in the voltage of half maximal charge movement in the presence of 5 microM toxin) confirmed that ProTxII-induced gating perturbations in this channel occur at the level of the voltage sensors, and kinetic modeling based on these findings suggested that reductions in current magnitude could be largely accounted for by kinetic perturbations of activation.


Subject(s)
Calcium Channel Blockers/pharmacology , Calcium Channels, T-Type/chemistry , Spider Venoms/chemistry , Calcium Channel Blockers/isolation & purification , Calcium Channels, T-Type/physiology , Cell Line , Electric Conductivity , Humans , Patch-Clamp Techniques , Spider Venoms/isolation & purification , Spider Venoms/pharmacology
6.
Toxicon ; 52(3): 489-500, 2008 Sep 01.
Article in English | MEDLINE | ID: mdl-18657562

ABSTRACT

The peptide toxin ProTxII, recently isolated from the venom of the tarantula spider Thrixopelma pruriens, modifies gating in voltage-gated Na+ and Ca2+ channels. ProTxII is distinct from other known Na+ channel gating modifier toxins in that it affects activation, but not inactivation. It shifts activation gating positively and decreases current magnitude such that the dose-dependence of toxin action measured at a single potential reflects both effects. To test the extent to which these effects were independent, we tracked several different measures of current amplitude, voltage-dependent activation, and current kinetics in Na(V)1.5 in a range of toxin concentrations. Changes in voltage dependence and a decrease in G(max) appeared at relatively low concentrations (40-100 nM) while a positive shift in the voltage range of activation was apparent at higher toxin concentrations (> or =500 nM). Because ProTxII carries a net +4 charge we tested whether electrostatic interactions contributed to toxin action. We examined the effects of ProTxII in the presence of high extracellular Ba2+, known to screen and/or bind to surface charge. Some, but not all aspects of ProTxII modification were sensitive to the presence of Ba2+ indicating the contribution of an electrostatic, surface charge-like mechanism and supporting the idea of a multi-faceted toxin-channel interaction.


Subject(s)
Ion Channel Gating/drug effects , Muscle Proteins/metabolism , Sodium Channels/metabolism , Spider Venoms/pharmacology , Animals , Dose-Response Relationship, Drug , Humans , Muscle Proteins/genetics , Mutation , NAV1.5 Voltage-Gated Sodium Channel , Sodium Channels/genetics , Spider Venoms/chemistry , Spider Venoms/metabolism , Spiders/physiology
7.
Circ Res ; 102(1): 103-12, 2008 Jan 04.
Article in English | MEDLINE | ID: mdl-17967786

ABSTRACT

Hibernating myocardium is accompanied by a downregulation in energy utilization that prevents the immediate development of ischemia during stress at the expense of an attenuated level of regional contractile function. We used a discovery based proteomic approach to identify novel regional molecular adaptations responsible for this phenomenon in subendocardial samples from swine instrumented with a chronic LAD stenosis. After 3 months (n=8), hibernating myocardium was present as reflected by reduced resting LAD flow (0.75+/-0.14 versus 1.19+/-0.14 mL x min(-1) x g(-1) in remote) and wall thickening (1.93+/-0.46 mm versus 5.46+/-0.41 mm in remote, P<0.05). Regionally altered proteins were quantified with 2D Differential-in-Gel Electrophoresis (2D-DIGE) using normal myocardium as a reference with identification of candidates using MALDI-TOF mass spectrometry. Hibernating myocardium developed a significant downregulation of many mitochondrial proteins and an upregulation of stress proteins. Of particular note, the major entry points to oxidative metabolism (eg, pyruvate dehydrogenase complex and Acyl-CoA dehydrogenase) and enzymes involved in electron transport (eg, complexes I, III, and V) were reduced (P<0.05). Multiple subunits within an enzyme complex frequently showed a concordant downregulation in abundance leading to an amplification of their cumulative effects on activity (eg, "total" LAD PDC activity was 21.9+/-3.1 versus 42.8+/-1.9 mU, P<0.05). After 5-months (n=10), changes in mitochondrial and stress proteins persisted whereas cytoskeletal proteins (eg, desmin and vimentin) normalized. These data indicate that the proteomic phenotype of hibernating myocardium is dynamic and has similarities to global changes in energy substrate metabolism and function in the advanced failing heart. These proteomic changes may limit oxidative injury and apoptosis and impact functional recovery after revascularization.


