Dissertations / Theses on the topic 'Single-ion channel'

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第19767号
農博第2163号
新制||農||1040(附属図書館)
学位論文||H28||N4983(農学部図書室)
32803
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 加納 健司, 教授 三芳 秀人, 教授 三上 文三
学位規則第4条第1項該当

This thesis describes a project in which algorithms are developed for the rapid and accurate solution of Poisson's equation in the presence of a dielectric boundary and multiple point charges. These algorithms are then used to perform Brownian dynamics simulations on realistic models of biological ion channels. An iterative method of solution, in which the dielectric boundary is tiled with variable sized surface charge sectors, provides the flexibility to deal with arbitrarily shaped boundaries, but is too slow to perform Brownian dynamics. An analytical solution is derived, which is faster and more accurate, but only works for a toroidal boundary. Finally, a method is developed of pre-calculating solutions to Poisson's equation and storing them in tables. The solution for a particular configuration of ions in the channel can then be assembled by interpolation from the tables and application of the principle of superposition. This algorithm combines the flexibility of the iterative method with greater speed even than the analytical method, and is fast enough that channel conductance can be predicted. The results of simulations for a model single-ion channel, based on the acetylcholine receptor channel, show that the narrow pore through the low dielectric strength medium of the protein creates an energy barrier which restricts the permeation of ions. They further show that this barrier can be removed by dipoles in the neck of the channel, but that the barrier is not removed by shielding by counter-ions. The results of simulations for a model multi-ion channel, based on a bacterial potassium channel, show that the model channel has conductance characteristics similar to those of real potassium channels. Ions appear to move through the model multi-ion channel via rapid transitions between a series of semi-stable states. This observation suggests a possible physical basis for the reaction rate theory of channel conductance, and opens up an avenue for future research.

A few studies support the idea that the theta rhythm modulates synaptic plasticity by demonstrating that the phase of the theta cycle at which the induction stimuli are delivered determines the nature of the resulting synaptic plasticity. These studies were conducted in urethane-anesthetized animals and in vitro slice preparations where the theta rhythm is artificially generated. Our goal was to find whether and how natural theta activity affects synaptic plasticity in the hippocampus of adult and old freely behaving animals. In both adult and aged, memory-impaired rats, LTP lasting at least 48 h was induced when stimuli were delivered at theta peak. No change in synaptic strength was observed when stimuli were delivered at theta trough. These observations indicate that the naturally occurring theta rhythm modulates synaptic plasticity, and suggest a mechanism by which the phase of firing could contain meaningful information. The degree of LTP, however, was significantly smaller in the old animals. To better understand the conformational changes and the dynamic interactions that govern ion-channel kinetics we developed a new approach using simultaneous single-channel patch-clamp recording and single-molecule fluorescence microscopy. Gramicidin monomers were tagged with a fluorescence dye and single-channel current was recorded from gramicidin channels in the bilayer that was formed at the tip of a patch pipette. Co-localization and fluorescence resonance energy transfer (FRET) within a single gramicidin dimer were probed. The new technique made it possible to directly capture the conformational dynamics between the two gramicidin monomers by observing the changes in the distance between the attached dye molecules. The molecular interactions of the NMDA receptor with its ligands determine the dynamic properties of activation and desensitization that in turn shape NMDA receptor mediated currents. We have monitored the occurrence and intensity changes of FRET between two fluorescence-labeled agonists at the glutamate binding site of the receptor, simultaneously with single channel current recording. These observations can be translated to dissociation/association rates and aid in our understanding of the mechanisms that underlie the transitions of the receptor between different kinetic states.

BACKGROUND:Aquaporin-1 (AQP1) functions as an osmotic water channel and a gated cation channel. Activation of the AQP1 ion conductance by intracellular cGMP was hypothesized to involve the carboxyl (C-) terminus, based on amino acid sequence alignments with cyclic-nucleotide-gated channels and cGMP-selective phosphodiesterases.RESULTS:Voltage clamp analyses of human AQP1 channels expressed in Xenopus oocytes demonstrated that the nitric oxide donor, sodium nitroprusside (SNP
3-14 mM) activated the ionic conductance response in a dose-dependent manner. Block of soluble guanylate cyclase prevented the response. Enzyme immunoassays confirmed a linear dose-dependent relationship between SNP and the resulting intracellular cGMP levels (up to 1700 fmol cGMP /oocyte at 14 mM SNP). Results here are the first to show that the efficacy of ion channel activation is decreased by mutations of AQP1 at conserved residues in the C-terminal domain (aspartate D237 and lysine K243).CONCLUSIONS:These data support the idea that the limited amino acid sequence similarities found between three diverse classes of cGMP-binding proteins are significant to the function of AQP1 as a cGMP-gated ion channel, and provide direct evidence for the involvement of the AQP1 C-terminal domain in cGMP-mediated ion channel activation.

