Flow-Dependent Mass Transfer May Trigger Endothelial Signaling Cascades

It is well known that fluid mechanical forces directly impact endothelial signaling pathways. But while this general observation is clear, less apparent are the underlying mechanisms that initiate these critical signaling processes. This is because fluid mechanical forces can offer a direct mechanical input to possible mechanotransducers as well as alter critical mass transport characteristics (i.e., concentration gradients) of a host of chemical stimuli present in the blood stream. However, it has recently been accepted that mechanotransduction (direct mechanical force input), and not mass transfer, is the fundamental mechanism for many hemodynamic force-modulated endothelial signaling pathways and their downstream gene products. This conclusion has been largely based, indirectly, on accepted criteria that correlate signaling behavior and shear rate and shear stress, relative to changes in viscosity. However, in this work, we investigate the negative control for these criteria. Here we computationally and experimentally subject mass-transfer limited systems, independent of mechanotransduction, to the purported criteria. The results showed that the negative control (mass-transfer limited system) produced the same trends that have been used to identify mechanotransduction-dominant systems. Thus, the widely used viscosity-related shear stress and shear rate criteria are insufficient in determining mechanotransduction-dominant systems. Thus, research should continue to consider the importance of mass transfer in triggering signaling cascades

Characterising chromosome rearrangements: recent technical advances in molecular cytogenetics

Genomic rearrangements can result in losses, amplifications, translocations and inversions of DNA fragments thereby modifying genome architecture, and potentially having clinical consequences. Many genomic disorders caused by structural variation have initially been uncovered by early cytogenetic methods. The last decade has seen significant progression in molecular cytogenetic techniques, allowing rapid and precise detection of structural rearrangements on a whole-genome scale. The high resolution attainable with these recently developed techniques has also uncovered the role of structural variants in normal genetic variation alongside single-nucleotide polymorphisms (SNPs). We describe how array-based comparative genomic hybridisation, SNP arrays, array painting and next-generation sequencing analytical methods (read depth, read pair and split read) allow the extensive characterisation of chromosome rearrangements in human genomes

Altered ionic selectivity of the sodium channel revealed by cysteine mutations within the pore

To explore the role of pore-lining amino acids in Na+ channel ion- selectivity, pore residues were replaced serially with cysteine in cloned rat skeletal muscle Na+ channels. Ionic selectivity was determined by measuring permeability and ionic current ratios of whole-cell currents in Xenopus oocytes. The rSkM1 channels displayed an ionic selectivity sequence Na+>Li+>NH4 +>>K+>>Cs+ and were impermeable to divalent cations. Replacement of residues in domain IV showed significantly enhanced current and permeability ratios of NH4 + and K+, and negative shifts in the reversal potentials recorded in the presence of external Na+ solutions when compared to cysteine mutants in domains I, II, and III (except K1237C). Mutants in domain IV showed altered selectivity sequences: W1531C (NH4 +>K+>Na+≤Li+≃Cs+), D1532C, and G1533C (Na+>Li+≤NH4 +>K+>Cs+). Conservative replacement of the aromatic residue in domain IV (W1531) with phenylalanine or tyrosine retained Na+ selectivity of the channel while the alanine mutant (W1531A) reduced ion selectivity. A single mutation within the third pore forming region (K1237C) dramatically altered the selectivity sequence of the rSkM1 channel (NH4 +>+K+>Na+≤Li+≃Cs+) and was permeable to divalent cations having the selectivity sequence Ca2+≤Sr2+>Mg2+>Ba2+. Sulfhydryl modification of K1237C, W1531C or D1532C with methanethiosulfonate derivatives that introduce a positively charged ammonium group, large trimethylammonium moiety, or a negatively charged sulfonate group within the pore was ineffective in restoring Na+ selectivity to these channels. Selectivity of D1532C mutants could be largely restored by increasing extracellular pH suggesting altering the ionized state at this position influences selectivity. These data suggest that K1237 in domain III and W1531, D1532, and G1533 in domain IV play a critical role in determining the ionic selectivity of the Na+ channel.link_to_subscribed_fulltex

P-loop flexibility in Na+ channel pores revealed by single- and double- cysteine replacements

Replacement of individual P-loop residues with cysteines in rat skeletal muscle Na+ channels (SkMI) caused an increased sensitivity to current blockade by Cd2+ thus allowing detection of residues lining the pore. Simultaneous replacement of two residues in distinct P-loops created channels with enhanced and reduced sensitivity to Cd2+ block relative to the individual single mutants, suggesting coordinated Cd2+ binding and cross- linking by the inserted sulfhydryl pairs. Double-mutant channels with reduced sensitivity to Cd2+ block showed enhanced sensitivity after the application of sulfhydryl reducing agents. These results allow identification of residue pairs capable of approaching one another to within less than 3.5 Å. We often observed that multiple consecutive adjacent residues in one P-loop could coordinately bind Cd2+ with a single residue in another P-loop. These results suggest that, on the time-scale of Cd2+ binding to mutant Na+ channels, P-loops show a high degree of flexibility.link_to_subscribed_fulltex

