Inactivating peptide of the Shaker B potassium channel: conformational preferences inferred from studies on simple model systems

José A. ENCINAR; Asia M. FERNÁNDEZ; Emilio GIL-MARTÍN; Francisco GAVILANES; Juan P. ALBAR; José A. FERRAGUT; José M. GONZÁLEZ-ROS

doi:10.1042/bj3310497

Inactivating peptide of the Shaker B potassium channel: conformational preferences inferred from studies on simple model systems

Biochemical Journal ◽

10.1042/bj3310497 ◽

1998 ◽

Vol 331 (2) ◽

pp. 497-504 ◽

Cited By ~ 7

Author(s):

José A. ENCINAR ◽

Asia M. FERNÁNDEZ ◽

Emilio GIL-MARTÍN ◽

Francisco GAVILANES ◽

Juan P. ALBAR ◽

...

Keyword(s):

Electrostatic Interactions ◽

Membrane Surface ◽

Freeze Fracture ◽

Structural Models ◽

Model Systems ◽

Peptide Sequence ◽

K Channel ◽

Anionic Phospholipid ◽

Fluorescence Studies ◽

Hydrogen Bonded

Previous studies on the interaction between the inactivating peptide of the Shaker B K+ channel (ShB peptide, H2N-MAAVAGLYGLGEDRQHRKKQ) and anionic phospholipid vesicles, used as model targets, have shown that the ShB peptide: (i) binds to the vesicle surface with high affinity; (ii) readily adopts a strongly hydrogen-bonded β-structure; and (iii) becomes inserted into the hydrophobic bilayer. We now report fluorescence studies showing that the vesicle-inserted ShB peptide is in a monomeric form and, therefore, the observed β-structure must be intramolecularly hydrogen-bonded to produce a β-hairpin conformation. Also, additional freeze–fracture and accessibility-to-trypsin studies, which aimed to estimate how deeply and in which orientation the folded monomeric peptide inserts into the model target, have allowed us to build structural models for the target-inserted peptide. In such models, the peptide has been folded near G6 to configure a long β-hairpin modelled to produce an internal cancellation of net charges in the stretch comprising amino acids 1–16. As to the positively charged C-terminal portion of the ShB peptide (RKKQ), this has been modelled to be in parallel with the anionic membrane surface to facilitate electrostatic interactions. Since the negatively charged surface and the hydrophobic domains in the model vesicle target may partly imitate those present at the inactivation ‘entrance ’ in the channel protein [Kukuljan, M., Labarca, P. and Latorre, R. (1995) Am. J. Physiol. Cell Physiol. 268, C535–C556], we believe that the structural models postulated here for the vesicle-inserted peptide could help to understand how the ShB peptide associates with the channel during inactivation and why mutations at specific sites in the ShB peptide sequence, such as that in the ShB-L7E peptide, result in non-inactivating peptide variants.

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Membrane-induced conformational change in human apolipoprotein H

Biochemical Journal ◽

10.1042/bj3480103 ◽

2000 ◽

Vol 348 (1) ◽

pp. 103-106 ◽

Cited By ~ 15

Author(s):

Shao-Xiong WANG ◽

Yu-Tong SUN ◽

Sen-Fang SUI

Keyword(s):

Conformational Change ◽

Lipid Membrane ◽

Membrane Surface ◽

Basic Mechanism ◽

Cd Spectroscopy ◽

Model Systems ◽

Anionic Phospholipid ◽

Human Apolipoprotein ◽

Apolipoprotein H ◽

Α Helix

The interaction of apolipoprotein H (Apo H) with lipid membrane has been considered to be a basic mechanism for the biological function of the protein. Previous reports have demonstrated that Apo H can interact only with membranes containing anionic phospholipids. Here we study the membrane-induced conformational change of Apo H by CD spectroscopy with two different model systems: anionic-phospholipid-containing liposomes [such as 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and cardiolipin], and the water/methanol mixtures at moderately low pH, which mimic the micro-physicochemical environment near the membrane surface. It is found that Apo H undergoes a remarkable conformational change on interaction with liposomes containing anionic phospholipid. To interact with liposomes containing DMPG, there is a 6.8% increase in α-helix in the secondary structures; in liposomes containing cardiolipin, however, there is a 12.6% increase in α-helix and a 9% decrease in β-sheet. The similar conformation change in Apo H can be induced by treatment with an appropriate mixture of water/methanol. The results indicate that the association of Apo H with membrane is correlated with a certain conformational change in the secondary structure of the protein.

