DOCSLIB.ORG

Studies of the Structure of Potassium Channel Kcsa in the Open Conformation and the Effect of Anionic Lipids on Channel Inactivation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Studies of the Structure of Potassium Channel Kcsa in the Open Conformation and the Effect of Anionic Lipids on Channel Inactivation Studies of the structure of potassium channel KcsA in the open conformation and the effect of anionic lipids on channel inactivation Dongyu (Allison) Zhang Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2019 © 2019 Dongyu Zhang All rights reserved Abstract Studies of the structure of potassium channel KcsA in the open conformation and the effect of anionic lipids on channel inactivation Dongyu Zhang Membrane proteins play a vital role in cellular processes. In this thesis, we use KcsA, a prokaryotic potassium channel, as a model to investigate the gating mechanism of ion channels and the effect of anionic lipids on the channel activity using solid-state NMR spectroscopy. KcsA activity is known to be highly dependent on the presence of negatively charged lipids. Multiple crystal structures combined with biochemistry assays suggest that KcsA is co-purified with anionic lipids with phosphatidylglycerol headgroup. Here, we identified this specifically bound, isotopically labeled lipid in the protein 13C-13C correlation spectra. Our results reveal that the lipid cross peaks show stronger intensity when the channel is in the inactivated state compared to the activated state, which indicates a stronger protein-lipid interaction when KcsA is inactivated. In addition, our data shows that including anionic lipids into proteoliposomes leads to a weaker potassium ion affinity at the selectivity filter. Considering ion loss as a model of inactivation, our results suggest anionic lipids promote channel inactivation. However, the surface charge is not the only physical parameter that regulates channel gating or conformational preference. We found that the channel adapted to different conformations when reconstituted into liposome either made of DOPC or DOPE, two zwitterionic lipids. Also, we were able to stabilize the open-conformation of KcsA in 3:1 DOPE/DOPG liposome at pH 4.0 and acquired several multi-dimensional solid-state NMR experiments for site-specific resonance assignments. This is the first time that we obtain wild-type full-length KcsA structural information on the transient state. The structure is not only important for understanding channel gating, but can also serve as a homology model for investigating drug binding with more complicated potassium channels such as human voltage gated channel (hERG). Table of Contents List of Figures ................................................................................................................................ iv 1. Introduction ............................................................................................................................. 1 1.1 Cell membrane and membrane proteins ......................................................................... 1 1.2 Potassium channel ........................................................................................................... 3 1.2.1 Pore-forming domain and selectivity ...................................................................... 3 1.2.2 Gating and inactivation ........................................................................................... 4 1.2.3 KcsA as a model ..................................................................................................... 5 1.3 Solid-state NMR studies of membrane proteins ............................................................. 6 1.3.1 Overview of structural biology techniques ............................................................. 6 1.3.2 SSNMR methodologies for membrane proteins ..................................................... 7 1.4 Contribution of this thesis ............................................................................................... 9 1.5 Reference ...................................................................................................................... 11 2. Site-specific Solid-state Resonance Assignments of KcsA in the open conformation ......... 13 2.1 Abstract ......................................................................................................................... 13 2.2 Introduction ................................................................................................................... 13 2.2.1 What is chemical shift and resonance assignments? ............................................. 13 2.2.2 The importance of the open conformation of KcsA ............................................. 16 2.3 Methods......................................................................................................................... 18 2.3.1 KcsA purification .................................................................................................. 18 2.3.2 NMR Spectroscopy ............................................................................................... 20 i 2.3.3 Data processing ..................................................................................................... 21 2.3.4 Assignment ........................................................................................................... 21 2.4 Results and Discussions ................................................................................................ 22 2.4.1 Assignment overview ............................................................................................ 22 2.4.2 Challenges in assignment ...................................................................................... 27 2.4.3 Comparison of SSNMR assigment of KcsA in open and deactivated state ........ 27 2.4.4 How do we know this open conformation is the open-conductive state? ............. 