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1
Qiu, Min Ru Clinical School of Medicine St Vincent's Hospital UNSW. "Functional and molecular aspects of ion channels in macrophages". Awarded by:University of New South Wales. Clinical School of Medicine, St. Vincent's Hospital, 2003. http://handle.unsw.edu.au/1959.4/20442.
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Monocytes/macrophages play very important roles in innate and adaptive immunity. Ion channels are small molecules embedded in the cell membrane and they play fundamental roles in cell function. Both topics have been extensively studied in isolation, however the role of ion channels in macrophage function is far less understood. In this thesis, the functional and molecular aspects of two ion channels expressed in macrophages, Kor, a potassium channel, and CLIC1, a chloride channel were studied. The biological function of Kor and CLIC1 in activated human macrophages was examined using ion channel blockers. In addition, the role of CLIC1 in the cell cycling of CHO-K1 cells was also investigated. The in vitro studies showed that Kor and CLIC1 are involved in cytokine production by PMA-activated human macrophages and that CLIC1 is also involved in the cell cycling of CHO-K1 cells. Despite providing interesting data, the results of the in vitro studies were difficult to interpret due to the uncertain specificities of the Cl- channel blockers. Therefore, to understand the biological role of CLIC1 in vivo, a gene targeting experiment was performed to create a CLIC1 knock-out (KO) mouse. This involved cloning the mouse CLIC1 gene, making a targeting vector, producing targeted ES cells, and generating a CLIC1 knock-in (KI) mouse which carries a flag tag at the N-terminal and three loxP sites in the targeted locus. Crossing the CLIC1 KI mouse with the TNAP-Cre mouse, a strain over expressing Cre recombinase under a TNAP promoter, a CLIC1 KO mouse was generated. The initial phenotype analysis showed no major development or growth abnormality in the CLIC1 KO mouse. Instead, hyperplasia of megakaryocytes and possible erythroid cells in the spleen and bone marrow was observed suggesting some degree of abnormality in the haematopoeitic system. Furthermore, a comparison of wild type mice with the CLIC1 KO mouse showed that CLIC1 protein expresses at high levels in monocytes, lymphocytes, platelets, and tissue macrophages of normal animals tissues, such as spleen, kidney (mesangial cell), and liver (kupffer cells). This further indicates that CLIC1 may play a significant role in regulating functions of platelets, lymphocytes, and specially tissue macrophages. More extensive studies can now be performed on the CLIC1 KO mouse to clarify the biological function of CLIC1. In summary, the generation of the CLIC1 KO mouse provides a valuable model to study the biological function of CLIC1 both in vivo and in vitro.
2
Corry, Ben Alexander. "Simulation studies of biological ion channels". View thesis entry in Australian Digital Theses Program, 2002. http://thesis.anu.edu.au/public/adt-ANU20030423.162927/index.html.
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Amiri, Shiva. "Computational modelling and molecular dynamics simulations of ligand-gated ion channels". Thesis, University of Oxford, 2006. http://ora.ox.ac.uk/objects/uuid:119c7ccb-e7b2-4da1-a137-40c3289c3ad8.
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Torpedo AChR structure was used to make models of other LGICs. Coarse-grain MD allowed the identification of residues in the TM domain interacting with the lipid-bilayer. Born energy profiles through LGIC pores reveal that the EC domain plays a key role in ion selectivity.
4
Hedley, Paula Louise. "Molecular and functional characterisation of Long QT Syndrome causing genes". Thesis, Stellenbosch : Stellenbosch University, 2014. http://hdl.handle.net/10019.1/86480.
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Thesis (PhD)-- Stellenbosch University, 2014.ENGLISH ABSTRACT: Ventricular arrhythmias are the most important cause of sudden cardiac death (SCD) among adults living in industrialised nations. Genetic factors have substantial effects in determining population-based risk for SCD and may also account for inter-individual variability in susceptibility. Great progress has been made in identifying genes underlying various Mendelian disorders associated with inherited arrhythmia susceptibility. The most well studied familial arrhythmia syndrome is the congenital long QT syndrome (LQTS) caused by mutations in genes encoding subunits of myocardial ion channels. Not all mutation carriers have equal risk for experiencing the clinical manifestations of disease (i.e. syncope, sudden death). This observation has raised the possibility that additional genetic factors may modify the risk of LQTS manifestations. This study establishes the genetic aetiology of LQTS in South Africa and Denmark through the identification and characterisation of LQTS-causative mutations in five previously identified genes, as well as examining possible novel genetic causes of LQTS in a cohort comprising Danish and British probands. We have functionally characterised several of the mutations identified in this study and examined other cardiac phenotypes that may be explained by variants causing repolarisation disorders.
AFRIKAANSE OPSOMMING: Ventrikulêre aritmie bly die enkele belangrikste oorsaak van skielike hart dood (SCD) onder volwassenes wat in geïndustrialiseerde lande woon. Genetiese faktore het aansienlike gevolge in die bepaling van bevolking-gebaseerde risiko vir SCD en kan ook verantwoordelik wees vir die inter-individuele variasie in vatbaarheid. Groot vordering is gemaak in die identifisering van gene onderliggende verskeie Mendeliese siektes wat verband hou met geërf aritmie vatbaarheid. Die mees goed bestudeerde familie aritmie sindroom is die aangebore lang QT-sindroom (LQTS) wat veroorsaak word deur mutasies in gene kode subeenhede van miokardiale ioonkanale. Nie alle mutasie draers het 'n gelyke risiko vir die ervaring van die kliniese manifestasies van die siekte (dws sinkopee, skielike dood). Hierdie waarneming het die moontlikheid genoem dat genetiese faktore anders as die primêre siekte-verwante mutasie kan die risiko van LQTS manifestasies verander. Hierdie studie stel die genetiese oorsake van LQTS in Suid-Afrika en Denemarke deur die identifisering en karakterisering van LQTS-veroorsakende mutasies in vyf voorheen geïdentifiseer gene, asook die behandeling van moontlike nuwe genetiese oorsake van LQTS in 'n groep wat bestaan uit van die Deense en die Britse probands. Ons het funksioneel gekenmerk verskeie van die mutasies wat in hierdie studie ondersoek en ander kardiovaskulêre fenotipes wat deur variante veroorsaak repolarisasie versteurings verduidelik word.
South African National Research Foundation
Harry and Doris Crossley Foundation
Danish Strategic Research Foundation.
5
Breed, Jason. "Molecular modelling of ion channels". Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308690.
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Zhou, Xin. "Towards voltage-gated ion channels, molecular diodes". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0012/NQ32730.pdf.
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Bjelkmar, Pär. "Modeling of voltage-gated ion channels". Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-63437.
