scholarly journals Mapping the interaction surface of scorpion β-toxins with an insect sodium channel

2021 ◽  
Author(s):  
Boris S Zhorov ◽  
Yuzhe Du ◽  
Weizhong Song ◽  
Ningguang Luo ◽  
Dalia Gordon ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Periplaneta Americana ◽  
Mutational Analysis ◽  
Structural Models ◽  
High Sensitivity ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Low Sensitivity ◽  
Voltage Sensing Domain ◽  
Interaction Surface

The interaction of insect-selective scorpion depressant β-toxins (LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus) with the Blattella germanica sodium channel, BgNav1-1a, was investigated using site-directed mutagenesis, electrophysiological analyses, and structural modeling. Focusing on the pharmacologically-defined binding site-4 of scorpion β-toxins at the voltage-sensing domain II (VSD-II), we found that charge neutralization of D802 in VSD-II greatly enhanced the channel sensitivity to Lqh-dprIT3. This was consistent with the high sensitivity of the splice variant BgNav2-1, bearing G802, to Lqh-dprIT3, and low sensitivity of BgNav2-1 mutant, G802D, to the toxin. Further mutational and electrophysiological analyses revealed that the sensitivity of the WT = D802E < D802G < D802A < D802K channel mutants to Lqh-dprIT3 correlated with the depolarizing shifts of activation in toxin-free channels. However, the sensitivity of single mutants involving IIS4 basic residues (K4E = WT << R1E < R2E < R3E) or double mutants (D802K = K4E/D802K = R3E/D802K > R2E/D802K > R1E/D802K > WT) did not correlate with the activation shifts. Using the cryo-EM structure of the Periplaneta americana channel, NavPaS, as template and the crystal structure of LqhIT2, we constructed structural models of LqhIT2 and Lqh-dprIT3-c in complex with BgNav1-1a. These models along with the mutational analysis suggest that depressant toxins approach the salt-bridge between R1 and D802 at VSD-II to form contacts with linkers IIS1-S2, IIS3-S4, IIIP5-P1 and IIIP2-S6. Elimination of this salt-bridge enables deeper penetration of the toxin into a VSD-II gorge to form new contacts with the channel, leading to increased channel sensitivity to Lqh-dprIT3.

scholarly journals Pathological turret mutations in the cardiac sodium channel cause long-range pore disruption

2021 ◽  
Author(s):  
Zaki F Habib ◽  
Manas Kohli ◽  
Samantha C Salvage ◽  
Taufiq Rahman ◽  
Christopher L-H Huang ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Salt Bridge ◽  
Complex Salt ◽  
Site Directed Mutagenesis ◽  
Salt Bridges ◽  
Ion Permeability ◽  
Aromatic Residues ◽  
Functional Roles ◽  
Cardiac Sodium Channel ◽  
Channel Structures

AbstractThe voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Germline mutations that disrupt Nav1.5 activity predispose affected individuals to inherited cardiopathologies. Some of these Nav1.5 mutations alter amino acids in extracellular turret domains DII and DIII. Yet the mechanism is unclear. In the rat Nav1.5 structure determined by cryogenic electron microscopy, the wild-type residues corresponding to these mutants form a complex salt-bridge between the DII and DIII turret interface. Furthermore, adjacent aromatic residues form cation-π interactions with the complex salt-bridge. Here, we examine this region using site-directed mutagenesis, electrophysiology and in silico modeling. We confirm functional roles for the salt-bridges and the aromatic residues. We show that their disruption perturbs the geometry of both the DEKA selectivity ring and the inner pore vestibule that are crucial for sodium ion permeability. Our findings provide insights into a class of pathological mutations occurring not only in Nav1.5 but also in other sodium channel isoforms too. Our work illustrates how the sodium channel structures now being reported can be used to formulate and guide novel functional hypotheses.