Subject(s)
Energy Metabolism/genetics , Gene Expression Regulation/physiology , Myocardial Stunning/genetics , Proteins/analysis , Proteomics/methods , Stress, Physiological/genetics , Adaptation, Physiological/genetics , Animals , Chronic Disease , Disease Models, Animal , Down-Regulation , Enzymes , Gene Expression Regulation, Enzymologic , Mitochondrial Proteins , Proteins/genetics , Swine , Up-Regulation
8.
J Biol Chem ; 282(17): 12687-97, 2007 Apr 27.
Article in English | MEDLINE | ID: mdl-17339321

ABSTRACT

Voltage-gated Na(+) channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Na(v) and Ca(v), but not K(v), channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Na(v)1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly K(v) channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Na(v)1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na(v) channels with potential to shed new light on this process.


Subject(s)
Ion Channel Gating/drug effects , Muscle Proteins/metabolism , Sodium Channels/metabolism , Spider Venoms/pharmacology , Amino Acid Substitution , Binding Sites/genetics , Cell Line , Humans , Membrane Potentials/drug effects , Mutagenesis , NAV1.5 Voltage-Gated Sodium Channel , Peptide Mapping , Spider Venoms/genetics
9.
Hear Res ; 226(1-2): 140-56, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17321087

ABSTRACT

To identify early changes in protein expression associated with cisplatin ototoxicity, we used two dimensional-difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometry to analyze proteins from P3 rat cochleae that were cultured for 3h with or without 1mM cisplatin. Replicate analysis of fluorescent images from six gels revealed significant (p<0.01) cisplatin-induced changes (greater than 1.5-fold) in expression of 22 cochlear proteins. These include increases in the expression of five proteins, four of which were identified as nucleobindin 1, a nuclear calcium signaling and homeostasis protein (2.1-fold), heterogeneous nuclear ribonucleoprotein C, an RNA processing protein (1.8-fold), a 55 kDa protein that is either endothelial differentiation-related factor 1 or alpha-6 tubulin (1.7-fold), and calreticulin, a calcium binding chaperone of the endoplasmic reticulum (ER, 1.6-fold). The expression of 17 proteins was significantly (p<0.01) decreased by greater than 1.5-fold. These include ribonuclease/angiogenin inhibitor 1 (1.6-fold), RAS-like, family 12 (predicted), ras association (RalGDS/AF-6) domain family 5 (4.5-fold), homologous the RAS family of GTPase signaling proteins (2.4-fold), and Protein tyrosine phosphatase domain containing 1 (predicted, 6.1-fold). We identified seven cochlear proteins with either smaller (1.2-1.5-fold) or less significant (p<0.05) cisplatin-induced changes in expression. Notably, heat shock 70 kDa protein 5 (Hspa5, Grp78, and BiP), an ER chaperone protein involved in stress response, decreased 1.7-fold. We observed changes consistent with phosphorylation in the level of isoforms of another ER stress-induced protein, glucose-regulated protein Grp58. Changes in cisplatin-induced protein expression are discussed with respect to known or hypothesized functions of the identified proteins.


Subject(s)
Cisplatin/toxicity , Cochlea/drug effects , Cochlea/metabolism , Proteins/metabolism , Animals , Antineoplastic Agents/toxicity , Calcium-Binding Proteins/metabolism , Cochlea/pathology , DNA-Binding Proteins/metabolism , Electrophoresis, Gel, Two-Dimensional , Heat-Shock Proteins/metabolism , Isoelectric Point , Molecular Chaperones/metabolism , Molecular Weight , Nerve Tissue Proteins , Nucleobindins , Peptide Mapping , Proteins/isolation & purification , Proteomics , Rats , Rats, Inbred F344 , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tissue Culture Techniques
10.
Toxicon ; 49(2): 194-201, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17087985

ABSTRACT

The tarantula venom peptides ProTx-I and ProTx-II inhibit voltage-gated sodium channels by shifting their voltage dependence of activation to a more positive potential, thus acting by a mechanism similar to that of potassium channel gating modifiers such as hanatoxin and VSTX1. ProTx-I and ProTx-II inhibit all sodium channel (Nav1) subtypes tested with similar potency and represent the first potent peptidyl inhibitors of TTX-resistant sodium channels. Like gating modifiers of potassium channels, ProTx-I and ProTx-II conform to the inhibitory cystine knot motif, and ProTx-II was demonstrated to bind to sodium channels in the closed state. Both toxins have been synthesized chemically, and ProTx-II, produced by recombinant means, has been used to map the interaction surface of the peptide with the Nav1.5 channel. In comparison, beta-scorpion toxins activate sodium channels by shifting the voltage dependence of activation to more negative potentials, and together these peptides represent valuable tools for exploring the gating mechanism of sodium channels.