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The recent determination of several crystal structures of voltage-gated ion channels has catalyzed computational efforts of studying these remarkable molecular machines that are able to conduct ions across biological membranes at extremely high rates without compromising the ion selectivity. Starting from the open crystal structures, we have studied the gating mechanism of these channels by molecular modeling techniques. Firstly, by applying a membrane potential, initial stages of the closing of the channel were captured, manifested in a secondary-structure change in the voltage-sensor. In a follow-up study, we found that the energetic cost of translocating this 310-helix conformation was significantly lower than in the original conformation. Thirdly, collaborators of ours identified new molecular constraints for different states along the gating pathway. We used those to build new protein models that were evaluated by simulations. All these results point to a gating mechanism where the S4 helix undergoes a secondary structure transformation during gating. These simulations also provide information about how the protein interacts with the surrounding membrane. In particular, we found that lipid molecules close to the protein diffuse together with it, forming a large dynamic lipid-protein cluster. This has important consequences for the understanding of protein-membrane interactions and for the theories of lateral diffusion of membrane proteins. Further, simulations of the simple ion channel antiamoebin were performed where different molecular models of the channel were evaluated by calculating ion conduction rates, which were compared to experimentally measured values. One of the models had a conductance consistent with the experimental data and was proposed to represent the biological active state of the channel. Finally, the underlying methods for simulating molecular systems were probed by implementing the CHARMM force field into the GROMACS simulation package. The implementation was verified and specific GROMACS-features were combined with CHARMM and evaluated on long timescales. The CHARMM interaction potential was found to sample relevant protein conformations indifferently of the model of solvent used.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.
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Adcock, Charlotte. "Molecular modelling and electrostatic properties of ion channels". Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297941.
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Bahnasi, Yahya Mohamed. "Molecular physiology and pharmacolgy of TRPC5 ion channels". Thesis, University of Leeds, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.496554.
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Elliott, David James Stuart. "Molecular mechanisms of voltage sensing by ion channels". Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406206.
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Verdin, Paul Stephen. "Molecular interactions of pyrethroid insecticides with insect ion channels". Thesis, University of Nottingham, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.495529.
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Much of the present knowledge of the activity of pyrethroids on the insect Na⁺ channel has come from the electrophysiological investigation of Na⁺ channels expressed heterologously in the Xenopus laevis oocyte expression system. The effects of the naturally occurring pyrethroid resistance mutations, located within the Na⁺ channel gene(s), have also been studied in this way. These studies have yielded a wealth of information, but make several assumptions - that insect Na⁺ channels behave normally in this alien environment and that the expressed channels accurately represent the range channels present in neurons.
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Dayl, Sudad Amer. "Molecular modelling of ATP-gated P2X receptor ion channels". Thesis, University of Leicester, 2018. http://hdl.handle.net/2381/42761.
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P2X receptors (P2XRs) are trimeric cation channels activated by extracellular ATP. Human P2XRs (P2X1-7) are expressed in nearly all mammalian tissues, and they are an important drug target because of their involvement in inflammation and neuropathic pain. The aim of this thesis is to address the following questions. P2XR crystal structures have revealed an unusual U-shape conformation for bound ATP; how does the U-shape conformation of ATP and its derivatives affect channel activation? Where and how do the selective, non-competitive inhibitors AZ10606120 and A438079 bind to P2X7R? What is the structure of the hP2X1R intracellular domain in the closed state? Molecular modelling and bioinformatics were used to answer these questions, hypotheses resulting from this work were tested in collaboration with Prof. Evans. Investigating the binding modes of ATP and its deoxy forms in hP2X1R showed that the ribose 2′-hydroxyl group is stabilising the U-shape conformation by a hydrogen bond to the γ-phosphate. The reduced ability of 2′-deoxy ATP to adopt the U-shape conformation could explain its weak agonist action in contrast to full agonists ATP and 3′-deoxy ATP. Ligand docking of AZ10606120 and A438079 into the hP2X7R predicted an allosteric binding site, this site has meanwhile been confirmed by P2X7R/antagonist X-ray structures. MD simulations suggested that unique P2X7R regions (residues 73-79 and T90/T94) contribute to an increase of the allosteric pocket volume compared to the hP2X1R. This difference in size might be the key for selectivity. The hP2X1R intracellular domain in the closed state was modelled ab initio, and interpreted in context of chemical cross-links (collaboration with Prof. Evans). This suggests a symmetrical arrangement of two short b-antiparallel strands within the Nterminal region and short a-helix in the C-terminal region and additional asymmetrical states.
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Ta, Chau My. "Molecular pharmacology of native and exogenous vascular ion channels". Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:798b4303-12d5-40b2-a850-0a68a03865a1.
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Vascular Ca2+-activated chloride channels (CaCCs), are a class of ligand- and voltage-gated channels that couple agonist binding and inositol triphosphate (IP3) signalling to depolarisation of the cell membrane and smooth muscle contraction. The elucidation of the precise physiological role and the pharmacological profile of this channel has been accelerated by the recent discovery of the gene (TMEM16A) coding for these channels. Based on the rationale that depolarising membrane potential (Vm) causes contraction in vascular SMCs, the possibility of controlling vessel tone via the exogenous light-activated, depolarising Channelrhodopsin 2 (ChR2) channels was also investigated. The overall aim of this thesis is two folds: i) to define mechanisms of regulation of CaCCs by endogenous signalling molecules and pharmacological agents, and ii) to develop a new method of controlling vascular contraction by exogenous ion channels. The main findings of this thesis are: 1) Arteries (large conduit, systemic and pulmonary) obtained from mice lacking of one allele of TMEM16A gene (heterozygous knockouts) presented reduced response to α1-adrenergic receptor activation. This finding reinforced the view that TMEM16A proteins serve as key mechanism of modulation of artery tone. 2) Phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid involved in the IP3 signalling cascade, is a key activator of native and cloned vascular TMEM16A channel. Furthermore, the closely related TMEM16B was inhibited by PIP2. These modulatory effect of PIP2 on these channels were especially pronounced in the physiological range of [Ca2+]i. 3) Anthracene-9-carboxylic acid (A9C), a general Cl- blocker, exhibited bimodal effects on TMEM16A-mediated currents. The inhibiting effect occurred via open-channel block mechanism while the activating effect was due to an increase in Po and a leftward shift in the steady-state activation curve. These mechanistic insights may help the design of novel drug (activators and inhibitors) that could be used to modulate blood vessel tone. 4) ChR2, a light-gated and non-selective cation channel, was introduced specifically into the SMC in mice (ChR2-SMC). Isolated arterial SMCs obtained from these mice showed light-dependent inward currents. ChR2-SMC artery rings also contracted in a light-dependent manner. The mechanism of lightinduced contraction involved voltage-gated Ca2+ channels (VGC) activation and Ca2+ influx. To conclude, this work of thesis has shed light on the functional role of TMEM16A in various vascular SMCs types. The regulation of TMEM16A by membrane lipid will lay the foundation for the identification of the PIP2 binding site on these channels, which could also be exploited as a site for pharmacological intervention. The study of A9C mechanisms of activation and inhibition may aid the development of selective blockers and activators for TMEM16A channels. Lastly, the control of vascular tone by ChR2 in mice can be used to control blood perfusion to organs and tissues for experimental purposes.
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Mustafa, Morad. "Ion Permeation through Membrane Channels: Molecular Dynamics Simulations Studies". Diss., CLICK HERE for online access, 2008. http://contentdm.lib.byu.edu/ETD/image/etd2477.pdf.
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Nilsson, Johanna. "Molecular mechanisms of local anaesthetic action on voltage-gated ion channels /". Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-748-7/.
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Broomand, Amir. "Molecular aspects on voltage-sensor movement". Doctoral thesis, Linköping : Univ, 2007. http://www.bibl.liu.se/liupubl/disp/disp2007/med1028s.pdf.
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Gwan, Jean-Fang. "The molecular mechanism of multi-ion conduction in K+ channels". [S.l.] : [s.n.], 2007. http://deposit.ddb.de/cgi-bin/dokserv?idn=983151253.
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Wei, Xiaomei. "Investigating Meningeal Ion Channels As New Molecular Targets For Migraine". Diss., The University of Arizona, 2014. http://hdl.handle.net/10150/565832.