Pore residues critical for μ-CTX binding to rat skeletal muscle Na+ channels revealed by cysteine mutagenesis

We have studied μ-conotoxin (μ-CTX) block of rat skeletal muscle sodium channel (rSkM1) currents in which single amine acids within the pore (P-loop) were substituted with cysteine. Among 17 cysteine mutants expressed in Xenopus oocytes, 7 showed significant alterations in sensitivity to μ- CTX compared to wild-type rSkM 1 channel (IC50- = 17.5 ± 2.8 nM). E758C and D1241C were less sensitive to μ-CTX block (IC50 = 220 ± 39 nM and 112 ± 24 nM, respectively), whereas the tryptophan mutants W402C, W1239C, and W1531C showed enhanced μ-CTX sensitivity (IC50 = 1.9 ± 0.1, 4.9 ± 0.9, and 5.5 ± 0.4 nM, respectively). D400C and Y401C also showed statistically significant yet modest (approximately twofold) changes in sensitivity to μ- CTX block compared to WT (p 1 μM) and increased the IC50 of D1241C by about threefold. Applications of MTSEA, MTSES, and the neutral MTSBN (benzyl methanethiosulfonate) to the tryptophan- to-cysteine mutants partially or fully restored the wild-type μ-CTX sensitivity, suggesting that the bulkiness of the tryptophan's indole group is a determinant of toxin binding. In support of this suggestion, the blocking IC50 of W1531A (7.5 ± 1.3 nM) was similar to W1531C, whereas W1531Y showed reduced toxin sensitivity (14.6 ± 3.5 nM) similar to that of the wild-type channel. Our results demonstrate that charge at positions 758 and 1241 are important for μ-CTX toxin binding and further suggest that the tryptophan residues within the pore in domains I, III, and IV negatively influence toxin-channel interaction.link_to_subscribed_fulltex

A multifunctional aromatic residue in the external pore vestibule of Na+ channels contributes to the local anesthetic receptor

Voltage-gated Na+ (Nav) channels are responsible for initiating action potentials in excitable cells and are the targets of local anesthetics (LA). The LA receptor is localized to the cytoplasmic pore mouth formed by the S6 segments from all four domains (DI-DIV) but several outer pore-lining residues have also been shown to influence LA block (albeit somewhat modestly). Many of the reported amino acid substitutions, however, also disrupt the inactivated conformations that favor LA binding, complicating the interpretation of their specific effects on drug block. In this article, we report that an externally accessible aromatic residue in the Nav channel pore, DIV-Trp1531, when substituted with cysteine, completely abolished LA block (e.g., 300 μM mexiletine induced a use-dependent block with 65.0 ± 2.9% remaining current and -11.0 ± 0.6 mV of steady-state inactivation shift of wild-type (WT) channels versus 97.4 ± 0.7% and -2.4 ± 2.1 mV of W1531C, respectively; p < 0.05) without destabilizing fast inactivation (complete inactivation at 20 ms at -20 mV; V1/2 = -70.0 ± 1.6 mV versus -48.6 ± 0.5 mV of WT). W1531C also abolished internal QX-222 block (200 μM; 98.4 ± 3.4% versus 54.0 ± 3.2% of WT) without altering drug access. It is interesting that W1531Y restored WT blocking behavior, whereas W1531A channels exhibited an intermediate phenotype. Together, our results provide novel insights into the mechanism of drug action, and the structural relationship between the LA receptor and the outer pore vestibule.link_to_subscribed_fulltex

Local anesthetic anchoring to cardiac sodium channels: Implications into tissue-selective drug targeting

