Die exzitatorische Neurotransmission im gesamten Zentralnervensystem (ZNS) der Wirbeltiere wird weitgehend durch die α-Amino-3-hydroxy-5-methyl-4-isoxazolpropionsäure-Rezeptoren (AMPARs) vermittelt. AMPARs sind Glutamat-gesteuerte Ionenkanäle, die sich an der postsynaptischen Membran befinden, wo sie den Kern makromolekularer Komplexe mit einer Reihe von Hilfsproteinen bilden, die die Rezeptorfunktion konzertiert regulieren. Die bekanntesten dieser Proteine sind die transmembranen AMPA-Rezeptor-Regulierungsproteine (TARPs). TARPs zeigen eine verwirrende Reihe von Effekten auf den Handel, die synaptische Verankerung, die Gate-Kinetik und die Pharmakologie von AMPARs. Über die strukturellen Merkmale des AMPAR-TARP-Komplexes wurde zunehmendes Wissen gesammelt. Die molekularen Mechanismen, die der TARP-Modulation der AMPARs zugrunde liegen, sind jedoch noch nicht vollständig aufgeklärt. In der vorliegenden Studie wurden die AMPAR-TARP-Interaktionen mit Hilfe der Elektrophysiologie in 293 Zellen der menschlichen embryonalen Niere (HEK) untersucht. Die Rolle der extrazellulären TARP-Schleifen, Loop1 (L1) und Loop2 (L2), bei der Modulation der AMPAR-Ansteuerung wurde analysiert. Es wurde ein Modell für die TARP-Modulation vorgeschlagen, das auf vorhergesagten zustandsabhängigen Wechselwirkungen von TARP L1 und L2 mit dem AMPAR basiert. Da die nativen AMPARs im Gehirn hauptsächlich aus heterotetrameren Zusammensetzungen von vier verschiedenen Untereinheiten (GluA1-4) bestehen, wurden außerdem verschiedene Zusammensetzungen von AMPAR-Untereinheiten getestet. Es wurden sowohl gemeinsame als auch von den Untereinheiten abhängige Mechanismen der AMPAR-Modulation durch TARPs beobachtet. Zusammenfassend liefern diese Experimente den Nachweis, dass TARP L1 und L2 nicht an der Assoziation von AMPAR-TARP-Komplexen beteiligt sind und die Modulation der AMPAR-Ansteuerung durch TARPs vollständig erklären können. / Excitatory neurotransmission throughout the vertebrate central nervous system (CNS) is largely mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). AMPARs are glutamate-gated ion channels located at the postsynaptic membrane, where they compose the hub of macromolecular complexes with a number of auxiliary proteins that concertedly regulate the receptor function. Among these proteins the most known ones are the transmembrane AMPA receptor regulatory proteins (TARPs). TARPs show a bewildering array of effects on the trafficking, synaptic anchoring, gating kinetics and pharmacology of AMPARs. Growing knowledge has been gathered about the structural features of the AMPAR-TARP complex. However, the molecular mechanisms underlying TARP modulation of AMPARs have not been fully revealed yet. Given that higher brain functions rely upon AMPAR activity and dysregulation of AMPARs has been associated to life-threatening CNS disorders, big efforts are being made to unravel the molecular machinery behind AMPAR regulation and to identify AMPAR auxiliary proteins as potential pharmacological targets. In the present study, AMPAR-TARP interactions were investigated using electrophysiology in human embryonic kidney (HEK) 293 cells. The role of TARP extracellular loops, Loop1 (L1) and Loop2 (L2), in the modulation of AMPAR gating was analysed. A model for TARP modulation has been proposed, based on predicted state-dependent interactions of TARP L1 and L2 with the AMPAR. Moreover, considering that native AMPARs in the brain mainly consist of heterotetrameric assemblies of four distinct subunits (GluA1-4), different AMPAR subunit compositions were tested. Common as well as subunit-dependent mechanisms of AMPAR modulation by TARPs have been observed. In summary, these experiments provided evidence that TARP L1 and L2 are not involved in association of AMPAR-TARP complexes and can entirely account for the modulation of AMPAR gating by TARPs.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / As mass production of carbon nanoparticles (CNPs) continues to rise, the likelihood of occupational and environmental exposure raises the potential for exposure‐related health hazards. Although many groups have studied the effects of CNPs on biological systems, very few studies have examined the effects of exposure of cells, tissues or organisms to low, physiologically relevant concentrations of CNPs. Three of the most common types of CNPs are single wall nanotubes (SWNT), multi wall nanotubes (MWNT) and fullerenes (C60). We used electrophysiological techniques to test the effects of CNP exposure (40 μg/cm2 – 4 ng/cm2) on barrier function and hormonal responses of well characterized cell lines representing barrier epithelia from the kidney (mpkCCDcl4) and airways (Calu‐3). mpkCCDcl4 is a cell line representing principal cell type that lines the distal nephron in an electrically tight epithelia that aids in salt and water homeostasis and Calu‐3 is one of the few cell lines that produces features of a differentiated, functional human airway epithelium in vivo. These cell lines respond to hormones that regulate salt/water reabsorption (mpkCCDcl4) and chloride secretion (Calu‐3). In mpkCCDcl4 cells, after 48 hour exposure, the transepithelial electrical resistance (TEER) was unaffected by high concentrations (40 – 0.4 μg/cm2) of C60 or SWNT while lower, more relevant levels (< 0.04 μg/cm2) caused a decrease in TEER. MWNT decreased TEER at both high and low concentrations. CNT exposure for 48 hour did not change the transepithelial ion transport in response to anti‐diuretic hormone (ADH). In Calu‐3 cells, after 48 h of exposure to CNPs, fullerenes did not show any effect on TEER whereas the nanotubes significantly decreased TEER over a range of concentrations (4 μg/cm2‐0.004 ng/cm2). The ion transport response to epinephrine was also significantly decreased by the nanotubes but not by fullerenes. To look at the effect of exposure times, airway cells were exposed to same concentrations of CNPs for 24 and 1h. While the 48 h and 24 h exposures exhibited similar effects, there was no effect seen after 1h in terms of TEER or hormonal responses. In both the cell lines the magnitude of the transepithelial resistance change does not indicate a decrease in cellular viability but would be most consistent with more subtle changes (e.g., modifications of the cytoskeleton or changes in the composition of the cellular membrane). These changes in both the cell lines manifested as an inverse relationship with CNP concentration, were further corroborated by an inverse correlation between dose and changes in protein expression as indicated by proteomic analysis. These results indicate a functional impact of CNPs on epithelial cells at concentrations lower than have been previously studied and suggest caution with regard to increasing CNP levels due to increasing environmental pollution.