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This dissertation will present the four manuscripts I published or am ready to publish on the study of the pathophysiology of migraine headache. The first chapter will discuss the background of the current understanding of migraine pathophysiology. Chapter 2 is focused on studying how Transient receptor potential vanilloid 4 (TRPV4) might play a role in migraine headache. Chapter 3 is the study of a novel cell type: dural fibroblasts might also play an active role in migraine headache. Chapter 4 is discussing Norepinephrine's role in headache pathophysiology. Chapter 5 is studying the combined effect of Acid and ATP in the pathophysiology of migraine headache. The dissertation will end in a conclusion in Chapter 6.
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Liang, Jieming, e 梁捷明. "Physiology of acupuncture: a study of mechanosensitive ion channels". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45451709.
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Mullapudi, Laxmi. "A PARALLEL MOLECULAR DYNAMICS PROGRAM FOR SIMULATION OF WATER IN ION CHANNELS". VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/1789.
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With a modest beginning from developing a model of dynamics of hard liquid spheres (Alder et al., 1957), molecular dynamics (MD) simulations have come to a point where complex biomolecules can be simulated with precision close to reality (Noskov et al., 2007). In this context, a parallel molecular dynamics program for simulation of ion channels associated with cellular membranes has been developed. The parallel MD code developed is simple, efficient, and easily coupled to other codes such as the hybrid molecular dynamics/ brownian dynamics (MD/BD) code developed for the study of protein interactions (Ying et al., 2005). The Atom Decomposition (AD) Method was used in partitioning calculations on atoms to processors. One of the major impediments in using AD was the relatively large size of data that had to be communicated by the processes (Plimpton et al., 1995). Replicating only positions of atoms eased the congestion created by communication of both force terms and positions of atoms between processes. The performance of the code was tested on KcsA, a bacterial potassium channel. The program was written in Fortran 90 with parallel functions from the library of mpich-1.2.7. The idle time of processes was optimized by message driven ordering of communication. The scaling of the parallel program with 2000 – 60,000 atoms was determined and compared with the results obtained from the serial program. As expected, the parallel program scaled better than the serial program as the number of atoms included in the simulation increased from 2000 - 60000. The performance of the parallel program was tested on 4-15 processes, for a system comprising 20,000 atoms. The results obtained were compared with results from the serial program. It was observed that the parallel program scaled better than the serial program as the number of processes increased from 4 to 15. When compared with serial program, which had application of Newton’s Third Law in calculating force terms once per each pair of atoms, it was observed that the parallel program scaled better on 6-15 processes for a physical system comprising of 20,000 atoms.
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Forster, Christine. "Aspects of charge exchange in ion-atom collisions". Thesis, Durham University, 1990. http://etheses.dur.ac.uk/6189/.
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The straight line semi-classical Impact Parameter method has been modified for use with classical trajectories. Ion-atom collisions have been modelled using wavefunctions expanded in terms of atomic basis states which were centred on either the target or projectile ions. Total and differential charge exchange cross-sections are presented for (^4)He(^++) and (^4)He(^+) collisions within the centre of mass energy range 0.21 kev < E(_em) < 2.5 keV. Results using curved and straight line paths are compared with data from other authors. Significant trajectory effects were found at the lower energies in the range. The curved trajectory results were lower than those from the straight line model and also lower than previous calculations carried out. At higher energies in the range there was good agreement between straight line and curved trajectory models and previous work. Differential cross-sections were found to be sensitive to the trajectories employed, and comparisons have been made with previous work. Total, state specific and differential cross- sections for charge exchange are presented for Be(^++) and H collisions using a five state basis, within the centre of mass energy range 0.111 keV < E(_em) < 0.4444 keV. There was reasonable agreement between the straight line results and previous work. There were significant trajectory effects for all the final charge transfer states. Results are presented for low-energy collisions between positively charged muons and atomic hydrogen. An eight state basis has been used. Direct excitation cross sections for n = 2 atomic states and charge transfer cross sections to Is and n = 2 have been calculated. The effect on the cross sections of using different internuclear potentials has been examined. Trajectory effects were small for charge transfer to Is but were more pronounced in the direct excitation and charge exchange cross- sections to n = 2. These results have been compared to those obtained for curved trajectory H(^+) and H collisions at the same relative velocity, to assess the validity of velocity scaling. It was found that velocity scaling was reliable for charge transfer to Is and for total electron capture cross-sections. However, it was progressively inaccurate for direct excitation and for electron capture into excited states for µ(^+) impact energies of less than 300 eV. These results are discussed and suggestions for further work are made.
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Lange, Michael. "Competition between reaction channels in electron collisions of the hydrogen molecular ion HD+". [S.l. : s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=962794775.
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Araújo, Rúbia Aparecida de. "Molecular actions of pyrethroids on ion channels in the maize weevil, Sitophilus zeamais". Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/11604/.
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Previous studies on the mechanism of action of pyrethroids have confirmed that voltage-gated sodium channels (VGSC) in the axon membrane are the major target site of these compounds. The use of pyrethroids to control maize weevils, Sitophilus zeamais, a major pest of stored maize in Brazil, has led to the occurrence of resistance. The work described here seeks to establish whether changes in VGSC of S.zeamais can explain pyrethroid resistance. The S. zeamais homologue of the Drosophila para VGSC was identified using degenerate primers and sequenced. Resistance mutations were examined by sequencing the IIS4-IIS6 region of the gene from laboratory strains of susceptible and resistant insects, revealing one amino acid replacement (T929I). The T929I mutation has been identified in other insects but always associated with a second mutation together producing a highly resistant phenotype. The occurrence of T929I in isolation is rare. DNA-based diagnostic assays were designed to screen weevils for the T929I mutation and analyse Brazilian field populations revealing a low frequency of heterozygous individuals carrying the mutation. The effect of the T929I mutation on VGSC function was investigated using whole cell patch clamping on cultured neurons isolated from thoracic ganglia of wild-type and resistant weevils. Inward currents were recorded by depolarizing the neuron to test potentials in the range -70mV to +70mV in 10mV increments for 25ms from a holding potential of -80mV. Current amplitudes were similar in cells from resistant weevils however other changes were apparent, notably a significant depolarizing shift in the voltage-dependence of activation of sodium currents in the resistant animals (P<0.05). Mutant neurons are also less sensitive to deltamethrin than the wild types.
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Cifelli, Carlo. "Impairment of force development in K(ATP) channel deficient skeletal muscle involves calcium ion influx through L-type calcium ion channels". Thesis, University of Ottawa (Canada), 2006. http://hdl.handle.net/10393/27342.
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ATP-sensitive potassium (KATP) channels link membrane excitability to metabolism. They are regulated by intracellular nucleotides and other factors, and have been shown to play a role in development of skeletal muscle force, but controversy surrounds their role during fatigue. The aim of this research project was to determine the role of KATP channel under conditions that allow for better assessment of changes in force during fatigue, by virtue of using a smaller whole muscle model less subject to anoxia. Thus, the first objective was to determine the effect of the loss of KATP channel activity on force during fatigue in small FDB muscle bundles. KATP channel deficient fibers had faster and greater decreases in peak tetanic force during fatigue, developed greater resting tension, and had lower force recovery following fatigue compared to control wild type muscles. The second objective was to determine whether the functional impairment in skeletal muscle without KATP channel activity was due to an increase in Ca 2+ influx. When [Ca2+]e was reduced or L-type Ca2+ channels partially blocked, Kir6.2-/- FDB muscle had slower fatigue development, less resting tension, and had an improved force recovery.