28 2.5 Reference ...................................................................................................................... 39 3. The effect of anionic lipids on the structure and function of KcsA ...................................... 41 3.1 Abstract ......................................................................................................................... 41 3.2 Introduction ................................................................................................................... 41 3.2.1 Effect of phospholipids on potassium channel KcsA ........................................... 41 3.3 Results ........................................................................................................................... 45 3.3.1 Observation of the specific binding lipid with PG headgroup .............................. 45 3.3.2 Lipids affect the conformational preferences of the gates in KcsA and the open conformation is stable in the absence of added anionic lipids .............................................. 48 3.3.3 Loss of ion affinity upon reconstitution in liposome with anionic head groups at acidic pH 53 3.4 Discussion ..................................................................................................................... 55 3.5 Material and Method ..................................................................................................... 57 3.5.1 Protein overexpression and purification ............................................................... 57 3.5.2 NMR spectroscopy ................................................................................................ 58 ii 3.6 Reference ...................................................................................................................... 59 4. TmDOTP : An NMR- based Thermometer for Magic Angle Spinning NMR Experiments 62 4.1 Abstract ......................................................................................................................... 62 4.2 Introduction ................................................................................................................... 63 4.2.1 NMR sample preparation ...................................................................................... 65 4.2.2 NMR Spectroscopy ............................................................................................... 65 4.3 Results and Discussion ................................................................................................. 67 1 4.3.1 H NMR signals of H2O and TmDOTP provide precise temperature measurements ........................................................................................................................ 67 4.3.2 Sample heating due to spinning is proportional to the rotor frequency squared .. 68 4.3.3 The influence of TmDOTP on hydrated proteoliposome samples ....................... 69 4.3.4 RF heating is linear with pulse power, pulse length and duty cycle ..................... 70 4.3.5 Inequivalent RF heating on pellet vs. supernatant ................................................ 71 4.3.6 Application and significance ................................................................................. 73 4.4 Conclusion .................................................................................................................... 73 4.5 Reference ...................................................................................................................... 75 Appendix 1 .................................................................................................................................... 77 A1.1 Working with SMALPs KcsA ......................................................................................... 82 Reference .................................................................................................................................
Load more

Recommended publications
  • Suramin Alters Phosphoinositide Synthesis and Inhibits Growth Factor Receptor Binding in HT-29 Cells'
    (CANCER RESEARCH 50. 6490-6496. October 15. 1990] Suramin Alters Phosphoinositide Synthesis and Inhibits Growth Factor Receptor Binding in HT-29 Cells' Reinhard Kopp2 and Andreas Pfeiffer Departments of Surgery and Internal Medicine II, Klinikum (irosshadern. University of Munich, West Germany ABSTRACT levels and a stimulation of calcium/calmodulin kinases. Di acylglycerol activates protein kinase C, a family of Ca2+-sensi- Initiation of cell growth frequently involves activation of growth factor tive and phospholipid-dependent isoenzymes, known to phos- receptor-coupled u rosine kinases and stimulation of the phosphoinositide phorylate regulatory proteins and to elevate cytosolic pH levels second messenger system. The antitrypanosomal and antifiliarial drug suramin has been shown to exert antiproliferative activities by inhibition (5, 6). Activation of protein kinase C by phorbol esters and of growth factor receptor binding. We therefore investigated the effect of elevation of intracellular calcium levels by calcium ionophores suramin on epidermal growth factor receptor-binding characteristics and, have been shown to be mitogenically active cofactors during the additionally, searched for effects on basal or cholinergically stimulated initiation of DNA synthesis (7-10). phospholipid metabolism in HT-29 cells. HT-29 colon carcinoma cells have recently been shown to Suramin caused a dose-dependent and noncompetitive inhibition of produce EGF/transforming growth factor «and insulin-like '"I-epidermal growth factor binding (concentration producing 50% inhi growth factor 1-like activities (11), indicating a possible auto bition, 44.2 Mg/ml)but did not alter muscarinic receptor binding. Suramin did not affect the basal '-'I* incorporation into phosphoinositides at crine proliferative effect of these growth factors.