Hybrid technologies, thanks to the convergence of integrated microelectronic devices and new class of microfluidic structures could open new perspectives to the way how nanoscale events are discovered, monitored and controlled. The key point of this thesis is to evaluate the impact of such an approach into applications of ion-channel High Throughput Screening (HTS)platforms. This approach offers promising opportunities for the development of new classes of sensitive, reliable and cheap sensors. There are numerous advantages of embedding microelectronic readout structures strictly coupled to sensing elements. On the one hand the signal-to-noise-ratio is increased as a result of scaling. On the other, the readout miniaturization allows organization of sensors into arrays, increasing the capability of the platform in terms of number of acquired data, as required in the HTS approach, to improve sensing accuracy and reliabiity. However, accurate interface design is required to establish efficient communication between ionic-based and electronic-based signals. The work made in this thesis will show a first example of a complete parallel readout system with single ion channel resolution, using a compact and scalable hybrid architecture suitable to be interfaced to large array of sensors, ensuring simultaneous signal recording and smart control of the signal-to-noise ratio and bandwidth trade off. More specifically, an array of microfluidic polymer structures, hosting artificial lipid bilayers blocks where single ion channel pores are embededed, is coupled with an array of ultra-low noise current amplifiers for signal amplification and data processing. As demonstrating working example, the platform was used to acquire ultra small currents derived by single non-covalent molecular binding between alpha-hemolysin pores and beta-cyclodextrin molecules in artificial lipid membranes.

A method is described for preset-count irradiations between 1 and 100 ions singling-out individual ions from an ion beam with more than a billion ions arriving per second. The ion tracks are etched in a conductometric system with real-time evaluation of the acquired data. The etch process can be interrupted when reaching a preset channel diameter. Cylindrical channels are obtained by adding surfactants to the etch solution forming a self-assembled barrier between etching medium and polymer. Asymmetric etching of single ion tracks leads to pH sensitive conical pores with diode-like properties. Using etched channels as template, homogeneous and multilayer magnetic single-wires are electrodeposited. The magnetoresistivity of the wires is studied. Single-track applications comprise critical apertures (cylindric, conic, necked), asymmetric pores (pH sensitive, biospecific), Giant Magneto Resistance sensors, and spintronic devices. On the basis of studies with individual ion tracks we tackled tilted multiporous systems such as ion beam lithography with a masked ion beam leading to micro-structures with inclined walls and anisotropic superhydrophobic ion track textures, analogous to biological shingle structures on butterfly wings. We demonstrated qualitatively, that the asymmetry of the texture translates into motion under ultrasonic agitation. This could lead to the development of rotary drives.

Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane.