A novel phenomenon was observed while studying the effect of KATP channel activity in vitro. During a second bout of fatigue the decrease in peak tension was significantly lower than the decrease during the first bout of fatigue. Furthermore, the deleterious effects of the loss of KATP channel activity during an initial fatigue were absent during the second fatigue in FDB exposed to glibenclamide.
It is concluded (i) that the KATP channel is important to prevent impairment of function during fatigue, (ii) that this impairment of function is due to an increase in Ca2+ influx through L-type Ca2+ channels, causing Ca2+ overload, and (iii) that fatigue resistance increases while the dependency on the KATP channel to prevent function impairment and fiber damage decreases following one fatigue bout at 37°C; a phenomenon here termed fatigue pre-conditioning.
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Schmidt, Matthias Rene. "K+ channels : gating mechanisms and lipid interactions". Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:51dc4149-d943-4dcd-bf5b-f04130456d84.
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Computational methods, including homology modelling, in-silico dockings, and molecular dynamics simulations have been used to study the functional dynamics and interactions of K+ channels. Molecular models were built of the inwardly rectifying K+ channel Kir2.2, the bacterial homolog K+ channel KirBac3.1, and the twin pore (K2P) K+ channels TREK-1 and TRESK. To investigate the electrostatic energy profile of K+ permeating through these homology models, continuum electrostatic calculations were performed. The primary mechanism of KirBac3.1 gating is believed to involve an opening at the helix bundle crossing (HBC). However, simulations of Kir channels have not yet revealed opening at the HBC. Here, in simulations of the new KirBac3.1-S129R X-ray crystal structure, in which the HBC was trapped open by the S129R mutation in the inner pore-lining helix (TM2), the HBC was found to exhibit considerable mobility. In a simulation of the new KirBac3.1-S129R-S205L double mutant structure, if the S129R and the S205L mutations were converted back to the wild-type serine, the HBC would close faster than in the simulations of the KirBac3.1-S129R single mutant structure. The double mutant structure KirBac3.1-S129R-S205L therefore likely represents a higher-energy state than the single mutant KirBac3.1-S129R structure, and these simulations indicate a staged pathway of gating in KirBac channels. Molecular modelling and MD simulations of the Kir2.2 channel structure demonstrated that the HBC would tend to open if the C-linker between the transmembrane and cytoplasmic domain was modelled helical. The electrostatic energy barrier for K+ permeation at the helix bundle crossing was found to be sensitive to subtle structural changes in the C-linker. Charge neutralization or charge reversal of the PIP2-binding residue R186 on the C-linker decreased the electrostatic barrier for K+ permeation through the HBC, suggesting an electrostatic contribution to the PIP2-dependent gating mechanism. Multi-scale simulations determined the PIP2 binding site in Kir2.2, in good agreement with crystallographic predictions. A TREK-1 homology model was built, based on the TRAAK structure. Two PIP2 binding sites were found in this TREK-1 model, at the C-terminal end, in line with existing functional data, and between transmembrane helices TM2 and TM3. The TM2-TM3 site is in reasonably good agreement with electron density attributed to an acyl tail in a recently deposited TREK-2 structure.
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Naylor, S. "Applications and mechanistic aspects of fast atom bombardment mass spectroscopy". Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.234018.
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Rosenberg, Madelaine. "Factors that influence the expression of neurotransmitter-gated ion channels on developing peripheral neurons". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0019/NQ44563.pdf.
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Livesey, Matthew Robert. "Molecular determinants of single channel conductance and ion selectivity in cationic Cys-loop receptor channels". Thesis, University of Dundee, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510623.
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Sunderman, Elizabeth R. "Single-channel kinetic analysis of the allosteric transition of rod cyclic nucleotide-gated channels /". Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/10526.
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Richards, Ryan. "Molecular and structural determinants that contribute to channel function and gating in channelrhodopsin-2". Digital WPI, 2016. https://digitalcommons.wpi.edu/etd-dissertations/481.
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The green algae Chlamydomonas reinhardtii senses light through two photosensory proteins, channelrhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2). The initial discovery of these two photoreceptors introduced a new class of light-gated ion channels. ChR2 is an inwardly-rectified ion channel that is selective for cations of multiple valencies. Similar to microbial-rhodopsin ion pumps, ChR2 has a seven transmembrane domain motif that binds the chromophore all-trans retinal through a protonated Schiff base linkage. Physiologically, ChR2 functions to depolarize the membrane which initiates a signaling cascade triggering phototactic response. This fundamental property has been pivotal in pioneering the field of optogenetics, where excitable cells can be manipulated by light. ChR2 reliably causes neuronal spiking with high spatial and temporal control. Moreover, the recent discovery of new chloride-conducting channelrhodopsins (ChloCs) has further expanded the optogenetic toolbox. Although structurally similar to microbial-rhodopsin ion pumps, ChR2 undergoes more complex conformational rearrangements that lead to ion conductance. Currently, the molecular basis for ChR2 gating remains unresolved. Revealing the specific structural interactions that modulate ChR2 function have important implications in understanding the intricacies of ion transport and molecular differences between ion pumps, channels, and transporters. Here we describe a combined computational and experimental approach to elucidate the mechanism of ion conductance, channel gating, and structure-function relationship of ChR2. Our results have contributed to expanding our understanding of the fundamental properties of ion channels.
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Englund, Ulrika. "The role of ion channels and intracellular metal ions in apoptosis of Xenopus oocytes". Doctoral thesis, Linköpings universitet, Avdelningen för cellbiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-111045.
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Apoptosis is one type of programmed cell death, important during tissue development and to maintain the tissue homeostasis. Apoptosis comprises a complex network of internal signaling pathways, and an important part of this signaling network is the action of voltage‐gated ion channels. The aim of this thesis was to explore the role of ion channels and the role of intracellular metal ions during apoptosis in Xenopus laevis oocytes. The reasons for using these oocytes are that they are large, robust, easy to handle, and easy to study electrophysiologically. Apoptosis was induced either chemically by incubation of the oocytes in staurosporine (STS) or mechanically by centrifugation of the oocytes. Ion currents were measured by a two‐electrode voltage clamp technique, intracellular ion concentrations were measured either directly by in‐house developed K+‐selective microelectrodes or indirectly by the electrophysiological technique, and apoptosis was measured by caspase‐3 activation. Paper I describes that the intracellular K+ concentration was reduced by about 30 % during STS‐induced apoptosis. However, this reduction was prevented by excessive expression of exogenous ion channels. Despite the magnitude of the intracellular K+ concentration, either normal or reduced level, the oocytes displayed normal signs of apoptosis, suggesting that the intracellular K+ reduction was not required for the apoptotic process. Because the intracellular K+ concentration was not critical for apoptosis we searched for other ion fluxes by exploring the electrophysiological properties of X. laevis oocytes. Paper II, describes a non‐inactivating Na+ current activated at positive membrane voltages that was upregulated by a factor of five during STS‐induced apoptosis. By preventing influx of Na+, the apoptotic signaling network involving capsase‐3 was prevented. To molecularly identify this voltage‐gated Na channel, the X. tropicalis genome and conserved regions of the human SCNA genes were used as a map. Paper III, shows that the voltage‐gated Na channel corresponds to the SCN2A gene ortholog and that supression of this SCN2A ortholog using miRNA prevented cell death. In conclusion, this thesis work demonstrated that a voltage‐gated Na channel is critical for the apoptotic process in X. laevis oocytes by increasing the intracellular Na+ concentration.