    [Show full text]
  • Inactivation in the Potassium Channel Kcsa T ⁎ Yunyao Xu, Ann E
    Journal of Structural Biology: X 3 (2019) 100009 Contents lists available at ScienceDirect Journal of Structural Biology: X journal homepage: www.journals.elsevier.com/journal-of-structural-biology-x This article is part of the Special Issue on Solid-State NMR of Biomolecular Assemblies ☆ Inactivation in the potassium channel KcsA T ⁎ Yunyao Xu, Ann E. McDermott Department of Chemistry, Columbia University, New York, NY 10027, United States ARTICLE INFO ABSTRACT Keywords: Inactivation, the slow cessation of transmission after activation, is a general feature of potassium channels. It is Potassium channel essential for their function, and malfunctions in inactivation leads to numerous pathologies. The detailed me- C-type inactivation chanism for the C-type inactivation, distinct from the N-type inactivation, remains an active area of investiga- Solid state NMR tion. Crystallography, computational simulations, and NMR have greatly enriched our understanding of the Allosteric coupling process. Here we review the major hypotheses regarding C-type inactivation, particularly focusing on the key KcsA role played by NMR studies of the prokaryotic potassium channel KcsA, which serves as a good model for voltage gated mammalian channels. 1. Introduction treatment in a wide range of disorders including vasodilation and blood pressure regulation, arrhythmias, neurodegenerative diseases and psy- Potassium channels form the second largest family of membrane chiatric treatments (Parekh et al., 2018). Deletion of one of the back- proteins, and control numerous metabolic processes. Their primary ground potassium channels TREK-1 results in a depression-resistant function is to facilitate passive diffusion of potassium ions through low phenotype (Heurteaux et al., 2006). Potassium channel mutations and dielectric membrane, with great selectivity with respect to other ions malfunction (“channelopathies”) result in heart timing defects such as (McCoy and Nimigean, 2012; Yellen, 2002).
    [Show full text]
  • Structural Characterization of Bacterial Collagenases Ryan Bauer University of Arkansas, Fayetteville
    University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 8-2017 Structural Characterization of Bacterial Collagenases Ryan Bauer University of Arkansas, Fayetteville Follow this and additional works at: http://scholarworks.uark.edu/etd Part of the Biochemistry Commons Recommended Citation Bauer, Ryan, "Structural Characterization of Bacterial Collagenases" (2017). Theses and Dissertations. 2431. http://scholarworks.uark.edu/etd/2431 This Dissertation is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. Structural Characterization of Bacterial Collagenases A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry by Ryan Bauer University of Arkansas Bachelor of Science in Chemistry, 2006 August 2017 University of Arkansas This dissertation is approved for recommendation to the Graduate Council ________________________________ Dr. Joshua Sakon Dissertation Director _______________________________ _______________________________ Dr. Colin Heyes Dr. Wesley Stites Committee Member Committee Member _______________________________ Dr. Suresh Thallapuranam Committee Member Abstract Clostridium histolyticum secretes collagenases ColG and ColH to cause extensive tissue destruction during myonecrosis. The collagenases are multi-domain enzymes consisting of a N- terminal collagenase module, s1, polycystic kidney disease (PKD)-like domains (s2 in ColG; s2a and s2b in ColH) and collagen-binding domains (CBD) (s3a and s3b in ColG; s3 in ColH). The individual CBD and PKD-like domains chelate calcium to modulate stability and domain rearrangement. Though used by bacteria to break down collagen in the extracellular matrix during infection, therapeutic use of the enzyme to break down excess connective tissue has been approved by the FDA.