The combination of fluoroquinolones and non-steroidal anti-inflammatory drugs (NSAIDs) in patients increases the incidence of convulsions. The molecular mechanism underlying this interaction is not fully understood. The primary aim of this study was to investigate the pharmacological mechanisms that might underlie convulsions associated with the use of fluoroquinolones and NSAIDs. In this study, the interaction of ciprofloxacin (cipro; a prototype fluoroquinolone) and biphenylacetic acid (BPAA; a NSAID) was investigated using patch-clamp recording techniques. The specific aims of this study were to investigate the actions of cipro and BPAA on: (1) neuronal GABA A receptors, (2) spontaneous synaptic activity in cultured neurons; (3) voltage-gated ion channels; (4) GABA-gated single channels, 5) the association and dissociation rate kinetics of cipro and BPAA at GABA A -receptors, and 6) their effects at different human GABA A receptor isoforms. BPAA acted as an allosteric modulator to enhance the antagonist effect of cipro at GABA A receptors. Cipro + BPAA abolished inhibitory synaptic activity, whereas excitatory synaptic events were enhanced. These data suggest that this combination of drugs inhibits GABA A receptor-mediated inhibition and secondarily increases excitability in neuronal networks. Cipro and BPAA had little or no effects at voltage-gated Na + , K + or Ca 2+ ion channels, which suggests that the main effects of cipro + BPAA are at GABA A receptors. The kinetics study showed that BPAA increased the stability of the cipro-receptor complex to enhance the action of cipro. Cipro + BPAA inhibited the GABA A receptor by decreasing channel opening frequency and increasing the mean channel closed time. Subunit selectivity studies suggest that the β1/2 or γ2 subunits of GABA A receptors may not be a critical for the interaction of cipro + BPAA. Interestingly, the potentiation of cipro by BPAA was significantly less (p < 0.001) in GABA A receptors containing the α6 subunit compared to potentiation in the non-α6 contained receptor. The α subunit, therefore, appear to be a critical site for the synergistic antagonism of GABA A receptors by Cipro + BPAA. These data also suggest that there may be a novel binding site for BPAA at the a subunit of GABA A receptors.