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Bush, Elizabeth Rosina. "Molecular biology and expression of some ligand-gated ion channels from the nematodes Ascaris suum and Caenorhabditis elegans". Thesis, University of Southampton, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246225.
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Kirk, Kirsty-Anne. "The expression of potential molecular candidates for chloride ion channels in primary human granulocytes and granulocytic cell lines". Thesis, University of East Anglia, 2014. https://ueaeprints.uea.ac.uk/50544/.
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Introduction and hypothesis: The project aims to identify potential candidates for chloride (Cl-) ion channels in granulocytes, and granulocytic cell lines. It is hypothesised that Cl- ion channels, in particular hBest1, are implicated in the role of granulocytes in response to inflammation. Methodology: Two main methodologies were used; laboratory techniques and systematic review. Laboratory techniques included RT-PCR, flow cytometry and western blot analysis to characterise the expression of hANO1, hBest1 and hCLCA1 as potential chloride ion channels in granulocytes and granulocytic cell lines. Systematic review was performed to identify whether chloride ion channels are up-regulated in COPD and asthma. Results: RT-PCR demonstrated hCLCA1 expression in granulocytes and eosinophils but not HL60. hBest1 and hBest3 was expressed in all 3 cell types. In granulocytes, flow cytometry demonstrated greater hCLCA1 protein expression intracellularly, compared to hBest1 protein, and greater hBest1 plasma membrane expression compared to hCLCA1 (P<0.05). There was a negative correlation between hBest1, and hCLCA1 but also a weak negative correlation between hBest1 and hANO1 (P<0.05). Granulocytes stimulated with IL-13 over 24 hours, had a greater protein expression both intracellularly and at the plasma membrane. There was increased migration of HL60s when transfected with hBest1, in response to fMLP (P<0.05). Systematic review did not support the project due to limitations. Conclusions: There is a complex relationship between hBest1, hCLCA1 and hANO1 which may contribute to the function of granulocytes. HBest1 protein expression peaked 24 hours after continuous stimulation with IL-13. This correlates with peak symptom expression in diseases such as COPD and asthma. It is suggested that hBest1 has a role in migration and activation of granulocytes, through regulation of cell shape and volume. It is concluded that hBest1 is a novel therapeutic target in the control of symptoms in chronic inflammatory lung diseases.
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Jiang, Ruotian. "Molecular modus operandi of ligand-gated ion channels : Studies of trimeric P2X receptors and pentameric GABA A receptors". Strasbourg, 2011. http://www.theses.fr/2011STRA6086.
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Le principal objectif de cette thèse a été d’utiliser divers outils chimiques et biologiques afin de mieux décortiquer le mécanisme d’action au niveau moléculaire de deux membres des récepteurs appartenant à deux superfamilles différentes des récepteur canaux activés par les ligands: les récepteurs P2XRs et GABAARs. Le récepteur P2XR est un canal ionique sélectif aux cations activé par fixation de l'ATP extracellulaire. Mon objectif a été d’étudier les mécanismes moléculaires pour la liaison de l’ATP et l'ouverture rapide d’un canal ionique suite à la fixation de l’ATP. En utilisant l’approche d’ingénierie de marquage d’affinité combinés à patch-clamp eletrophysiologie, nous avons défini un site interfacial très large et dynamique. Dans une autre étude, nous avons identifié un pont salin dans une région inexplorée du récepteur impliqué dans la régulation du mouvement d’ouverture du canal. Les récepteurs GABAARs sont impliqués dans la transmission synaptique inhibitrice au niveau du système nerveux central. En utilisant l’électrophysiologie patch-clamp, nous avons décrit sur ces récepteurs la modulation allostérique d’une série de composés synthétiques qui sont les trans-retrochalcones et qui appartiennent à la famille des flavonoïdes. Nous avons mis en évidence que le site d'action de ces nouveaux composés est distinct non seulement du site de liaison des benzodiazépines classiques, mais aussi d'autres sites de modulation connus. Nos données révèlent un mode d'action original et fournissent une base rationnelle pour la découverte de nouveaux médicaments afin de traiter les désordres physiologiques liés aux dysfonctionnements des récepteurs GABAAThis thesis, by using various chemical and biological tools, focuses on the molecular modus operandi of two different superfamilies of ligand-gated ion channels: P2XRs and GABAARs. P2XR is a cation-selective ion channel gated by extracellular ATP (and is implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. Here I studied the molecular mechanism underlying ATP binding and channel opening of the P2X receptors. In the ATP-binding site study, we definitely localized the ATP-binding sites in P2X2 receptor through affnity labeling. Our results thus define a large and dynamic inter-subunit ATP-binding pocket. In the “gating”†part, an inter-subunit salt bridge located at the “body” domain that regulates channel gating movement was identified by using charge reversal and charge swapping combined with double mutant cycle analysisPentameric GABAARs form chloride permeable ion channels and mediate inhibitory synaptic transmission in the central nervous system. The modulation of their action is critical for brain normal function and for various pathophysiological conditions. In the GABAARs part, using patch-clamp electrophysiology, we described the allosteric modulation of GABAARs by a series of synthetic compounds that are trans-retrochalcones belonging to the flavonoids family. We characterized their subunit-dependent positive modulations at both synaptic and extrasynaptic GABAARs. Our data reveal an original mode of action and provide a rational basis for hypothesis-driven drug discovery efforts with emphasis on the retrochalcone scaffold for treating GABAA-related central nervous system disorders
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Martin, Nicolas. "Allosteric modulation of pentameric ligand gated ion channels : from the jiggling of atoms to neuropharmacological strategies". Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAF079/document.
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Les récepteurs pentamériques canaux (pLGICs) sont des récepteurs neuronaux impliqués dans la neurotransmission rapide et qui comprennent les récepteurs suivants : nAchR, GABAR, GlyR or 5HT3R. Lorsqu’ils ne fonctionnent pas correctement ils sont impliqués dans des pathologies comme Alzheimer ou Parkinson. Dans cette étude, nous avons réalisé des simulations de dynamique moléculaire d’un homologue procaryote des pLGICs. Grâce à l’analyse de 2.5 us de simulation nous avons pu capturer la fermeture complète dudit récepteur et décrire un mécanisme de gating. Ce mécanisme en deux étapes, 1) twisting puis 2) blooming semble compatible avec tous les pLGICs. Dans un second temps, nous avons utilisé notre connaissance du mécanisme de gating afin de faire des calculs d’énergie libre le long du twisting, pour différents complexes protéine/ligands. De cette façon, nous avons pu discriminer entre des ligands actifs et inactifs et ainsi fournir des pistes pour le design de nouveaux traitementsPentameric ligand gated ion channels (pLGICs) are brain receptors involved in fast neurotransmission and include nAchR, GABAR, GlyR or 5HT3R. When dysfunctioning, they are involved in diseases such as Alzheimer’s and Parkinson’s. In this study we have performed molecular dynamic simulations of an eukaryotic homologue of the pLGICs (GluCl) to understand the gating mechanism of pLGICs. Thanks to the analysis of two 2.5 us long simulations in which we could capture the full closing of the receptor we described in great details a gating mechanism in two steps, first twisting then blooming, that we believe applicable to the whole pLGICs family. In a second time we used our description of the gating mechanism to perform free energy calculations along the twisting reaction coordinate, for various ligands in complex with GluCl. Doing so we could show a significant difference between IVM-bound and non-bound states and provide hints for the design of new treatments
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Crozier, Paul S. "Slab-geometry molecular dynamics simulations : development and application to calculation of activity coefficients, interfacial electrochemistry, and ion channel transport /". Diss., CLICK HERE for online access:, 2001. http://contentdm.lib.byu.edu/ETD/image/etd15.pdf.