    [Show full text]
  • Inositol Triphosphate-Triggered Calcium Release Blocks Lipid Exchange at Endoplasmic Reticulum- Golgi Contact Sites
    ARTICLE https://doi.org/10.1038/s41467-021-22882-x OPEN Inositol triphosphate-triggered calcium release blocks lipid exchange at endoplasmic reticulum- Golgi contact sites Mouhannad Malek 1, Anna M. Wawrzyniak 1, Peter Koch1, Christian Lüchtenborg2, Manuel Hessenberger1, ✉ Timo Sachsenheimer2, Wonyul Jang 1, Britta Brügger 2 & Volker Haucke 1,3 fi 1234567890():,; Vesicular traf c and membrane contact sites between organelles enable the exchange of proteins, lipids, and metabolites. Recruitment of tethers to contact sites between the endo- plasmic reticulum (ER) and the plasma membrane is often triggered by calcium. Here we reveal a function for calcium in the repression of cholesterol export at membrane contact sites between the ER and the Golgi complex. We show that calcium efflux from ER stores induced by inositol-triphosphate [IP3] accumulation upon loss of the inositol 5-phosphatase INPP5A or receptor signaling triggers depletion of cholesterol and associated Gb3 from the cell surface, resulting in a blockade of clathrin-independent endocytosis (CIE) of Shiga toxin. This phenotype is caused by the calcium-induced dissociation of oxysterol binding protein (OSBP) from the Golgi complex and from VAP-containing membrane contact sites. Our findings reveal a crucial function for INPP5A-mediated IP3 hydrolysis in the control of lipid exchange at membrane contact sites. 1 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany. 2 Heidelberg University Biochemistry Center (BZH), Heidelberg ✉ University, Heidelberg, Germany. 3 Faculty of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Berlin, Germany. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:2673 | https://doi.org/10.1038/s41467-021-22882-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22882-x ellular membrane homeostasis and the exchange of Results material between compartments can occur by vesicular INPP5A is required for Gb3-mediated Shiga toxin cell entry.
    [Show full text]
  • Regulation of Signaling and Metabolism by Lipin-Mediated Phosphatidic Acid Phosphohydrolase Activity
    biomolecules Review Regulation of Signaling and Metabolism by Lipin-mediated Phosphatidic Acid Phosphohydrolase Activity Andrew J. Lutkewitte and Brian N. Finck * Center for Human Nutrition, Division of Geriatrics and Nutritional Sciences, Department of Medicine, Washington University School of Medicine, Euclid Avenue, Campus Box 8031, St. Louis, MO 63110, USA; [email protected] * Correspondence: bfi[email protected]; Tel: +1-3143628963 Received: 4 September 2020; Accepted: 24 September 2020; Published: 29 September 2020 Abstract: Phosphatidic acid (PA) is a glycerophospholipid intermediate in the triglyceride synthesis pathway that has incredibly important structural functions as a component of cell membranes and dynamic effects on intracellular and intercellular signaling pathways. Although there are many pathways to synthesize and degrade PA, a family of PA phosphohydrolases (lipin family proteins) that generate diacylglycerol constitute the primary pathway for PA incorporation into triglycerides. Previously, it was believed that the pool of PA used to synthesize triglyceride was distinct, compartmentalized, and did not widely intersect with signaling pathways. However, we now know that modulating the activity of lipin 1 has profound effects on signaling in a variety of cell types. Indeed, in most tissues except adipose tissue, lipin-mediated PA phosphohydrolase activity is far from limiting for normal rates of triglyceride synthesis, but rather impacts critical signaling cascades that control cellular homeostasis. In this review, we will discuss how lipin-mediated control of PA concentrations regulates metabolism and signaling in mammalian organisms. Keywords: phosphatidic acid; diacylglycerol; lipin; signaling 1. Introduction Foundational work many decades ago by the laboratory of Dr. Eugene Kennedy defined the four sequential enzymatic steps by which three fatty acyl groups were esterified onto the glycerol-3-phosphate backbone to synthesize triglyceride [1].