QC 20151217

Ion channels are fundamental molecules in the nervous system that catalyze the flux of ions across the cell membrane. There are mounting evidences suggesting that the kinetic properties of ion channels undergo activity-dependent changes in various pathophysiological conditions. Here such activity-dependent changes were studied in case of two different ion channels; the rat brain derived voltage-gated Na+ channel, rNav1.2 and the human background leak K+ channel, hTREK1 using the single channel patch-clamp technique. Our results on the voltage-gated Na+ channel (Chapter III) illustrated that sustained membrane depolarization, as seen in pathophysiological conditions like epilepsy, induced a defined non-linear variation in the unitary conductance, activation, inactivation and recovery kinetic properties of the channel. Signal processing tools attributed a pseudo-oscillatory nature to the non-linearity observed in the channel properties. Prolonged membrane depolarization also induced a “molecular memory” phenomenon, characterized by clustering of dwell time events and strong autocorrelation in the dwell time series. The persistence of such molecular memory was found to be dependent on the duration of depolarization. Similar plastic changes were observed in case of the hTREK1 channel in presence of saturating concentrations of agonist, trichloroethanol (TCE) (Chapter IV). TREK1 channel behaves similar to single enzyme molecules with a single binding site for the substrate K+ ion whereas TCE acts as an allosteric activator of the channel. We observed that with increasing concentration of TCE (10 M to 10 mM) the catalytic turnover rate exhibited progressive departure from monoexponential to multi-exponential distribution suggesting the presence of ‘dynamic disorder’ analogous to single enzyme molecules. In addition, we observed the induction of strong correlation in successive waiting times and flux intensities, exemplified by distinct mode switching between high and low flux activity, which implied the induction of memory in single ion channel. Our observation of such molecular memory in two different ion channels in different experimental conditions highlights the importance and generality of the phenomenon which is normally hidden under the ensemble behaviour of ion channels. In the final part of the work (chapter V) we observed strong negative cooperativity and half-of-sites saturation kinetics in the interaction of local anesthetic, lidocaine with hTREK1 channel. We also mapped the specific anesthetic binding site in the c-terminal domain of the channel. Further, single channel analysis and the heterodimer studies enabled us to propose a model for this interaction and provide a plausible paradigm for the inhibitory action of lidocaine on hTREK1.

This thesis describes a project in which algorithms are developed for the rapid and accurate solution of Poisson's equation in the presence of a dielectric boundary and multiple point charges. These algorithms are then used to perform Brownian dynamics simulations on realistic models of biological ion channels. An iterative method of solution, in which the dielectric boundary is tiled with variable sized surface charge sectors, provides the flexibility to deal with arbitrarily shaped boundaries, but is too slow to perform Brownian dynamics. An analytical solution is derived, which is faster and more accurate, but only works for a toroidal boundary. Finally, a method is developed of pre-calculating solutions to Poisson's equation and storing them in tables. The solution for a particular configuration of ions in the channel can then be assembled by interpolation from the tables and application of the principle of superposition. This algorithm combines the flexibility of the iterative method with greater speed even than the analytical method, and is fast enough that channel conductance can be predicted. The results of simulations for a model single-ion channel, based on the acetylcholine receptor channel, show that the narrow pore through the low dielectric strength medium of the protein creates an energy barrier which restricts the permeation of ions. They further show that this barrier can be removed by dipoles in the neck of the channel, but that the barrier is not removed by shielding by counter-ions. The results of simulations for a model multi-ion channel, based on a bacterial potassium channel, show that the model channel has conductance characteristics similar to those of real potassium channels. Ions appear to move through the model multi-ion channel via rapid transitions between a series of semi-stable states. This observation suggests a possible physical basis for the reaction rate theory of channel conductance, and opens up an avenue for future research.