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Österberg, Fredrik. "Exploring Ligand Binding in HIV-1 Protease and K+ Channels Using Computational Methods". Doctoral thesis, Uppsala universitet, Strukturell molekylärbiologi, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6167.
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Understanding protein-ligand interactions is highly important in drug development. In the present work the objective is to comprehend the link between structure and function using molecular modelling. Specifically, this thesis has been focused on implementation of receptor flexibility in molecular docking and studying structure-activity relationships of potassium ion channels and their blockers. In ligand docking simulations protein motion and heterogeneity of structural waters are approximated using an ensemble of protein structures. Four methods of combining multiple target structures within a single grid-based lookup table of interaction energies are tested. Two weighted average methods permit consistent and accurate ligand docking using a single grid representation of the target protein structures. Quaternary ammonium ions (QAIs) are well known K+ channel blockers. Conformations around C–N bonds at the quaternary centre in tetraalkylammonium ions in water solution are investigated using quantum mechanical methods. Relative solvation free energies of QAIs are further estimated from molecular dynamics simulations. The torsion barrier for a two-step interconversion between the conformations D2d and S4 is calculated to be 9.5 kcal mol–1. Furthermore D2d is found to be more stable than the S4 conformation which is in agreement with experimental studies. External QAI binding to the K+ channel KcsA is also studied. Computer simulations and relative binding free energies of the KcsA complexes with QAIs are calculated. This is done with the molecular dynamics free energy perturbation approach together with automated ligand docking. In agreement with experiment, the Et4N+ blocker in D2d symmetry has better binding than the other QAIs. Binding of blockers to the human cardiac hERG potassium channel is studied using a combination of homology modelling, automated docking and molecular dynamics simulations. The calculations reproduce the relative binding affinities of a set of drug derivatives very well and indicate that both polar interactions near the intracellular opening of the selectivity filter as well as hydrophobic complementarity in the region around F656 are important for blocker binding. Hence, the derived model of hERG should be useful for further interpretations of structure-activity relationships.
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Pirri, Jennifer K. "The Role of Ion Channels in Coordinating Neural Circuit Activity in Caenorhabditis elegans: A Dissertation". eScholarship@UMMS, 2013. http://escholarship.umassmed.edu/gsbs_diss/662.
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Despite the current understanding that sensorimotor circuits function through the action of transmitters and modulators, we have a limited understanding of how the nervous system directs the flow of information necessary to orchestrate complex behaviors. In this dissertation, I aimed to uncover how the nervous system coordinates these behaviors using the escape response of the soil nematode, Caenorhabditis elegans, as a paradigm. C. elegans exhibits a robust escape behavior in response to touch. The worm typically moves forward in a sinusoidal pattern, which is accompanied by exploratory head movements. During escape, the worm quickly retreats by moving backward from the point of stimulus while suppressing its head movements. It was previously shown that the biogenic amine tyramine played an important role in modulating the suppression of these head movmemetns in response to touch. We identified a novel tyramine-gated chloride channel, LGC-55, whose activation by tyramine coordinates motor programs essential for escape. Furthermore, we found that changing the electrical nature of a synapse within the neural circuit for escape behavior can reverse its behavioral output, indicating that the C. elegans connectome is established independent of the nature of synaptic activity or behavioral output. Finally, we characterized a unique mutant, zf35 , which is hyperactive in reversal behavior. This mutant was identified as a gain of function allele of the C. elegans P/Q/N-type voltage-gated calcium channel, UNC-2. Taken together, this work defines tyramine as a genuine neurotransmitter and completes the neural circuit that controls the initial phases of the C. elegans escape response. Additionally, this research further advances the understanding of how the interactions between transmitters and ion channels can precisely regulate neural circuit activity in the execution of a complex behavior.
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Kaprielian, Roger. "Molecular and cellular mechanisms associated with cardiac hypertrophy following myocardial infarction in rats, studies on ion channels and intracellular calcium". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0024/NQ49955.pdf.
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Newton, Hannah S. "Potassium channels and adenosine signaling in T cells of head and neck cancer patients". University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1603713656776019.
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Boukharta, Lars. "Computational Modelling of Ligand Complexes with G-Protein Coupled Receptors, Ion Channels and Enzymes". Doctoral thesis, Uppsala universitet, Beräknings- och systembiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-212103.
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Accurate predictions of binding free energies from computer simulations are an invaluable resource for understanding biochemical processes and drug action. The primary aim of the work described in the thesis was to predict and understand ligand binding to several proteins of major pharmaceutical importance using computational methods. We report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 G-protein coupled receptor and a series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. Site-directed mutagenesis, homology modelling and docking were further used to characterize agonist binding to the human neuropeptide Y2 receptor, which is important in feeding behavior and an obesity drug target. In a separate project, homology modelling was also used for rationalization of mutagenesis data for an integron integrase involved in antibiotic resistance. Blockade of the hERG potassium channel by various drug-like compounds, potentially causing serious cardiac side effects, is a major problem in drug development. We have used a homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations are in good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships with implications for design of new compounds. Docking, scoring, molecular dynamics, and the linear interaction energy method were also used to predict binding modes and affinities for a large set of inhibitors to HIV-1 reverse transcriptase. Good agreement with experiment was found and the work provides a validation of the methodology as a powerful tool in structure-based drug design. It is also easily scalable for higher throughput of compounds.
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Moomaw, Andrea Sue. "Structure-Function Studies of the CorA Magnesium Channel". Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1295621689.
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Idikuda, Vinaykumar. "REGULATION OF HCN CHANNEL FUNCTION BY DIRECT cAMP BINDING AND SINGLET OXYGEN". VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5455.
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Hyperpolarization-activated, cyclic-nucleotide gated ion channels (HCN channels) are activated by membrane hyperpolarization and modulated by cyclic nucleotides. HCN channels are important to maintain the resting membrane potential and input resistance in neurons and have important physiological functions in the brain and heart. Four mammalian HCN isoforms, HCN1-4, and the isoform cloned from sea urchin, spHCN, have been extensively studied. Among these, only spHCN channel shows a voltage dependent inactivation. Previous studies have shown that the ligand binding in mHCN2 channel is activity dependent: cAMP binding increases along with channel opening or channels in the open state have higher binding affinity for cAMP. But to date, information pertaining to the ligand binding to an inactivated ion channel or desensitized receptor is lacking. To address this gap, we used fluorescently labelled cAMP analogues in conjunction with patch clamp fluorometry (PCF) to study the ligand binding to the spHCN channel in various conformational states. We show that inactivated spHCN channel shows reduced binding affinity for cAMP, compared to that of the closed or open channel. Parallelly, we noticed significant changes to channel function when a combination of laser and photosensitizer was used to study ligand binding. A reactive oxygen species called singlet oxygen has been confirmed to be the major player in this process. Both photo-dynamically generated and chemically generated singlet oxygen modifies spHCN channel by removing the inactivation. The effect of singlet oxygen on channel can be abolished by the mutation of a key histidine (H462) residue in the ion conducting pore. Taken together, these two projects expanded our understanding about the physicochemical nature of fluorophores from two aspects: (i) the release of photon as a valuable tool to study the conformational dynamics in proteins; (ii) the generation of singlet oxygen as an effective modulator of protein function.
