The voltage-gated sodium channel Na v

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The polypeptide neurotoxin anthopleurin B (ApB) isolated from the venom of the sea anemone Anthopleura xanthogrammica is one of a family of toxins that bind to the extracellular face of voltage-dependent sodium channels and retard channel inactivation. Because most regions of the sodium channel known to contribute to inactivation are located intracellularly or within the membrane bilayer, identification of the toxin/channel binding site is of obvious interest. Recently, mutation of a glutamic acid residue on the extracellular face of the fourth domain of the rat neuronal sodium channel (rBr2a) was shown to disrupt toxin/channel binding (Rogers, J. C., Qu, Y. S., Tanada, T. N., Scheuer, T., and Catterall, W. A. (1996) J. Biol. Chem. 271, 15950 -15962). A negative charge at this position is highly conserved between mammalian sodium channel isoforms. We have constructed mutations of the corresponding residue (Asp-1612) in the rat cardiac channel isoform (rH1) and shown that the lowered affinity occurs primarily through an increase in the toxin/channel dissociation rate k off . Further, we have used thermodynamic mutant cycle analysis to demonstrate a specific interaction between this anionic amino acid and Lys-37 of ApB (⌬⌬G ‫؍‬ 1.5 kcal/mol), a residue that is conserved among many sea anemone toxins. Reversal of the charge at Asp-1612, as in the mutant D1612R, also affects channel inactivation independent of toxin (؊14 mV shift in channel availability). Binding of the toxin to Asp-1612 may therefore contribute both to toxin/channel affinity and to transduction of the effects of the toxin on channel kinetics.

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 (nϭ8), hibernating myocardium was present as reflected by reduced resting LAD flow (0.75Ϯ0.14 versus 1.19Ϯ0.14 mL ⅐ min Ϫ1 ⅐ g Ϫ1 in remote) and wall thickening (1.93Ϯ0.46 mm versus 5.46Ϯ0.41 mm in remote, PϽ0.05). 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 (PϽ0.05). 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 21.9Ϯ3.1 versus 42.8Ϯ1.9 mU, PϽ0.05). After 5-months (nϭ10), 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. Key Words: metabolism Ⅲ proteomics Ⅲ hibernating myocardium Ⅲ ischemic heart disease H ibernating myocardium is characterized by viable, chronically dysfunctional myocardium that develops in response to repetitive myocardial ischemia. 1,2 We have previously demonstrated that the relation between regional oxygen consumption, coronary flow, and function in response to stress is attenuated in hibernating myocardium and thus dissociated from the usual determinants of myocardial oxygen demand. 3 By reducing regional energy utilization, hibernation prevents the development of ischemia after submaximal stress. This is supported by a lack of biochemical markers of ischemia and preservation of total ATP and creatine phosphate content in swine with hibernating myocardium 3,4 as well as human biopsies from patients without significant fibrosis. 5 Although there has been interest in identifying the role of increased glucose uptake in these responses, maximal insulin stimulated glucose uptake is unchanged in chronic hibernating myocardium, and alterations in other metabolic pathways responsible for the attenuated increase...

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.Chemically diverse neurotoxins have historically been of great value in defining the overall architecture of voltage-dependent Na ϩ (Na V ) 1 and K ϩ (K V ) channels. The pores of such channels have been mapped by analysis of their interactions with conotoxins (Na V ) and a variety of polypeptides from scorpion venoms, such as charybdotoxin and agitoxins (1-4). More recently, regions of these channels involved in gating have been identified by using gating modifier toxins derived from scorpion (5), sea anemone (6), and spider (7-9) venoms. Most interestingly, gating modifier toxins appear to interact with the same channel region, designated the S3-S4 linker, irrespective of the type of channel being studied (10).Gating modifier toxins can also be important probes for the accessibility of defined regions of a given channel. Very recently, the MacKinnon laboratory has employed a novel spider toxin, VSTX, to probe the accessibility of defined regions of the voltage sensor of the archaebacterial K V AP channel (9). The resulting data were interpreted in the context of a channel three-dimensional structure (11) in which the K V AP S3-S4 linker was located either near the cytoplasmic surface or buried within the bilayer, depending on whether the channel was in the resting or activated state (9). The observation that VSTX possessed phospholipid binding activity (12) provided a potential explanation for the ability of this toxin to modify channels via interaction with sequences that were never exposed at the extracellular surface.To understand the extent to which this model could ...

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In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa. The ankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location of ankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is ϳ65% ␣-helical. RNase protection and KatB-and AnkB-LacZ translational fusion analyses indicated that katB and ankB are part of a small operon whose transcription is induced dramatically by H 2 O 2 , and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of an ankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H 2 O 2 , phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H 2 O 2 detoxification.

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.