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Liposomes with cerasome-forming lipids as gene therapy vectors

10.31219/osf.io/zjn6v ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

Electrostatic Interactions ◽

Membrane Surface ◽

Immunological Response ◽

Basic Research ◽

Systemic Circulation ◽

Cationic Lipids ◽

Transport Systems ◽

Component Selection ◽

Polar Groups ◽

Gene Therapy Vectors

Basic research focuses on liposome transport systems as non-viral ways of transferring genetic and therapeutic substances. Due to electrostatic interactions between positive polar groups of cationic lipids and the negatively charged cell membrane surface, nucleic acids may readily be transported into cells as part of lipoplexes. Cationic liposomes, however, have a number of well-known problems, including low stability, toxicity, and a potential for severe immunological response. Recent research has revealed, however, that by increasing the system with additional components such as cerasome-forming lipids, these problems may be solved. They aid in the prolongation of the release of a beneficial liposome load in cells by increasing vesicle stability in the systemic circulation. This circumstance broadens the applications for liposomal systems. As a consequence, using varied lipids can create numerous liposomes for use in a range of medical fields. Nonviral delivery approaches based on hybrid liposomes with suitable structural component selection may be a possible response to a number of medical concerns, and further study is urgently needed.

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Calcium accumulated by sickle cell anemia red cells does not affect their potassium (86Rb+) flux components

Blood ◽

10.1182/blood.v67.3.710.bloodjournal673710 ◽

1986 ◽

Vol 67 (3) ◽

pp. 710-715

Author(s):

OE Ortiz ◽

VL Lew ◽

RM Bookchin

Keyword(s):

Sickle Cell ◽

Sickle Cell Anemia ◽

Membrane Surface ◽

Red Cells ◽

Cell Fraction ◽

K Channel ◽

Ionophore A23187 ◽

Na Pump ◽

Before And After ◽

86Rb Influx

We investigate here the hypothesis that the high Ca content of sickle cell anemia (SS) red cells may produce a sustained activation of the Ca2+-dependent K+ permeability (Gardos effect) and that the particularly high Ca levels in the dense SS cell fraction rich in irreversibly sickled cells (ISCs) might account for the Na pump inhibition observed in these cells. We measured active and passive 86Rb+ influx (as a marker for K+) in density-fractionated SS cells before and after extraction of their excess Ca by exposure to the Ca ionophore (A23187) and ethylene glycol tetra-acetic acid and with or without adenosine triphosphate depletion or addition of quinine. None of these maneuvers revealed any evidence of a Ca2+-dependent K leak in SS discocytes or dense cells. Na pump inhibition in the dense SS cells was associated with normal activation by external K+ and a low Vmax that persisted after Ca extraction from the cells. These results are consistent with our recent findings that the excess Ca in these cells is compartmentalized in intracellular inside-out vesicles and unavailable as free Ca2+ to the inner membrane surface. Although the steady-state free cytoplasmic Ca2+ in oxygenated SS cells must be below the levels needed to activate the K+ channel, possible brief activation of the channels of some SS cells resulting from transient elevations of cell Ca2+ during deoxygenation-induced sickling cannot be excluded. The dense, ISC-rich SS cell fraction showed a Ca2+-independent increase in the ouabain-resistant, nonsaturable component of 86Rb+ influx that, if uncompensated by Na+ gain, could contribute to the dehydration of these cells.

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The Hob Proteins: Putative, Novel Lipid Transfer Proteins at ER-PM Contact Sites

Contact ◽

10.1177/25152564211052376 ◽

2021 ◽

Vol 4 ◽

pp. 251525642110523

Author(s):

Sarah D. Neuman ◽

Amy T. Cavanagh ◽

Arash Bashirullah

Keyword(s):

Structural Models ◽

Model Systems ◽

Lipid Transfer ◽

Lipid Transfer Proteins ◽

Membrane Contact Sites ◽

Contact Sites ◽

Bona Fide ◽

Membrane Contact ◽

Mutant Cells ◽

Critical Process

Nonvesicular transfer of lipids at membrane contact sites (MCS) has recently emerged as a critical process for cellular function. Lipid transfer proteins (LTPs) mediate this unique transport mechanism, and although several LTPs are known, the cellular complement of these proteins continues to expand. Our recent work has revealed the highly conserved but poorly characterized Hobbit/Hob proteins as novel, putative LTPs at endoplasmic reticulum-plasma membrane (ER-PM) contact sites. Using both S. cerevisiae and D. melanogaster model systems, we demonstrated that the Hob proteins localize to ER-PM contact sites via an N-terminal ER membrane anchor and conserved C-terminal sequences. These conserved C-terminal sequences bind to phosphoinositides (PIPs), and the distribution of PIPs is disrupted in hobbit mutant cells. Recently released structural models of the Hob proteins exhibit remarkable similarity to other bona fide LTPs, like VPS13A and ATG2, that function at MCS. Hobbit is required for viability in Drosophila, suggesting that the Hob proteins are essential genes that may mediate lipid transfer at MCS.