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Carstens, Johanna J. "Identification of the modulators of cardiac ion channel function". Thesis, Stellenbosch : University of Stellenbosch, 2009. http://hdl.handle.net/10019.1/2163.
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Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009.The human ether-à-go-go-related gene (HERG) encodes the protein underlying the cardiac potassium current IKr. Mutations in HERG may produce defective channels and cause Long QT Syndrome (LQTS), a cardiac disease affecting 1 in 2500 people. The disease is characterised by a prolonged QT interval on a surface electrocardiogram and has a symptomatic variability of sudden cardiac death in childhood to asymptomatic longevity. We hypothesised that genetic variation in the proteins that interact with HERG might modify the clinical expression of LQTS. Yeast two-hybrid methodology was used to screen a human cardiac cDNA library in order to identify putative HERG N-terminus ligands. Successive selection stages reduced the number of putative HERG ligandcontaining colonies (preys) from 268 to 8. Putative prey ligands were sequenced and identified by BLAST-search. False positive ligands were excluded based on their function and subcellular location. Three strong candidate ligands were identified: Rhoassociated coiled-coil containing kinase 1 (ROCK1), γ-sarcoglycan (SGCG) and microtubule-associated protein 1A (MAP1A). In vitro co-immunoprecipitation (Co-IP) and mammalian two-hybrid (M2H) analyses were used to validate these proposed interactions, but failed to do so. This should be further investigated. Analysis of confirmed interactions will shed light on their functional role and might contribute to understanding the symptomatic variability seen in LQTS.
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Hanson, Sonya M. "Structural, biochemical and computational studies of TRP channel transmembrane domain modularity". Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:328269a9-11c0-4d5b-9cb7-d7433cf4d6c4.
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Transient receptor potential (TRP) channels are expressed throughout the central nervous system and have a unique ability to detect a wide range of stimuli including changes in voltage, temperature, pH, lipid environment, small molecule agonists, and mechanical stress. While it is known that TRP channels contain the same six transmembrane helix (S1-S6), tetrameric architecture as voltage-gated channels, the degree to which functional and structural analogies are relevant remains poorly understood. This thesis describes a multidisciplinary approach toward understanding the structure and function of TRP channel transmembrane domains by focusing on the S1-S4 transmembrane helices of the TRPV1. This focus is inspired by the voltage-sensor domain (VSD) of the S1-S4 helices of voltage-gated channels, for which a range of studies show functional and structural independence. While some TRP channels are voltage-sensitive, their S4 helix does not contain the positive string of amino acids of canonical VSDs. However, the S1-S4 helices are functionally significant as the binding site of small molecule ligands in both TRPV1 and TRPM8 (for capsaicin and menthol, respectively). The question of TRP channel transmembrane domain modularity is addressed in this thesis by expression and purification trials as well as radioligand-binding assays. It is demonstrated that the S1-S4 and S1-S6 helices of TRPV1 can be properly inserted, overexpressed, and show signs of stability upon detergent-extraction from Saccharomyces cerevisiae membranes. However the TRPV1 S1-S4 and S1-S6 helices do not show wildtype (WT)-like binding in [3H]-RTX binding assays. These results indicate that the TRPV1 transmembrane domains are likely structural but not functional domains. The S. cerevisiae expression system remained promising for the overexpression of TRP transmembrane domains as well as the production of functional, though not stable upon detergent-extraction, WT TRPV1. This WT TRPV1 was subsequently found to functionally bind both RTX, used in ligand binding assays, as well as the double-knot toxin (DkTx), targeted to the pore domain (the S5-S6 helices). An effect of DkTx on RTX binding affinity demonstrates an allosteric interaction indicative of a possible tighter packing between the two transmembrane domains than is seen in voltage-gated channels containing the canonical VSD. Computational approaches additionally allowed for the investigation of the intramembrane capsaicin binding site in the TRPV1 S1-S4 helices, crucial to the initial motivations of this study. While the literature locates the capsaicin binding site to the TRPV1 S1-S4 helices, a `binding pocket' has yet to be defined, with regards to the orientation of bound capsaicin and its access route to the site via the bilayer. Using molecular dynamics (MD) simulations the preferred location of capsaicin within the bilayer is defined, as well as the elucidiation of capsaicin flip-flop between bilayer leaflets as a key event prior to TRPV1 binding. A transient binding was also observed between a homology model of the TRPV1 S1-S4 helices and capsaicin, possibly encouraging the idea that the S1-S4 helices still contain a partial binding site, though of too low affinity to be observed in the binding experiments performed here.
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Cosseddu, Salvatore M. "Structure and dynamics of protein in the permeation and gating of potassium ion channels : identifying molecular determinants and developing coarse-grained approaches". Thesis, University of Warwick, 2013. http://wrap.warwick.ac.uk/62117/.
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Ion channels are transmembrane proteins which allow small ions to flow across the membrane downhill along the electrochemical gradient, with high effciency and selectivity over the different ion species, and which play crucial roles in a wide range of vital physiological functions. Research on the channels selective for potassium ions have attracted a great deal of attention over the past decade. This is because of the availability of three dimensional microscopical structures and because they provide a paradigm for the study of the complex superposition of the permeation of the ions and structural rearrangements which is responsible for the regulation of the current in ion channels. In the present work a thorough study of the strong coupling between permeation and the dynamics of the protein in the potassium ion channel KcsA is presented, based on Molecular Dynamics and Metadynamics calculations, which reveals the clear links between the function and the structure of the protein. The molecular determinants for the conformational changes of the pore region have been identified and described in details. The relationship between these rearrangements and the gating process known as "C-type inactivation", found in a variety of potassium ion channels, have been investigated and a mechanism has been proposed for the process. The knowledge acquired from these investigations is finally applied to unveil the driving forces and energetics associated with the permeation and selectivity properties of KcsA channel.
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Gutheim, Sabina. "Characterization of Alcohol Modulation of a Pentameric Ligand-gated Ion Channel with Electrophysiology and Molecular Dynamics Simulations". Thesis, KTH, Tillämpad fysik, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-296530.