Subject(s)
Ion Channel Gating , Sodium Channel Blockers/pharmacology , Sodium Channels/drug effects , Spider Venoms/pharmacology , Animals , Humans , Ion Channel Gating/drug effects , Ion Channel Gating/physiology
11.
Toxicon ; 49(2): 159-70, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17095031

ABSTRACT

Sea anemone toxins, whose biological function is the capture of marine prey, are invaluable tools for studying the structure and function of mammalian voltage-gated sodium channels. Their high degree of specificity and selectivity have allowed for detailed analysis of inactivation gating and assignment of molecular entities responsible for this process. Because of their ability to discriminate among channel isoforms, and their high degree of structural conservation, these toxins could serve as important lead compounds for future pharmaceutical design.


Subject(s)
Cnidarian Venoms/genetics , Cnidarian Venoms/pharmacology , Ion Channel Gating , Sea Anemones , Sodium Channels/drug effects , Amino Acid Sequence , Animals , Humans , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Molecular Probes , Molecular Sequence Data
12.
J Biol Chem ; 281(41): 30455-62, 2006 Oct 13.
Article in English | MEDLINE | ID: mdl-16912051

ABSTRACT

Epithelial Na+ channels (ENaCs) comprise three subunits that have been proposed to be arranged in either an alpha2betagamma or a higher ordered configuration. Each subunit has two putative membrane-spanning segments (M1 and M2), intracellular amino and carboxyl termini, and a large extracellular loop. We have used the TOXCAT assay (a reporter assay for transmembrane segment homodimerization) to identify residues within the transmembrane segments of ENaC that may participate in important structural interactions within ENaC, with which we identified a candidate site within alphaM1. We performed site-directed mutagenesis at this site and found that, although the mutants reduced channel activity, ENaC protein expression at the plasma membrane was unaffected. To deduce the role of alphaM1 in the pore structure of ENaC, we performed tryptophan-scanning mutagenesis throughout alphaM1 (residues 110-130). We found that mutations within the amino-terminal part of alphaM1 had effects on activity and selectivity with a periodicity consistent with a helical structure but no effect on channel surface expression. We also observed that mutations within the carboxyl-terminal part of alphaM1 had effects on activity and selectivity but with no apparent periodicity. Additionally, these mutants reduced channel surface expression. Our data support a model in which the amino-terminal half of alphaM1 is alpha-helical and packs against structural element(s) that contribute to the ENaC pore. Furthermore, these data suggest that the carboxyl-terminal half of alphaM1 may be helical or assume a different conformation and may be involved in tertiary interactions essential to proper channel folding or assembly. Together, our data suggest that alphaM1 is divided into two distinct regions.


Subject(s)
Epithelial Sodium Channels/chemistry , Amino Acid Sequence , Animals , Cell Membrane/metabolism , Epithelial Sodium Channels/genetics , Epithelial Sodium Channels/metabolism , Humans , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Patch-Clamp Techniques , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Tryptophan/chemistry
13.
Biochem Biophys Res Commun ; 339(1): 321-4, 2006 Jan 06.
Article in English | MEDLINE | ID: mdl-16297866

ABSTRACT

The TOXCAT assay system developed by Russ and Engelman [TOXCAT: a measure of transmembrane helix association in a biological membrane, Proc. Natl. Acad. Sci. USA 96 (1999) 863-868] provides an in vivo means of selecting for and evaluating the strength of interaction between identical transmembrane alpha-helices. In the course of utilizing TOXCAT to study the architecture of a sodium channel hNa(V)1.5, an apparently strong dimerization of two of its putative transmembrane segments was revealed. Following random mutagenesis of these regions, several amino acids critical for the observed dimerizations were identified. In order to develop a more efficient means of isolating mutations which specifically disrupt dimerization of these transmembrane segments without affecting their membrane-targeting properties, we developed a modification to the original TOXCAT design in which the C-terminal maltose binding protein moiety is replaced by the beta-lactamase. We show that this assay system is capable of simultaneously monitoring the integrity of the chimeric protein, its membrane insertion activity, and the ability of the transmembrane segment under study to dimerize.