Les canaux de potassium voltage-dépendant (Kv) sont des tétramères séparés en 12 familles. Chaque sous-unité est composée de six segments transmembranaires (S1-S6). Les quatre premiers (S1-S4) forment le senseur de voltage dont le rôle est de détecter des variations en potentiel membranaire grâce à des acides aminés chargés. Ces acides aminés vont bouger et ce mouvement va être transmis au second domaine, celui du pore (S5-S6). Les domaines du pore des quatre sous-unités vont se combiner pour créer le pore. Ces sous-unités peuvent former des canaux homomériques où chaque sous-unité est identique ou des canaux hétéromériques avec des membres de la même famille. Kv6.4 (KCNG4) est un membre de la famille de sous-unité silencieuse Kv6. Les familles de sous-unités silencieuses incluent également Kv5, Kv8 et Kv9. Ils ne peuvent pas former d’homomères. À la place, il doit former des hétéromères avec Kv2. Les canaux Kv2.1/Kv6.4 ont des propriétés différentes, lorsque comparées aux homomères de Kv2.1, particulièrement avec un décalage de l’inactivation vers les négatifs. Avec la technique du « cut-open voltage clamp fluorometry » (COVCF), nous avons pu déterminer que l’absence d’une charge positive à la position Kv6.4-Y345 est responsable pour une partie du décalage tout en étant capable de réduire ce décalage avec la mutation Kv6.4-Y345R. Nous avons également pu produire l’effet inverse dans Kv2.1 avec Kv2.1-R306Y. Également, nous avons déterminé que la mutation Kv6.4-L360P trouvée chez des patients souffrant de migraines mène à cette pathologie à cause d’un problème de trafic où les sous-unités mutées ne peuvent pas atteindre la surface et produire des canaux fonctionnels. Ce problème est causé par un bris dans l’hélice alpha du segment S4-S5. Uniquement des homomères de Kv2.1 se rendent à la surface ce qui réduit l’excitabilité membranaire. Nous proposons que lorsqu’exprimée dans le ganglion trigéminal, cette mutation mène à des migraines. / Voltage-gated potassium channels (Kv) are tetramers split into 12 families. Each subunit is composed of six transmembrane helices (S1-S6). The first four of those (S1-S4) form the voltage sensor domain whose role it is to detect variations in the membrane potential through charged amino acids. The movement of those amino acids will be transmitted to the second domain, the pore domain (S5-S6). The pore domain of all four subunits will combine to form the ion conducting pore. These subunits can form homomers where all four subunits are identical or heteromers with members of the same family. Kv6.4 (KCNG4) is a member of the silent subunit family Kv6, which also includes Kv5, Kv8 and Kv9. They cannot form functioning homomers. Instead, they form heteromers with Kv2. Kv2.1/Kv6.4 channels have different properties when compared to Kv2.1 homomers, particularly a negative shift of the voltage dependence of inactivation. With the cut-open voltage clamp fluorometry (COVC) technique, we were able to determine that the absence of a gating charge at position Kv6.4-Y345 is responsible for part of this shift. We were able to recover part of this shift with the mutation Kv6.4-Y345R. We were also able to produce the inverse effect in Kv2.1 with the mutation Kv2.1-R306Y. Also, we determined that the mutation Kv6.4-L360P. which is found in patients suffering from migraines, leads to this condition because of a trafficking defect caused by the mutation stopping the subunits from reaching the membrane and making functional channels. The defect is caused by a kink in the alpha helix of the S4-S5 linker. Only Kv2.1 homomers reach the membrane which reduces membrane excitability. We propose that when expressed in the trigeminal ganglion, this mutation leads to migraines because of this trafficking defect.

No description available.