Abstract 5317: Identification and Characterization of a Novel Susceptibility Gene, SCN3B, for Idiopathic Ventricular Fibrillation

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Carmen R Valdivia ◽  
Argelia Mereidos-Domingo ◽  
Thimothy J Algiers ◽  
Michael J Ackerman ◽  
Jonathan C Makielski
Keyword(s):  
Ventricular Fibrillation ◽  
Sodium Channel ◽  
Brugada Syndrome ◽  
Mutational Analysis ◽  
Sodium Current ◽  
Susceptibility Gene ◽  
Site Directed Mutagenesis ◽  
Macromolecular Complex ◽  
Patch Clamp Technique ◽  
Idiopathic Ventricular Fibrillation

Background: Mutations in the Na V 1.5 sodium channel macromolecular complex have been identified in some cases classified as idiopathic ventricular fibrillation (IVF). IVF and Brugada syndrome (BrS) are partially overlapping syndromes. Here, we report a mutation in SCN3B- encoded sodium channel β3 subunit as a novel pathogenic mechanism for IVF. Methods: Comprehensive open reading frame mutational analysis of SCN5A, GPD1L, and the beta subunit genes ( SCN1–4B ) was performed using PCR, DHPLC, and direct DNA sequencing of DNA extracted from a 20-year-old patient diagnosed with IVF. The SCN3B mutation was made by site directed mutagenesis and co-transfected with SCN5A into HEK-293 cells for functional chraracterization using the patch clamp technique. Results: A novel missense mutation, V54G-SCN3B, was identified in a 20-year-old male following collapse and external defibrillation from VF. After recovery, there was no detectable electrocardiographic abnormality. Imaging studies demonstrated a structurally normal heart, and the patient was diagnosed with IVF. The mutation was absent in 800 reference alleles and involved a highly conserved residue in the extracellular domain of the beta 3 subunit. No other mutations were identified in the 5 other genes. HEK cells expressing SCN5A and either WT-, or V54G-SCN3B were studied 24 hours after transfection. Cells expressing V54G-SCN3B showed significant decrease in sodium current density of 60±20 pA/pF compared to 203±35 pA/pF in WT-SCN3B (n=14–19). In addition V54G-SCN3B significantly shifted the activation curve +5 mV without affecting inactivation. Conclusions: This study provides the first molecular and cellular evidence implicating SCN3B in IVF. Given the marked loss-of-function to the sodium channel by V54G-SCN3B and the overlap between IVF and BrS, it will be interesting to determine whether mutations in SCN3B explain some cases of genotype negative Brugada syndrome.

scholarly journals Second harmonic generation correlation spectroscopy for characterizing translationally diffusing protein nanocrystals

2016 ◽  
Vol 72 (7) ◽  
pp. 849-859
Author(s):  
Ximeng Y. Dow ◽  
Christopher M. Dettmar ◽  
Emma L. DeWalt ◽  
Justin A. Newman ◽  
Alexander R. Dow ◽  
...  
Keyword(s):  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Unit Volume ◽  
Second Harmonic ◽  
Incident Beam ◽  
High Sensitivity ◽  
Correlation Spectroscopy ◽  
Optical Process ◽  
Active Nanoparticles ◽  
Low Sensitivity

Second harmonic generation correlation spectroscopy (SHG-CS) is demonstrated as a new approach to protein nanocrystal characterization. A novel line-scanning approach was performed to enable autocorrelation analysis without sample damage from the intense incident beam. An analytical model for autocorrelation was developed, which includes a correction for the optical scattering forces arising when focusing intense, infrared beams. SHG-CS was applied to the analysis of BaTiO3nanoparticles ranging from 200 to ∼500 nm and of photosystem I nanocrystals. A size distribution was recovered for each sample and compared with the size histogram measured by scanning electron microscopy (SEM). Good agreement was observed between the two independent measurements. The intrinsic selectivity of the second-order nonlinear optical process provides SHG-CS with the ability to distinguish well ordered nanocrystals from conglomerates and amorphous aggregates. Combining the recovered distribution of particle diameters with the histogram of measured SHG intensities provides the inherent hyperpolarizability per unit volume of the SHG-active nanoparticles. Simulations suggest that the SHG activity per unit volume is likely to exhibit relatively low sensitivity to the subtle distortions within the lattice that contribute to resolution loss in X-ray diffraction, but high sensitivity to the presence of multi-domain crystals.