Subject(s)
Cell Membrane/metabolism , Genes, Reporter , Muscle Proteins/metabolism , Sodium Channels/metabolism , Amino Acid Sequence , Dimerization , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Maltose/genetics , Molecular Sequence Data , Muscle Proteins/genetics , Mutation , NAV1.5 Voltage-Gated Sodium Channel , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sodium Channels/genetics , beta-Lactamases/genetics
14.
J Biol Chem ; 280(12): 11127-33, 2005 Mar 25.
Article in English | MEDLINE | ID: mdl-15632158

ABSTRACT

It has been shown recently that polypeptide toxins that modulate the gating properties of voltage-sensitive cation channels are able to bind to phospholipid membranes, leading to the suggestion that these toxins are able to access a channel-binding site that remains membrane-restricted (Lee, S.-Y., and MacKinnon, R. (2004) Nature 430, 232-235). We therefore examined the ability of anthopleurin B (ApB), a sea anemone toxin that selectively modifies inactivation kinetics of Na(V)1.x channels, and ProTx-II, a spider toxin that modifies activation kinetics of the same channels, to bind to liposomes. Whereas ProTx-II can be quantitatively depleted from solution upon incubation with phosphatidylcholine/phosphatidylserine liposomes, ApB displays no discernible phospholipid binding activity. We therefore examined the activities of structurally unrelated site 3 and site 4 toxins derived from Leiurus and Centruroides venoms, respectively, in the same assay. Like ApB, the site 3 toxin LqqV shows no lipid binding activity, whereas the site 4 toxin Centruroides toxin II, like ProTx-II, is completely bound. We conclude that toxins that modify inactivation kinetics via binding to Na(V)1.x site 3 lack the ability to bind phospholipids, whereas site 4 toxins, which modify activation, have this activity. This inherent difference suggests that the conformation of domain II more closely resembles that of the K(V)AP channel than does the conformation of domain IV.


Subject(s)
Muscle Proteins/chemistry , Peptides/metabolism , Phospholipids/metabolism , Scorpion Venoms/metabolism , Sodium Channels/chemistry , Spider Venoms/metabolism , Binding Sites , Cells, Cultured , Humans , Intercellular Signaling Peptides and Proteins , Muscle Proteins/drug effects , Muscle Proteins/metabolism , NAV1.5 Voltage-Gated Sodium Channel , Potassium Channels, Voltage-Gated/metabolism , Protein Conformation , Sodium Channels/drug effects , Sodium Channels/metabolism
15.
Biochemistry ; 43(22): 7082-9, 2004 Jun 08.
Article in English | MEDLINE | ID: mdl-15170345

ABSTRACT

Anthopleurin B (ApB) is a type 1 sea anemone toxin, which binds to voltage-sensitive sodium channels (Na(V)'s), thereby delaying channel inactivation. Previous work from our laboratories has demonstrated that the structurally unconstrained region involving residues 8-17 of this polypeptide, designated the Arg-14 loop, is important for full toxin affinity (Seibert et al., (2003) Biochemistry 42, 14515). Within this region, important contributions are made by residues Arg-12 and Leu-18 (Gallagher and Blumenthal, (1994) J. Biol. Chem. 269, 254; Dias-Kadambi et al., (1996) J. Biol. Chem. 271, 23828). Moreover, replacement of glycine residues found at positions 10 or 15 of the loop by alanine has been shown to have profound, isoform-selective effects on toxin-binding kinetics (Seibert et al., (2003)Biochemistry 42, 14515). To thoroughly understand the importance of this entire region, the work described here investigates the contribution of ApB residues Asn-16, Thr-17, and Ser-19 to toxin affinity and isoform selectivity. Our results demonstrate that residues within and proximal to the C terminus of the Arg-14 loop are important modulators of ApB affinity for Na(V) channels, indicating that the loop and channel site 3 are likely in close contact. A comparison of the effects of multiple replacements at each position reveals that Asn-16 and Ser-19 are involved in binding, whereas Thr-17 is not. The fact that anionic replacements for Asn-16 or Ser-19 are highly deleterious for toxin binding strongly suggests that site 3 contains either formal anionic residues or regions of high electron density, which could be formed by aromatic clusters. These data represent the first indication of the presence of such residues or regions within Na(V) site 3.