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Pentameric ligand-gated ion channels (pLGICs) are membrane receptors that play a crucial role in every living organism. The pLGIC protein structure forms a pore through the membrane of a cell that can let specific ions pass through, upon activation by endogenous agonists. pLGICs are allosterically modulated by ligands binding at allosteric sites, that either stabilize a certain conformation or change the binding affinity of the endogenous agonist. However, much remains unknown about the exact way in which these modulators bind to and affect pLGICs. An increased understanding could help in the search for novel and/or more effective target drugs. With this masters thesis, I hope to contribute by investigating the modulatory effect of ethanol on the bacterial Gloeobacter ligand-gated ion channel (GLIC). This has been done by performing oocyte electrophysiology recordings and analysis of molecular dynamics simulations, both with and without ethanol, and of four separate variants of GLIC that are either potentiated or inhibited by ethanol. Two possible allosteric sites were discovered in a transmembraneintrasubunit pocket: a potentiating allosteric site close to the M2 helix and residue V242, as well as an inhibitory membrane- and M4 helix-close intrasubunit site. Finally, evidence was found that could support a previously suggested inhibitory allosteric site in the pore around the 9’ hydrophobic gate.Pentameriska ligandstyrda jonkanaler (pLGICs) är membranreceptorer som utgör vitala delar av varje levande organism. pLGICs proteinstruktur formar en por genom cellmembranet, som kan släppa igenom specifika joner efter aktivering av endogena agonister. pLGICs är allostermodulerade av ligander som binder vid allostera säten och som därigenom antingen stabiliserar en viss form eller förändrar den endogena agonistens bindningsstyrka. Emellertid saknas fortfarande mycket kunskap på detaljnivå om hur dessa modulatorer binder sig till och påverkar kanalerna. En ökad förståelse skulle hjälpa forskningen efter nya och/eller mer effektiva mediciner. Mitt examensarbetehoppas bidra genom att studera hur etanol modulerar den bakteriella ligandstyrda jonkanalen GLIC från Gloeobacter. Det har gjorts genom elektrofysiologimätningar på oocyter och analys av molekulärdynamiksimuleringar, båda av fyra olika GLIC-varianter, som antingen potentieras eller hämmas av etanol, och med eller utan etanol. Två allostera säten upptäcktes i det transmembrana intrasubenhetområdet: ett säte för potentiering nära M2 helixen och aminosyran V242, och ett hämmande säte nära membranet och helix M4. Slutligen hittades tecken som kan styrka existensen av det tidigare föreslagna hämmande allostera sätet i poren kring den hydrophoba porten.
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Köpfer, David Alexander [Verfasser], Bert de [Akademischer Betreuer] Groot e Ralf [Akademischer Betreuer] Ficner. "Ion Conductance Through Potassium Channels : Studied by Molecular Dynamics Simulations / David Alexander Köpfer. Gutachter: Bert de Groot ; Ralf Ficner. Betreuer: Bert de Groot". Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://d-nb.info/107709678X/34.
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Makarewich, Catherine Anne. "MICRODOMAIN BASED CALCIUM INFLUX PATHWAYS THAT REGULATE PATHOLOGICAL CARDIAC HYPERTROPHY AND CONTRACTILITY". Diss., Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/266828.
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Molecular and Cellular PhysiologyPh.D.
Pathological cardiac stressors, including persistent hypertension or damage from ischemic heart disease, induce a chronic demand for enhanced contractile performance of the heart. The cytosolic calcium (Ca2+) transient that regulates myocyte contraction must be persistently increased in disease states in order to maintain cardiac output to sustain the metabolic requirements of the body. Associated with this enhanced intracellular Ca2+ ([Ca2+]i) state is pathological cardiac myocyte hypertrophy, which results in large part from the activation of Ca2+-dependent activation of calcineurin (Cn)-nuclear factor of activated T cells (NFAT) signaling. The puzzling feature of this hypertrophic signaling is that the cytosolic [Ca2+] that controls contractility appears to be separate from the [Ca2+] which activates Cn-NFAT signaling. The overarching theme of this dissertation is to explore the source and spatial constraints of pathological hypertrophic signaling Ca2+ and to investigate how it is possible that sensitive and finely tuned Ca2+-dependent signaling pathways are regulated in the background of massive Ca2+ fluctuations that oscillate between 100nM and upwards of 1-2μM during each cardiac contractile cycle. L-type Ca2+ channels (LTCCs) are a major source of Ca2+ entry in cardiac myocytes and are known to play an integral role in the initiation of myocyte excitation contraction-coupling (EC-coupling). We performed a number of experiments to show that a small population of LTCCs reside outside of EC-coupling domains within caveolin (Cav-3) signaling microdomains where they provide a local source of Ca2+ to activate Cn-NFAT signaling. We designed a Cav-targeted LTCC blocker that could eliminate Cn-NFAT activation but did not reduce myocyte contractility. The activity of Cav-targeted LTCCs could also be upregulated to enhance hypertrophic signaling without affecting contractility. Therefore, we believe that caveolae-localized LTCCs do not participate in EC-coupling, but instead act locally to control the coordinated activation of Cn-NFAT signaling that drives pathological remodeling. Transient Receptor Potential (TRP) channels are also thought to provide a source of Ca2+ for activation of hypertrophic signaling. The canonical family of TRP channels (TRPC) is expressed at low levels in normal adult cardiac tissue, but these channels are upregulated in disease conditions which implicates them as stress response molecules that could potentially provide a platform for hypertrophic Ca2+ signaling. We show evidence that TRPC channel abundance and function increases in cardiac stress conditions, such as myocardial infarction (MI), and that these channels are associated with hypertrophic responses, likely through a Ca2+ microdomain effect. While we found that TRPC channels housed in caveolae membrane microdomains provides a source of [Ca2+] for induction of cardiac hypertrophy, this effect also requires interplay with LTCCs. We also found that TRPC channels have negative effects on cardiac contractility, which we believe are due to local activation of Ca2+/calmodulin-dependent protein kinase (CaMKII) and subsequent modulation of ryanodine receptors (RyRs). Further, we found that inhibiting TRPC channels in a mouse model of MI led to increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling, as well as increased survival. Collectively, the data presented in this dissertation provides comprehensive evidence that Ca2+ regulation of Cn-NFAT signaling and resultant pathological hypertrophy can be coordinated by spatially localized and regulated Ca2+ channels. The compartmentalization of LTCCs and TRPC channels in caveolae membrane microdomains along with pathological hypertrophy signaling effectors makes for an attractive explanation for how Ca2+-dependent signaling pathways are regulated under conditions of continual Ca2+ transients that mediate cardiac contraction during each heart beat. Elucidation of additional Ca2+ signaling microdomains in adult cardiac myocytes will be important in more comprehensively resolving how myocytes differentiate between signaling versus contractile Ca2+.
Temple University--Theses
50
Mahajan, Rahul. "Gβγ acts at an inter-subunit cleft to activate GIRK1 channels". VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/3307.
Texto completo da fonteResumo:
Heterotrimeric guanine nucleotide-binding proteins (G-proteins) consist of an alpha subunit (Gα) and the dimeric beta-gamma subunit (Gβγ). The first example of direct cell signaling by Gβγ was the discovery of its role in activating G-protein regulated inwardly rectifying K+ (GIRK) channels which underlie the acetylcholine-induced K+ current responsible for vagal inhibition of heart rate. Published crystal structures have provided important insights into the structures of the G-protein subunits and GIRK channels separately, but co-crystals of the channel and Gβγ together remain elusive and no specific reciprocal residue interactions between the two proteins are currently known. Given the absence of direct structural evidence, we attempted to identify these functionally important channel-Gβγ interactions using a computational approach. We developed a multistage computational docking algorithm that combines several known methods in protein-protein docking. Application of the docking protocol to previously published structures of Gβγ and GIRK1 homomeric channels produced a clear signal of a favored binding mode. Analysis of this binding mode suggested a mechanism by which Gβγ promotes the open state of the channel. The channel-Gβγ interactions predicted by the model in silico could be disrupted in vitro by mutation of one protein and rescued by additional mutation of reciprocal residues in the other protein. These interactions were found to extend to agonist induced activation of the channels as well as to activation of the native heteromeric channels. Currently, the structural mechanism by which Gβγ regulates the functional conformations of GIRK channels or of any of its membrane-associated effector proteins is not known. This work shows the first evidence for specific reciprocal interactions between Gβγ and a GIRK channel and places these interactions in the context of a general model of intracellular regulation of GIRK gating.