Influence of Irrational Beliefs and Sensitivity to a Person on Future Psychology Students’ Propensity to Coercion or Nonviolence

2021 ◽  
pp. 143-165
Author(s):  
V.G. Maralov ◽  
◽  
V.A. Sitarov ◽  
Keyword(s):  
Irrational Beliefs ◽  
High Sensitivity ◽  
Daily Practice ◽  
Psychology Students ◽  
State Universities ◽  
Low Level ◽  
Level Of Understanding ◽  
Low Sensitivity ◽  
High Level ◽  
Dependent Type

The relevance of the problem is due to the importance of identifying factors that determine the propensity of students to coercion or nonviolence, creating psychological and pedagogical conditions for the formation of the socionomic sphere of nonviolent competencies for future specialists at universities. The theoretical basis of the study was the position of nonviolence as a daily practice of interaction, by which we understand the ability of a person to choose from a number of possible alternatives that carry the least charge of coercion. The aim of the work was to study the influence of irrational beliefs and sensitivity to a person (interest, empathy, understanding and assistance) on the students’ tendency to coercion, manipulation, non-violence and non-interference in the processes of interaction with people. The hypothesis was tested that the tendency of students to coercion, manipulation, and noninterference will be due to expressed irrational beliefs and low level of sensitivity to a person and the tendency to non-violence will be explained by the absence of irrational beliefs and a high level of sensitivity to a person. The study involved 125 students of pedagogical and psychological faculties of the Moscow Humanitarian and Cherepovets State universities. The authors used questionnaires to identify the positions of interaction among students and sensitivity to a person, as well as a list of irrational beliefs proposed by A. Beck and A. Freeman. It is established that the tendency to both coercion and manipulation are determined by the beliefs of anti-social type and low sensitivity to the person. The tendency to manipulate the narcissistic beliefs, high interest in people and understanding them, at the same time the tendency to non-violence and non-interference are determined by beliefs of avoidant and dependent types with a low level of understanding people. And a tendency to non-interference is determined by beliefs of dependent type with unexpressed orientation on helping. The tendency to nonviolence is determined by the high sensitivity to a person and the absence of irrational beliefs of antisocial, passive-aggressive and narcissistic types. As a result, the conclusion is made about the need to form purposefully the ability to nonviolent interaction among students, which should include the work on awareness and overcoming irrational beliefs and the development of sensitivity to a person. The obtained results can be used in practical work with students on the formation of their nonviolent competencies.

scholarly journals Modulation of Ligand Selectivity Associated with Activation of the Transmembrane Region of the Human Follitropin Receptor

2004 ◽  
Vol 18 (8) ◽  
pp. 2061-2073 ◽  
Author(s):  
Lucia Montanelli ◽  
Joost J. J. Van Durme ◽  
Guillaume Smits ◽  
Marco Bonomi ◽  
Patrice Rodien ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Hormone Receptors ◽  
Transmembrane Helix ◽  
High Sensitivity ◽  
Site Directed Mutagenesis ◽  
Constitutive Activity ◽  
Glycoprotein Hormone ◽  
Functional Specificity ◽  
Ligand Selectivity

Abstract Recently, three naturally occurring mutations in the serpentine region of the FSH receptor (FSHr) (D567N and T449I/A) have been identified in three families with spontaneous ovarian hyperstimulation syndrome (OHSS). All mutant receptors displayed abnormally high sensitivity to human chorionic gonadotropin and, in addition, D567N and T449A displayed concomitant increase in sensitivity to TSH and detectable constitutive activity. In the present study, we have used a combination of site-directed mutagenesis experiments and molecular modeling to explore the mechanisms responsible for the phenotype of the three OHSS FSHr mutants. Our results suggest that all mutations lead to weakening of interhelical locks between transmembrane helix (TM)-VI and TM-III, or TM-VI and TM-VII, which contributes to maintaining the receptor in the inactive state. They also indicate that broadening of the functional specificity of the mutant FSHr constructs is correlated to their increase in constitutive activity. This relation between basal activity and functional specificity is a characteristic of the FSHr, which is not shared by the other glycoprotein hormone receptors. It leads to the interesting suggestion that different pathways have been followed during primate evolution to avoid promiscuous stimulation of the TSHr and FSHr by human chorionic gonadotropin. In the hFSHr, specificity would be exerted both by the ectodomain and the serpentine portion.

scholarly journals Predicting Response to Total Neoadjuvant Treatment (TNT) in Locally Advanced Rectal Cancer Based on Multiparametric Magnetic Resonance Imaging: A Retrospective Study