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Molecular determinants of KA1 domain-mediated autoinhibition and phospholipid activation of MARK1 kinase

Biochemical Journal ◽

10.1042/bcj20160792 ◽

2017 ◽

Vol 474 (3) ◽

pp. 385-398 ◽

Cited By ~ 14

Author(s):

Ryan P. Emptage ◽

Mark A. Lemmon ◽

Kathryn M. Ferguson

Keyword(s):

Membrane Surface ◽

Kinase Domain ◽

Site Directed Mutagenesis ◽

Anionic Phospholipid ◽

Anionic Phospholipids ◽

Disease States ◽

C Terminus ◽

Regulatory Module ◽

Correct Location ◽

Mechanistic Basis

Protein kinases are frequently regulated by intramolecular autoinhibitory interactions between protein modules that are reversed when these modules bind other ‘activating’ protein or membrane-bound targets. One group of kinases, the MAP/microtubule affinity-regulating kinases (MARKs) contain a poorly understood regulatory module, the KA1 (kinase associated-1) domain, at their C-terminus. KA1 domains from MARK1 and several related kinases from yeast to humans have been shown to bind membranes containing anionic phospholipids, and peptide ligands have also been reported. Deleting or mutating the C-terminal KA1 domain has been reported to activate the kinase in which it is found — also suggesting an intramolecular autoinhibitory role. Here, we show that the KA1 domain of human MARK1 interacts with, and inhibits, the MARK1 kinase domain. Using site-directed mutagenesis, we identify residues in the KA1 domain required for this autoinhibitory activity, and find that residues involved in autoinhibition and in anionic phospholipid binding are the same. We also demonstrate that a ‘mini’ MARK1 becomes activated upon association with vesicles containing anionic phospholipids, but only if the protein is targeted to these vesicles by a second signal. These studies provide a mechanistic basis for understanding how MARK1 and its relatives may require more than one signal at the membrane surface to control their activation at the correct location and time. MARK family kinases have been implicated in a plethora of disease states including Alzheimer's, cancer, and autism, so advancing our understanding of their regulatory mechanisms may ultimately have therapeutic value.

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Properties of Hydrogen-Bonded Liquids at Interfaces

Zeitschrift für Physikalische Chemie ◽

10.1515/zpch-2018-1110 ◽

2018 ◽

Vol 232 (7-8) ◽

pp. 937-972 ◽

Cited By ~ 8

Author(s):

Gerd Buntkowsky ◽

Michael Vogel ◽

Roland Winter

Keyword(s):

Collaborative Research ◽

Research Unit ◽

Model Systems ◽

Experimental Characterization ◽

German Research Foundation ◽

Introductory Article ◽

German Research ◽

Fundamental Understanding ◽

Original Works ◽

Hydrogen Bonded

AbstractEffects of interfaces on hydrogen-bonded liquids play major roles in nature and technology. Despite their importance, a fundamental understanding of these effects is still lacking. In large parts, this shortcoming is due to the high complexity of these systems, leading to an interference of various interactions and effects. Therefore, it is advisable to take gradual approaches, which start from well designed and defined model systems and systematically increase the level of intricacy towards more complex mimetics. Moreover, it is necessary to combine insights from a multitude of methods, in particular, to link novel preparation strategies and comprehensive experimental characterization with inventive computational and theoretical modeling. Such concerted approach was taken by a group of preparative, experimentally, and theoretically working scientists in the framework of Research Unit FOR 1583 funded by the Deutsche Forschungsgemeinschaft (German Research Foundation). This special issue summarizes the outcome of this collaborative research. In this introductory article, we give an overview of the covered topics and the main results of the whole consortium. The following contributions are review articles or original works of individual research projects.

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Infrared Spectra of Crystalline Acetic Acid, a Hydrogen-Bonded Polymer

Applied Spectroscopy ◽

10.1366/000370277774463959 ◽

1977 ◽

Vol 31 (2) ◽

pp. 110-115 ◽

Cited By ~ 21

Author(s):

P. F. Krause ◽

J. E. Katon ◽

J. M. Rogers ◽

D. B. Phillips

Keyword(s):

Hydrogen Bonding ◽

Acetic Acid ◽

Infrared Spectra ◽

Group Analysis ◽

Structural Models ◽

Factor Group ◽

Vibrational Modes ◽

Cryogenic Temperatures ◽

Polymeric Chains ◽

Hydrogen Bonded

The polarized infrared spectra of crystalline acetic acid and two of its deuterated derivatives, CH3COOD and CD3COOD, have been recorded from 400 to 4000 cm−1 at cryogenic temperatures. The spectroscopic results have been interpreted on the basis of a factor group analysis based on two structural models: a crystallographic cell composed of four interacting monomer units some of whose vibrational modes are highly perturbed by hydrogen bonding and a unit cell composed of two noninteracting acetic acid chains. The results are discussed in terms of possible interactions between the hydrogen-bonded acetic acid polymeric chains.

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Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle.

The Journal of Cell Biology ◽

10.1083/jcb.99.3.875 ◽

1984 ◽

Vol 99 (3) ◽

pp. 875-885 ◽

Cited By ~ 388

Author(s):

A Saito ◽

S Seiler ◽

A Chu ◽

S Fleischer

Keyword(s):

Electron Microscopy ◽

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Thin Section ◽

Membrane Surface ◽

Freeze Fracture ◽

Morphological Characteristics ◽

Negative Staining ◽

Section Electron ◽

Terminal Cisternae

We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca2+ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend approximately 12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca2+ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca2+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.

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