Allostery is a regulatory process whereby a perturbation by an effector at one discrete locus creates a conformational change that stimulates a functional change at another. The two sites communicate through networks of interacting residues that respond in a concerted manner to the allosteric perturbation. These allosteric networks are traditionally mapped with high resolution structure determination techniques to understand the conformational changes that regulate protein function as well as its modulation by allosteric ligands and its dysfunction caused by disease-related mutations (DRMs). However, high resolution structural determination techniques, such as X-ray crystallography, cryo-electron microscopy and nuclear Overhauser effect NMR spectroscopy are not always amenable for systems plagued by poor solubility and line broadening caused by μs-ms dynamics or systems where allostery relies primarily on dynamical rather than structural changes. This dissertation discusses methodologies to map the allosteric sites and pathways for such challenging systems. The foundation of this approach is to model allosteric pathways in the context of their respective thermodynamic cycles. In chapter 2, the thermodynamic cycle of a DRM in the hyperpolarization-activated cyclic nucleotide-gated ion channel 4 (HCN4) is analyzed with respect to structure, dynamics and kinetics, revealing how the DRM remodels the free energy landscape of HCN4 and results in a loss-of-function disease phenotype. In chapter 3, the mechanism of action of an uncompetitive inhibitor for the exchange protein activated by cAMP is elucidated by characterizing its selectivity for distinct conformations within the thermodynamic cycle that are trapped using a combination of mutations and ligand analogs. In chapter 4, we discuss two new protocols for the chemical shift covariance analysis (CHESCA). The CHESCA is an approach that identifies allosteric signaling pathways by measuring concerted residue responses to a library of chemical perturbations that stabilize conformational equilibria at different positions. Overall, the approaches discussed in this dissertation are widely applicable for mapping the mechanisms of allosteric perturbations that arise from ligand binding, post-translational modifications and mutations, even in systems where traditional structure determination techniques remain challenging to implement. / Thesis / Doctor of Philosophy (PhD) / Allostery is a regulatory mechanism for proteins, which controls functional properties of one distinct site through the perturbation of another distinct, and often distant, site. The two sites are connected via a series of residues that undergo conformational changes once perturbed by the allosteric effector. Mapping these communication pathways reveals mechanisms of protein regulation, which are invaluable for developing pharmacological modulators to target these pathways or for understanding the mechanisms of disease mutations that disrupt these pathways. Allosteric pathways have been traditionally determined using structure determination approaches that provide a static snapshot of the protein’s structure. However, these approaches are typically not effective when allostery relies extensive changes in dynamics. The goal of this thesis was to develop methods to characterize systems that are dynamic or otherwise unsuitable for traditional structure determination. Herein, we utilize NMR spectroscopy to analyze the allosteric mechanisms of three cAMP-binding proteins involved in cardiovascular health.

Indiana University-Purdue University Indianapolis (IUPUI) / Carbon nanotubes (CNTs) are one of the most common nanoparticles (NP) found in workplace air. Therefore, there is a strong chance that these NP will enter the human body. They have similar physical properties to asbestos, a known toxic material, yet there is limited evidence showing that CNTs may be hazardous to human barrier epithelia. In previous studies done in our laboratory, the effects of CNTs on the barrier function in the human airway epithelial cell line (Calu-3) were measured. Measurements were done using electrophysiology, a technique which measures both transepithelial electrical resistance (TEER), a measure of monolayer integrity, and short circuit current (SCC) which is a measure of vectorial ion transport across the cell monolayer. The research findings showed that select physiologically relevant concentrations of long single-wall (SW) and multi-wall (MW) CNTs significantly decreased the stimulated SCC of the Calu-3 cells compared to untreated cultures. Calu-3 cells showed decreases in TEER when incubated for 48 hours (h) with concentrations of MWCNT ranging from 4µg/cm2 to 0.4ng/cm2 and SWCNT ranging from 4µg/cm2 to 0.04ng/cm2. The impaired cellular function, despite sustained cell viability, led us to investigate the mechanism by which the CNTs were affecting the cell membrane. We investigated the interaction of short MWCNTs with model lipid membranes using an ion channel amplifier, Planar Bilayer Workstation. Membranes were synthesized using neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoylphosphatidylserine (DPhPS) lipids. Gramicidin A (GA), an ion channel reporter protein, was used to measure changes in ion channel conductance due to CNT exposures. Synthetic membranes exposed to CNTs allowed bursts of currents to cross the membrane when they were added to the membrane buffer system. When added to the membrane in the presence of GA, they distorted channel formation and reduced membrane stability.

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.

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.