2020 ◽  
Author(s):  
Ganlu Ouyang ◽  
Xibiao Yang ◽  
Xiangbing Deng ◽  
Wenjian Meng ◽  
Yongyang Yu ◽  
...  
Keyword(s):  
Magnetic Resonance Imaging ◽  
Rectal Cancer ◽  
Lymph Node ◽  
Locally Advanced ◽  
Neoadjuvant Treatment ◽  
High Sensitivity ◽  
Advanced Rectal Cancer ◽  
Locally Advanced Rectal Cancer ◽  
Resonance Imaging ◽  
Low Sensitivity

Abstract Purpose: To investigate the potential value of magnetic resonance imaging (MRI) in predicting response relevance to total neoadjuvant treatment (TNT) in locally advanced rectal cancer.Methods: We analyzed MRI of 71 patients underwent TNT from 2015 to 2017 retrospectively. We categorized the response of TNT as CR (complete response) and non-CR, and high, moderate and low sensitivity. Logistic regression analysis was used to identify the best predictors of response. Diagnostic performance was assessed using receiver - operating characteristic curve analysis.Results: Post–ICT (induction chemotherapy) ∆TL (tumor length), post-CRT (concurrent chemoradiotherapy) ∆LNN (the numbers of lymph node metastases), post–CCT (consolidation chemotherapy) ∆SDWI (maximum cross-sectional area of tumor on diffusion-weighted imaging), post-CCT ADCT (the mean apparent diffusion coefficient values of tumor) and post-CCT ∆LNV (volume of lymph node) were the best CR predictors. Post-CRT EMVI (extramural vascular invasion) and post-CCT ∆ST2 (S on T2-weight) were the best significant factors for high sensitivity. Conclusions: Post-ICT ∆TL and post-CRT EMVI may an early predictor of CR and high sensitivity to TNT, respectively. The grouping scheme of CR and non–CR was more suitable for predicting response by MRI parameters than high, moderate and low sensitivity.Trial registration: retrospectively registered

scholarly journals The Effect of Oral Tactile Sensitivity on Texture Perception and Mastication Behavior in Humans

2018 ◽  
Author(s):  
Grace E. Shupe ◽  
Arran Wilson ◽  
Curtis R. Luckett
Keyword(s):  
High Sensitivity ◽  
Tactile Feedback ◽  
Interindividual Variation ◽  
Texture Perception ◽  
Tactile Acuity ◽  
Force Sensitivity ◽  
Significant Difference ◽  
Low Sensitivity ◽  
Oral Sensitivity ◽  
The Brain

AbstractMastication behavior is a notable source of interindividual variation in texture perception and could be linked to oral sensitivity. As oral sensitivity declines so does the amount of tactile feedback relayed to the brain, resulting in less effective manipulation or food and a reduced ability to discriminate differences. To address these hypotheses, we measured masticatory behavior and related this to texture discrimination and oral sensitivity. The study was performed on 41 participants in two groups, with high (n = 20) or low (n=21) sensitivity. Oral sensitivity was measured using a battery of tests that included: oral stereognosis, lingual tactile acuity, and bite force sensitivity. Sensitivity to texture changes was measured using a series of triangle tests with confectionaries of different hardness, with masticatory patterns and behaviors being video recorded and analyzed using jaw tracking software. Overall, there was no significant difference between high and low sensitivity participants and their ability to distinguish texture changes. But, there were significantly different trends found between the groups based on their masticatory behaviors including chewing pattern and overall number of chews. But, it was found that multiple masticatory behaviors were being modulated by oral sensitivity, including overall chewing cycles used (p < 0.0001). More, specifically those in the high sensitivity group used more stochastic chewing movements, while those in the low sensitivity group were found to use crescent-shaped chewing cycles. It was also noted that in the high sensitivity group the jaw moved further distances (p < 0.0001) in all phases and moved at a higher velocity when opening (p < 0.0001) but not when closing, when compared to the low sensitivity group. These results help bolster evidence that mastication and oral sensitivity are related.

