Inter-laboratory optimization of protein extraction, separation, and fluorescent detection of endogenous rice allergens.

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.

Gating-pore currents demonstrate selective and specific modulation of individual sodium channel voltage-sensors by biological toxins.

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 N-terminal basolateral targeting signal unlikely acts alone in the differential trafficking of membrane transporters in MDCK cells.

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.

The tarantula toxins ProTx-II and huwentoxin-IV differentially interact with human Nav1.7 voltage sensors to inhibit channel activation and inactivation.

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.

Inhibition of the activation pathway of the T-type calcium channel Ca(V)3.1 by ProTxII.

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.

Evidence for multiple effects of ProTxII on activation gating in Na(V)1.5.

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.

Persistent regional downregulation in mitochondrial enzymes and upregulation of stress proteins in swine with chronic hibernating myocardium.

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.

Molecular interactions of the gating modifier toxin ProTx-II with NaV 1.5: implied existence of a novel toxin binding site coupled to activation.

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.

Proteomic analysis of cisplatin-induced cochlear damage: methods and early changes in protein expression.

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.

Site-3 sea anemone toxins: molecular probes of gating mechanisms in voltage-dependent sodium channels.

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.

ProTx-I and ProTx-II: gating modifiers of voltage-gated sodium channels.

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.

Distinct structural elements in the first membrane-spanning segment of the epithelial sodium channel.

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.

A modified, dual reporter TOXCAT system for monitoring homodimerization of transmembrane segments of proteins.

The TOXCAT assay system developed by Russ and Engelman [TOXCAT: a measure of transmembrane helix association in a biological membrane, Proc. Natl. Acad.

Differential phospholipid binding by site 3 and site 4 toxins. Implications for structural variability between voltage-sensitive sodium channel domains.

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.

Role of Asn-16 and Ser-19 in anthopleurin B binding. Implications for the electrostatic nature of Na(V) site 3.

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).

Arg-14 loop of site 3 anemone toxins: effects of glycine replacement on toxin affinity.

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.

Voltage-gated sodium channel toxins: poisons, probes, and future promise.

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.