Subject(s)
Amino Acid Substitution/genetics , Asparagine/chemistry , Cnidarian Venoms/metabolism , Peptides/metabolism , Serine/chemistry , Sodium Channels/metabolism , Animals , Asparagine/genetics , Asparagine/metabolism , Binding Sites , Cells, Cultured , Cnidarian Venoms/genetics , Cnidarian Venoms/isolation & purification , Electrophysiology , Humans , Intercellular Signaling Peptides and Proteins , Models, Molecular , Mutagenesis, Site-Directed , Protein Binding , Sea Anemones/chemistry , Serine/genetics , Serine/metabolism , Static Electricity
16.
Biochemistry ; 42(49): 14515-21, 2003 Dec 16.
Article in English | MEDLINE | ID: mdl-14661964

ABSTRACT

Anthopleurin B (ApB) is a high-affinity sea anemone neurotoxin that interacts with voltage-sensitive sodium (Na(V)) channels, causing a delay in channel inactivation. The solution structures of all known anemone toxins having this activity include a poorly defined region encompassing ApB residues 8-17, which we call the Arg-14 loop. We propose that the inherent mobility of the Arg-14 loop is necessary for the toxins' ability to maintain a high-affinity channel complex throughout the continual conformational transitions experienced by the channel during its functional cycle. We have previously shown that Arg-12, located in this loop, and Leu-18, which is adjacent, are important for ApB activity. Here, we characterized the role of two glycines located within the loop (Gly-10 and Gly-15) and an additional glycine positioned immediately C-terminal to it (Gly-20). We used site-directed replacement by alanine to assess the functional contribution to toxin binding of each of these residues singly and in combination. Gly-20 was found to be an essential toxin folding determinant; Gly-10 and Gly-15 were important for determining toxin affinity. Compared to wild-type toxin, the G10A and G15A toxins displayed significantly higher K(D) values for both cardiac (Na(V)1.5) and neuronal (Na(V)1.2) channels, although both demonstrated greater isoform discrimination for Na(V)1.5 than did wild-type ApB. For both G10A and G15A, significant Na(V) isoform differences were evident for on- and off-rates, with the most dramatic effect of a single mutation being the 467-fold reduction in the on-rate for G10A binding to Na(V)1.2, suggestive of a more accommodating binding site on Na(V)1.5 as compared to Na(V)1.2. Because alanine replacement of glycines is known to be associated with reduced backbone freedom, these results suggest an essential role for Arg-14 loop flexibility in toxin function, although a direct steric effect of the mutant methyl group cannot be excluded.


Subject(s)
Amino Acid Substitution/genetics , Arginine/genetics , Cnidarian Venoms/metabolism , Glycine/genetics , Peptides/metabolism , Alanine/genetics , Animals , Arginine/metabolism , Binding Sites/genetics , Cell Line , Cell Line, Tumor , Cnidarian Venoms/genetics , Cnidarian Venoms/isolation & purification , Glycine/metabolism , Humans , Intercellular Signaling Peptides and Proteins , Kinetics , Mice , Models, Molecular , Muscle Proteins/metabolism , Mutagenesis, Site-Directed , NAV1.2 Voltage-Gated Sodium Channel , NAV1.5 Voltage-Gated Sodium Channel , Nerve Tissue Proteins/metabolism , Patch-Clamp Techniques , Peptides/genetics , Peptides/isolation & purification , Protein Structure, Tertiary/genetics , Sodium Channels/metabolism
17.
Cell Biochem Biophys ; 38(2): 215-38, 2003.
Article in English | MEDLINE | ID: mdl-12777715

ABSTRACT

Neurotoxins have served as invaluable agents for identification, purification, and functional characterization of voltage-gated ion channels. Multiple classes of these toxins, which target voltage- gated Na+ channels via high-affinity binding to distinct but allosterically coupled sites, have been identified. The toxins are chemically diverse, including guanidinium heterocycles, a variety of structurally unrelated alkaloids, and multiple families of nonhomologous polypeptides having either related or distinct functions. This review describes the biochemistry and pharmacology of these agents, and summarizes the structure-function relationships underlying their interaction with molecular targets. In addition, we explore recent advances in the use of these toxins as molecular scaffolding agents, drugs, and insecticides.


Subject(s)
Ion Channel Gating/physiology , Neurotoxins/chemistry , Neurotoxins/metabolism , Potassium Channels, Voltage-Gated/chemistry , Potassium Channels, Voltage-Gated/physiology , Amino Acid Sequence , Animals , Binding Sites , Cnidarian Venoms/chemistry , Drug Design , Extracellular Matrix/chemistry , Insecticides/chemical synthesis , Insecticides/chemistry , Ion Channel Gating/drug effects , Macromolecular Substances , Molecular Sequence Data , Neurotoxins/classification , Neurotoxins/pharmacology , Potassium Channels, Voltage-Gated/drug effects , Protein Binding , Protein Conformation , Protein Subunits , Scorpion Venoms/chemistry , Spider Venoms/chemistry , Structure-Activity Relationship
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