    [Show full text]
  • Phosphatidylglycerol Incorporates Into Cardiolipin to Improve
    www.nature.com/scientificreports OPEN Phosphatidylglycerol Incorporates into Cardiolipin to Improve Mitochondrial Activity and Inhibits Received: 3 July 2017 Accepted: 7 March 2018 Infammation Published: xx xx xxxx Wei-Wei Chen1, Yu-Jen Chao1, Wan-Hsin Chang1, Jui-Fen Chan1 & Yuan-Hao Howard Hsu1,2 Chronic infammation and concomitant oxidative stress can induce mitochondrial dysfunction due to cardiolipin (CL) abnormalities in the mitochondrial inner membrane. To examine the responses of mitochondria to infammation, macrophage-like RAW264.7 cells were activated by Kdo2-Lipid A (KLA) in our infammation model, and then the mitochondrial CL profle, mitochondrial activity, and the mRNA expression of CL metabolism-related genes were examined. The results demonstrated that KLA activation caused CL desaturation and the partial loss of mitochondrial activity. KLA activation also induced the gene upregulation of cyclooxygenase (COX)-2 and phospholipid scramblase 3, and the gene downregulation of COX-1, lipoxygenase 5, and Δ-6 desaturase. We further examined the phophatidylglycerol (PG) inhibition efects on infammation. PG supplementation resulted in a 358- fold inhibition of COX-2 mRNA expression. PG(18:1)2 and PG(18:2)2 were incorporated into CLs to considerably alter the CL profle. The decreased CL and increased monolysocardiolipin (MLCL) quantity resulted in a reduced CL/MLCL ratio. KLA-activated macrophages responded diferentially to PG(18:1)2 and PG(18:2)2 supplementation. Specifcally, PG(18:1)2 induced less changes in the CL/MLCL ratio than did PG(18:2)2, which resulted in a 50% reduction in the CL/MLCL ratio. However, both PG types rescued 20–30% of the mitochondrial activity that had been afected by KLA activation.
    [Show full text]
  • Twisting Conformational Changes of Single Kcsa Potassium Channel Upon Gating Using Diffracted X-Ray Tracking
    Life science : Medical Biology Twisting Conformational Changes of Single KcsA Potassium Channel upon Gating using Diffracted X-ray Tracking Ion channels are signal transduction molecules were exploited in this study to contrast the changes in that selectively permeate ions and generate electrical motion in a different gating status. signals through turning on and off of ion permeation When channels were in the closed state at neutral pathways. Channels respond to various stimuli, such pH, diffraction spots moved radially. This represents as membrane voltage and chemical substances. With bending motions of channels in the range of a few sensor domains, channel molecules transduce various degrees. When channels were actively gated at acidic stimuli into conformational changes of ion permeation pH, diffraction spots moved circumferentially (Fig. pathways, leading to ion-conductive (open) and non- 3(a,b)). These motions of the spots correspond to the conductive (closed) states. Single-channel current rotation of the channel around the longitudinal axis. recordings have elucidated the random nature of Random clockwise and counter-clockwise rotations opening and closing transitions (gating; Fig. 1(a)). were recorded. We found that the range of the Genes of potassium channels exist from bacteria to rotations was several tens of degrees, which are humans and constitute one of the largest gene family. unexpectedly large values. The direction of the Ten years ago, the crystal structure of a potassium rotational motions was sometimes reversed during channel from Streptomyces lividans (KcsA channel) recordings. We call this type of motion as twisting. was resolved (Fig. 1(b)). The three-dimensional This is the first observation of a large twisting in the structure provided the architecture of the ion conformational change of channel molecules upon permeation pathway and insights on how ions are gating.
    [Show full text]
  • Lipid–Protein and Protein–Protein Interactions in the Pulmonary Surfactant System and Their Role in Lung Homeostasis
    International Journal of Molecular Sciences Review Lipid–Protein and Protein–Protein Interactions in the Pulmonary Surfactant System and Their Role in Lung Homeostasis Olga Cañadas 1,2,Bárbara Olmeda 1,2, Alejandro Alonso 1,2 and Jesús Pérez-Gil 1,2,* 1 Departament of Biochemistry and Molecular Biology, Faculty of Biology, Complutense University, 28040 Madrid, Spain; [email protected] (O.C.); [email protected] (B.O.); [email protected] (A.A.) 2 Research Institut “Hospital Doce de Octubre (imasdoce)”, 28040 Madrid, Spain * Correspondence: [email protected]; Tel.: +34-913944994 Received: 9 May 2020; Accepted: 22 May 2020; Published: 25 May 2020 Abstract: Pulmonary surfactant is a lipid/protein complex synthesized by the alveolar epithelium and secreted into the airspaces, where it coats and protects the large respiratory air–liquid interface. Surfactant, assembled as a complex network of membranous structures, integrates elements in charge of reducing surface tension to a minimum along the breathing cycle, thus maintaining a large surface open to gas exchange and also protecting the lung and the body from the entrance of a myriad of potentially pathogenic entities. Different molecules in the surfactant establish a multivalent crosstalk with the epithelium, the immune system and the lung microbiota, constituting a crucial platform to sustain homeostasis, under health and disease. This review summarizes some of the most important molecules and interactions within lung surfactant and how multiple lipid–protein and protein–protein interactions contribute to the proper maintenance of an operative respiratory surface. Keywords: pulmonary surfactant film; surfactant metabolism; surface tension; respiratory air–liquid interface; inflammation; antimicrobial activity; apoptosis; efferocytosis; tissue repair 1.