Local anesthetics inhibit Na + channels in a variety of tissues, leading to potentially serious side effects when used clinically. We have created a series of novel local anesthetics by connecting benzocaine (BZ) to the sulfhydryl-reactive group methanethiosulfonate (MTS) via variable-length polyethylether linkers (L) (MTS-LX-BZ [X represents 0, 3, 6, or 9]). The application of MTS-LX-BZ agents modified native rat cardiac as well as heterologously expressed human heart (hH1) and rat skeletal muscle (rSkM1) Na + channels in a manner resembling that of free BZ. Like BZ, the effects of MTS-LX-BZ on rSkM1 channels were completely reversible. In contrast, MTS-LX- BZ modification of heart and mutant rSkM1 channels, containing a pore cysteine at the equivalent location as cardiac Na + channels (ie, Y401C), persisted after drug washout unless treated with DTT, which suggests anchoring to the pore via a disulfide bond. Anchored MTS-LX-BZ competitively reduced the affinity of cardiac Na + channels for lidocaine but had minimal effects on mutant channels with disrupted local anesthetic modification properties. These results establish that anchored MTS-LX-BZ compounds interact with the local anesthetic binding site (LABS). Variation in the linker length altered the potency of channel modification by the anchored drugs, thus providing information on the spatial relationship between the anchoring site and the LABS. Our observations demonstrate that local anesthetics can he anchored to the extracellular pore cysteine in cardiac Na + channels and dynamically interact with the intracellular LABS. These results suggest that nonselective agents, such as local anesthetics, might be made more selective by linking these agents to target-specific anchors.link_to_subscribed_fulltex

Local anesthetic anchoring to cardiac sodium channels: Implications into tissue-selective drug targeting

Local anesthetics inhibit Na + channels in a variety of tissues, leading to potentially serious side effects when used clinically. We have created a series of novel local anesthetics by connecting benzocaine (BZ) to the sulfhydryl-reactive group methanethiosulfonate (MTS) via variable-length polyethylether linkers (L) (MTS-LX-BZ [X represents 0, 3, 6, or 9]). The application of MTS-LX-BZ agents modified native rat cardiac as well as heterologously expressed human heart (hH1) and rat skeletal muscle (rSkM1) Na + channels in a manner resembling that of free BZ. Like BZ, the effects of MTS-LX-BZ on rSkM1 channels were completely reversible. In contrast, MTS-LX- BZ modification of heart and mutant rSkM1 channels, containing a pore cysteine at the equivalent location as cardiac Na + channels (ie, Y401C), persisted after drug washout unless treated with DTT, which suggests anchoring to the pore via a disulfide bond. Anchored MTS-LX-BZ competitively reduced the affinity of cardiac Na + channels for lidocaine but had minimal effects on mutant channels with disrupted local anesthetic modification properties. These results establish that anchored MTS-LX-BZ compounds interact with the local anesthetic binding site (LABS). Variation in the linker length altered the potency of channel modification by the anchored drugs, thus providing information on the spatial relationship between the anchoring site and the LABS. Our observations demonstrate that local anesthetics can he anchored to the extracellular pore cysteine in cardiac Na + channels and dynamically interact with the intracellular LABS. These results suggest that nonselective agents, such as local anesthetics, might be made more selective by linking these agents to target-specific anchors.link_to_subscribed_fulltex

Role of phosphatidylinositol 3-kinase gamma in the beta-cell: Interactions with glucagon-like peptide-1

Glucagon-like peptide-1 (GLP-1) increases beta-cell function and growth through protein kinase A- and phosphatidylinositol-3-kinase (PI3-K)/protein kinase B, respectively. GLP-1 acts via a G protein-coupled receptor, and PI3-K gamma is known to be activated by G(beta gamma). Therefore, the role of PI3-K gamma in the chronic effects of GLP-1 on the beta-cell was investigated using PI3-K gamma knockout (KO) mice treated with the GLP-1 receptor agonist, exendin-4 (Ex4; 1 nmol/kg sc every 24 h for 14 d). In vivo, glucose and insulin responses were similar in PBS- and Ex4-treated KO and wild-type (WT) mice. However, glucose-stimulated insulin secretion was markedly impaired in islets from PBS-KO mice (P < 0.05), and this was partially normalized by chronic Ex4 treatment (P < 0.05). In contrast, insulin content was increased in PBS- KO islets, and this was paradoxically decreased by Ex4 treatment, compared with the stimulatory effect of Ex4 on WT islets (P < 0.05-0.01). Transfection of INS-1E beta-cells with small interferingRNAfor PI3-K gamma similarly decreased glucose-stimulated insulin secretion (P < 0.01) and increased insulin content. Basal values for beta-cell mass, islet number and proliferation, glucose transporter 2, glucokinase, and insulin receptor substrate-2 were increased in PBS- KO mice (P < 0.05-0.001) and, although they were increased by Ex4 treatment of WT animals (P < 0.05), they were decreased in Ex4-KO mice (P < 0.05-0.01). These findings indicate that PI3-K gamma deficiency impairs insulin secretion, resulting in compensatory islet growth to maintain normoglycemia. Chronic Ex4 treatment normalizes the secretory defect, thereby relieving the pressure for expansion of beta-cell mass. These studies reveal a new role for PI3-K gamma as a positive regulator of insulin secretion, and reinforce the importance of GLP-1 for the maintenance of normal beta-cell function