Functional investigation of a highly conserved aspartate residue in the anti -cancer drug efflux transport protein MRP1

2018 ◽  
Author(s):  
Graeme Mullins
Keyword(s):  
Abc Transporters ◽  
Double Mutant ◽  
Transmembrane Helix ◽  
Salt Bridge ◽  
Multidrug Resistant ◽  
Site Directed Mutagenesis ◽  
Cancer Drug ◽  
Wide Range ◽  
Anti Cancer ◽  
Membrane Spanning

Multidrug Resistant Protein 1 (MRP1 or ABCC1) belongs to a subclass of ATP-binding cassette (ABC) transporters that export a wide range of metabolites and xenobiotics across the plasma membrane. Increased expression of MRP1 in cancer cells enhances efflux of many anti-cancer agents, giving rise to multidrug resistant tumours. The purpose of this study was to investigate the function of an aspartate (Asp) amino acid that is highly conserved in all MRP-related proteins by mutating it and determining the consequences of doing so. Asp430 lies at the interface of the cytoplasm and a transmembrane helix in the first membrane-spanning domain of MRP1. Previous studies have shown that when Asp430 is mutated, the protein becomes unstable and is degraded.Because this Asp430 is highly conserved in many MRP-related ABC transporters and because structural homology models of human MRP1 predict that Asp430 is in close proximity to Arg433, we hypothesized that a salt bridge between these two a mino acids could be essential for proper folding and stability of the protein during its biosynthesis. Using site -directed mutagenesis, these two amino acids were interchanged to probe the existence of such an interaction. Thus a double mutant where Asp430 was mutated to Arg, and Arg433 was mutated to Asp was created, and the resultant mutant protein (D430R/R433D) was tested for its ability to be detected in mammalian cells by gel electrophoresis and immunoblotting. Our results show differences between the migration patterns of double and single mutants that are compatible with differences in the glycosylation levels of MRP1. However the fact that D430R and the R433D mutants don’t share the same migration pattern, together with the variation in migration bet ween D430 wild type and Supported by CIHR MOP-10519the double mutant D430R/R433D indicate that the possibility of a salt bridge can be discarded.Supported by CIHR MOP-10519

Abstract 20627: Modulation of Cardiac Sodium Channel by Palmitoylation and Its Association With Cardiac Disease Mutations

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Zifan Pei ◽  
Andy Hudmon ◽  
Theodore R Cummins
Keyword(s):  
Steady State ◽  
Sodium Channel ◽  
Functional Activity ◽  
Site Directed Mutagenesis ◽  
Significant Shift ◽  
Biophysical Properties ◽  
Disease Mutations ◽  
Cardiac Sodium Channel ◽  
Steady State Inactivation

Cardiac sodium channel (Nav1.5) is responsible for the generation and propagation of the cardiac action potential, which underlies cardiac excitability. It can be modified by a variety of post-translational modifications. Palmitoylation is one of the most common post-translational lipid modifications that can dynamically regulate protein life cycle and functional activity. In our study, we identified palmitoylation on Nav1.5 and its alteration in channel biophysical properties. Nav1.5 palmitoylation was identified in both HEK 293 cells stably expressing Nav1.5 and cardiac tissues using acyl-biotin exchange assay. Nav1.5 palmitoylation was inhibited by pre-incubating the cells with the inhibitor 2-Br-Palmitate (2BP, 25uM, 24hrs). Biophysically, 2BP treatment drastically shifted the channel steady-state inactivation to more hyperpolarized voltages, suggesting palmitoylation altering channel functional activity. In addition, four predicted endogenous palmitoylation sites were identified using CSS-Palm 3.0. Site-directed mutagenesis method was used to generate a cysteine removing background of wt Nav1.5 to study the role of predicted sites. Patch clamp analysis of wt and cysteine-removed Nav1.5 revealed a significant change in channel biophysics. 2BP treatment significantly shifted steady-state inactivation of wt Nav1.5 while not affecting cysteine-removed Nav1.5 significantly, indicating the important role of these four cysteine sites in modulating channel palmitoylation. Moreover, several LQT disease mutations were identified to potentially add or remove palmitoylation sites. Further analysis of these disease mutations revealed a significant shift in channel steady-state inactivation and this alteration cannot be seen with the substitution of other residues on the same site, suggesting the specific role of cysteine residue in causing the functional alteration. For the LQT mutation that removes potential palmitoylation site, 2BP treatment did not affect channel biophysical properties, indicating the essential role of this cysteine in channel palmitoylation. These results suggest that palmitoylation on Nav1.5 regulates channel functional activity and its modulation may contribute to new cardiac channelopathies.

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