    [Show full text]
  • The Role of Phosphatidylserine in Recognition of Apoptotic Cells by Phagocytes
    Cell Death and Differentiation (1998) 5, 551 ± 562 1998 Stockton Press All rights reserved 13509047/98 $12.00 http://www.stockton-press.co.uk/cdd Review The role of phosphatidylserine in recognition of apoptotic cells by phagocytes Valerie A. Fadok1,2, Donna L. Bratton1, S. Courtney Frasch1, epithelial cells and vascular smooth muscle cells). To date, Mary L. Warner1 and Peter M. Henson1 there have been a number of receptors described for macrophages and other cells which bind to apoptotic cells 1 Department of Pediatrics, National Jewish Medical and Research Center, 1400 and mediate their uptake. These include lectin-like receptors Jackson Street, Denver, Colorado 80206 USA (Duvall et al, 1985, Dini et al, 1992; 1995; Hall et al, 1994; 2 corresponding author: tel: 1-303-398-1281 fax: 1-303-398-1381 Morris et al, 1994; Falasca et al, 1996), the vitronectin receptor email: [email protected] avb3 (Savill et al, 1990; Hall et al, 1994; Hughes et al, 1997), CD36 (Savill et al, 1992), an uncharacterized phosphatidyl- Received: 15.10.97; revised: 23.3.98; accepted: 2.4.98 Edited by M. Piacentini serine-recognizing receptor (Fadok et al, 1992a,b, 1993; Pradhan et al, 1997), CD14 (Flora and Gregory 1994; Devitt et al, 1998), and scavenger receptors (Sambrano and Steinberg, Abstract 1995; Fukasawa et al, 1996; Platt et al, 1996; Murao et al, 1997). The ABC1 transporter, also involved in uptake of Exposure of phosphatidylserine on the outer leaflet of the mammalian apoptotic cells, has recently been shown to plasma membrane is a surface change common to many mediate anion transport (Luciani and Chimini, 1996; Becq et apoptotic cells.
    [Show full text]
  • Selectivity Filter Modalities and Rapid Inactivation of the Herg1 Channel
    Selectivity filter modalities and rapid inactivation of the hERG1 channel Williams E. Mirandaa, Kevin R. DeMarcob, Jiqing Guoc, Henry J. Duffc, Igor Vorobyovb,d, Colleen E. Clancyb,d,1, and Sergei Yu. Noskova,1 aCentre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada; bDepartment of Physiology and Membrane Biology, University of California, Davis, CA 95616; cLibin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; and dDepartment of Pharmacology, University of California, Davis, CA 95616 Edited by Michael L. Klein, Temple University, Philadelphia, PA, and approved December 19, 2019 (received for review May 28, 2019) The human ether-á-go-go–related gene (hERG1) channel conducts slow-inactivating current observed from its bacterial counterparts small outward K+ currents that are critical for cardiomyocyte mem- (15–17) and voltage-gated channels from the Shaker family brane repolarization. The gain-of-function mutation N629D at the (18–20). Moreover, the electrophysiological properties of the outer mouth of the selectivity filter (SF) disrupts inactivation and hERG1 channel are highly sensitive to mutations altering local K+-selective transport in hERG1, leading to arrhythmogenic pheno- electrical fields or changing the interaction network topology in + types associated with long-QT syndrome. Here, we combined com- the vicinity of canonical K binding sites in the SF region. For putational electrophysiology with Markov state model analysis to example, the mutation N629D adjacent to the SF signature se- investigate how SF-level gating modalities control selective cation quence 626GFG628 of the hERG1 channel was the first reported to transport in wild-type (WT) and mutant (N629D) hERG1 variants.
