Charybdotoxin, belonging to the group of so-called scorpion toxins, is a short peptide able to block many voltage-gated potassium channels, such as mKv1.3, with high affinity. We use a reliable homology model based on the high-resolution crystal structure of the 94% sequence identical homologue Kv1.2 for charybdotoxin docking followed by molecular dynamics simulations to investigate the mechanism and energetics of unbinding, tracing the behavior of the channel protein and charybdotoxin during umbrella-sampling simulations as charybdotoxin is moved away from the binding site. The potential of mean force is constructed from the umbrella sampling simulations and combined with K(d) and free energy values gained experimentally using the patch-clamp technique to study the free energy of binding at different ion concentrations and the mechanism of the charybdotoxin-mKv1.3 binding process. A possible charybdotoxin binding mechanism is deduced that includes an initial hydrophobic contact followed by stepwise electrostatic interactions and finally optimization of hydrogen bonds and salt bridges.

ObjectiveWe previously demonstrated that promoting Beclin-1–dependent autophagy is cardiac protective during endotoxemia shock, suggesting that autophagy-based approaches may become a promising therapeutic strategy for sepsis. In this study, we applied both genetic and pharmacological approaches to evaluate whether Beclin-1 activation improves sepsis outcomes in a model of pneumonia-induced sepsis.MethodsSepsis was induced in mice by Klebsiella pneumoniae infection via intubation, and outcomes of clinical sickness scores, systemic infection, inflammation, survival, and pulmonary pathology were examined. Evaluation of Beclin-1 activation was achieved by comparing strains of C57BL/6J wild type and Becn1F121A that carries a transgenic expression of Beclin-1–active mutant F121A, and by comparing animal groups treated with Beclin-1–activating peptide, Tat-beclin-1 peptide (TB-peptide), or with vehicle control. The status of autophagy in the lung tissue was examined in autophagy reporter mice, CAG-RFP-EGFP-LC3, by fluorescence microscopy.ResultsPulmonary infection by K. pneumoniae produced an insufficient, maladaptive autophagy in the lung. Activation of Beclin-1 by forced expression of active mutant Becn1F121A or by treatment with TB-peptide enhanced autophagy and significantly reduced sickness scores, systemic infection, and circulating and pulmonary cytokine production. Both approaches demonstrated notable benefits in limiting post-infection pathogenesis in the lung, such as decreases in alveolar congestion, hemorrhage, infiltration of inflammatory cells, and alveolar wall thickness.ConclusionData suggest that targeted activation of Beclin-1 alleviates adverse outcomes of pneumonia-induced sepsis, and thus, possess a therapeutic potential.

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Background: Mitochondrial deficiency is a known pathology in sepsis-induced organ failure. We previously found that mitochondria-associated membranes (MAMs), a subcellular domain supporting mitochondrial status, are impaired in the heart during endotoxemia, suggesting a mechanism of mitochondrial damage occurred in sepsis. Mitophagy pathway via E3 ubiquitin ligase Parkin and PTEN-induced kinase 1 (PINK1) controls mitochondrial quality. Studies described here examined the impact of Parkin on cardiac MAMs and endotoxemia-induced cardiomyopathy. Additionally, point mutation W403A in Parkin was previously identified as a constitutively active mutation in vitro. In vivo effects of forced expression of this mutation were evaluated in the endotoxemia model.Methods: Mice of wild type (WT), Parkin-deficiency (Park2−/−), and knock-in expression of Parkin W402A (human Parkin W403A) were given lipopolysaccharide (LPS) challenge. Cardiac function was evaluated by echocardiography. In the harvested heart tissue, MAM fractions were isolated by ultracentrifugation, and their amount and function were quantified. Ultrastructure of MAMs and mitochondria was examined by electron microscopy. Mitochondrial respiratory activities were measured by enzyme assays. Myocardial inflammation was estimated by levels of pro-inflammatory cytokine IL-6. Myocardial mitophagy was assessed by levels of mitophagy factors associated with mitochondria and degrees of mitochondria-lysosome co-localization. Parkin activation, signified by phosphorylation on serine 65 of Parkin, was also evaluated.Results: Compared with WT, Park2−/− mice showed more severely impaired cardiac MAMs during endotoxemia, characterized by disrupted structure, reduced quantity, and weakened transporting function. Endotoxemia-induced cardiomyopathy was intensified in Park2−/− mice, shown by worsened cardiac contractility and higher production of IL-6. Mitochondria from the Park2−/− hearts were more deteriorated, indicated by losses in both structural integrity and respiration function. Unexpectedly, mice carrying Parkin W402A showed similar levels of cardiomyopathy and mitochondrial damage when compared with their WT counterparts. Further, Parkin W402A mutation neither enhanced mitophagy nor increased Parkin activation in myocardium under the challenge of endotoxemia.Conclusion: our results suggest that Parkin/PINK1 mitophagy participates in the regulation of cardiac MAMs during endotoxemia. Point mutation W402A (human W403A) in Parkin is not sufficient to alleviate cardiomyopathy induced by endotoxemia in vivo.

The effect of the pore-blocking peptides charybdotoxin and margatoxin, both scorpion toxins, on currents through human voltage-gated hK(v)1.3 wild-type and hK(v)1.3_H399N mutant potassium channels was characterized by the whole-cell patch clamp technique. In the mutant channels, both toxins hardly blocked current through the channels, although they did prevent C-type inactivation by slowing down the current decay during depolarization. Molecular dynamics simulations suggested that the fast current decay in the mutant channel was a consequence of amino acid reorientations behind the selectivity filter and indicated that the rigidity-flexibility in that region played a key role in its interactions with scorpion toxins. A channel with a slightly more flexible selectivity filter region exhibits distinct interactions with scorpion toxins. Our studies suggest that the toxin-channel interactions might partially restore rigidity in the selectivity filter and thereby prevent the structural rearrangements associated with C-type inactivation.