    [Show full text]
  • New Insights Into Multiple Sclerosis Mechanisms: Lipids on the Track to Control Inflammation and Neurodegeneration
    International Journal of Molecular Sciences Review New Insights into Multiple Sclerosis Mechanisms: Lipids on the Track to Control Inflammation and Neurodegeneration Maria Podbielska 1,2,* , Joan O’Keeffe 3 and Anna Pokryszko-Dragan 4 1 Department of Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA 2 Laboratory of Microbiome Immunobiology, Ludwik Hirszfeld Institute of Immunology & Experimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland 3 Department of Analytical, Biopharmaceutical and Medical Sciences, School of Science & Computing, Galway-Mayo Institute of Technology, Galway, Ireland; [email protected] 4 Department of Neurology, Wroclaw Medical University, 50-556 Wroclaw, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-71-370-9912 Abstract: Multiple sclerosis (MS) is a central nervous system disease with complex pathogenesis, including two main processes: immune-mediated inflammatory demyelination and progressive degeneration with axonal loss. Despite recent progress in our understanding and management of MS, availability of sensitive and specific biomarkers for these both processes, as well as neuroprotec- tive therapeutic options targeted at progressive phase of disease, are still being sought. Given their abundance in the myelin sheath, lipids are believed to play a central role in underlying immunopatho- genesis in MS and seem to be a promising subject of investigation in this field. On the basis of our previous research and a review of the literature, we discuss the current understanding of lipid-related mechanisms involved in active relapse, remission, and progression of MS. These insights highlight Citation: Podbielska, M.; O’Keeffe, J.; potential usefulness of lipid markers in prediction or monitoring the course of MS, particularly in its Pokryszko-Dragan, A.
    [Show full text]
  • Formation of Lipophilic Complexes with Phosphatidic Acid And
    476 Notizen Action of Polymyxin B on Bacterial Membranes, II Mono-A^-acetyl-14C-derivatives of polymyxin B (PX) Formation of Lipophilic Complexes with were prepared as previously reported 6. The specific activity was 14.7 juCi/jumole. Polymyxin B sulfate Phosphatidic Acid and Phosphatidylglycerol (sterile powder) was a gift of Pfizer GmbH (Karls­ M. Teuber ruhe, Germany). Radioactivity was determined in a Abteilung Mikrobiologie, Institut für Botanik, Technische toluene based scintillation liquid with a Beckman Universität München LSI00 spectrometer. (Z. Naturforsch. 28 c, 476 — 477 [1973] ; received Since it is generally assumed that phospholipids April 9, 1973) are arranged in biological membranes in bilayers Salmonella typhimurium, phospholipids, cell membrane, forming the hydrophobic permeability barrier7, it polymyxin B, antibiotic was of interest to search for a polymyxin-phospho- The bactericidal mechanism of action of poly­ lipid complex which would be stable in a hydro- myxin B against susceptible bacteria has been sug­ phobic environment. Because the polymyxin mole­ gested to result from electrostatic interactions of the cule is very polar at neutral pH (5 free amino- basic polypeptide antibiotic with acidic cell envelope groups) it does not migrate away from the origin phospholipids 1,2. This process causes the breakdown when chromatographed with chloroform-methanol- of the selective permeability barrier of the cyto­ water on Whatman No. 1 paper. However, when plasmic membrane, and finally cell death. The mixed with PA or PG, part of the radioactive anti- identities of the polymyxin receptor molecules in sensitive bacteria, however, have never been deter­ mined with certainty. The problem is further com­ plicated by the fact that polymyxin exhibits a strikingly selective activity against Gram-negative bacteria3 which, in addition to the plasma mem­ brane, possess a second membrane structure, the outer membrane or plastic layer composed of pro­ teins, lipopolysaccharide and phospholipids4.
    [Show full text]