Glycine receptors (GlyRs) are ligand-gated ion channels (LGICs) that, along with other members of the cys-loop superfamily of receptors, mediate a considerable portion of fast neurotransmission in the central nervous system (CNS). GlyRs are pentameric channels, organized quasi-symmetrically around an ion-conducting pore. Opening of the integral ion pore depends on ligand binding and transduction of this binding signal to the channel gate. Research presented in this dissertation describes a number of critical electrostatic interactions that play a role in conserving the closed-state stability of the receptor in the absence of ligand, ensuring that receptor activation occurs only upon neurotransmitter binding. These amino acids, aspartic acid at position 97 (D97), lysine 116 (K116), arginine 119 (R119) and arginine R131 (R131) are charged residues that interact with one another through electrostatic attraction. When D97 is replaced with any other amino acid this destabilizes the closed state of the channel and causes spontaneous GlyR channel opening. I show that restoration of this electrostatic interaction in GlyR bearing double mutations in which the charges are swapped (D97R/R119E and D97R/R131D) markedly decreases this spontaneous current. In addition, I investigate how these residues that interact at the interfaces between receptor subunits affect the efficacies of GlyR partial agonists. My work shows that the partial agonist taurine is converted into a full agonist at both D97R and R131D receptors. Furthermore, I analyzed the structure of the more extracellular part of the transmembrane (TM) 2 segment that lines the ion channel pore, showing that it is unlikely that this fragment (stretching from T13’ to S18’) is constrained in a true alpha helical conformation. From this work, using disulfide trapping and whole cell electrophysiology, I conclude that a significant level of flexibility characterizes this part of the TM2 domain. This segment includes residue S267, previously shown to be significant for alcohol and anesthetic actions, as well as residue Q266 that, when mutated, produces a hyperekplexia-like phenotype. The range of movement of residues in this region may therefore play an important role not only in channel gating but also in how modulators of GlyR function exert their actions.
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Les canaux ioniques sont des complexes macromoléculaires clés exprimés dans tous les types de cellules et sont impliqués dans divers processus physiologiques, y compris la génération et la propagation de potentiels d'action. Des canaux défectueux conduisent à des maladies graves, notamment l'épilepsie, des arythmies et des syndromes douloureux, ce qui en fait une cible potentielle intéressante pour le développement de médicaments. Pour améliorer notre compréhension de ces assemblages biologiques et éventuellement trouver des traitements spécifiques pour les canalopathies, il est crucial d'étudier la structure et la fonction des canaux ioniques. L'objectif principal de cette thèse a été d'étudier ce type de détails structurels et fonctionnels pour trois canaux ioniques associés aux domaines des capteurs de douleur et des canaux potassiques voltage-dépendants en utilisant des techniques de fluorescence et d'électrophysiologie. Dans le premier projet, nous avons étudié la stœchiométrie des canaux hétéromères Kv2.1 / 6.4 (chapitre trois). La technique du décompte de sous-unités isolées (single subunit counting :ssc) permet de compter les sous-unités marquées par fluorescence d’un complexe isolé en déterminant le nombre d'événements de photoblanchiment, qui apparaissent en sauts irréversibles vers le bas sur les traces de fluorescence. Pour désigner la stœchiométrie la plus probable, nous avons utilisé des calculs de probabilités pondérées et avons constaté que les canaux Kv2.1 / 6.4 s'expriment dans un arrangement 2 : 2. Plus précisément, les études fonctionnelles des canaux concatémériques montrent que les sous-unités Kv6.4 et 2.1 doivent être disposées de manière alternée. Le deuxième projet était également basé sur des expériences de SSC et visait à déterminer l'état oligomérique du nouveau canal ionique TACAN (chapitre quatre). Nous avons trouvé une portion significative de canaux intracellulaires, ce qui a provoqué une fluorescence de fond dans les expériences de SSC traditionnelles réalisées avec les cellules mammifères. Pour améliorer le rapport du signal sur bruit de fond, nous avons effectué des expériences de SSC sur des canaux purifiés qui ont été immobilisés sur des lamelles de verre fonctionnalisées Ni-NTA. En utilisant la méthode de calcul décrite dans le premier projet, nous avons trouvé différents états oligomériques et proposons que les canaux TACAN natifs s'assemblent en tétramères qui sont instables lorsqu'ils sont solubilisés dans un détergent. Dans le dernier projet, nous avons étudié la relation structure-fonction de la sous-unité auxiliaire DPP6 pour les canaux Kv4.2 (chapitre cinq). Ici, nous avons progressivement tronqué le grand domaine extracellulaire de 700 acides aminés de DPP6 et étudié son effet sur les courants macroscopiques en utilisant la technique du cut-open voltage clamp. Nous avons constaté que les sous-unités DPP6 avec un domaine extracellulaire court ne parviennent pas à moduler les propriétés du canal aussi efficacement que la DPP6 pleine longueur. Plus précisément, la seconde moitié du domaine extracellulaire b-propeller de DPP6 est responsable d'une inactivation du canal considérablement accélérée. Sur la base de la structure cristalline du domaine extracellulaire, nous avons proposé qu'un domaine b-propeller stable et possiblement la formation de dimères DPP6 sont responsables de la déstabilisation efficace de l'état du canal ouvert.
Ion channels are key macromolecular complexes expressed in all cell types and are involved in various physiological processes including the generation and propagation of action potentials. Defective channels lead to severe diseases including epilepsy, arrhythmias and pain syndromes making them an interesting potential drug target. To improve our understanding of these biological assemblies and eventually find specific treatments for channelopathies, it is crucial to study the structure and function of ion channels. The main purpose of this thesis has been to investigate such structural and functional details of three ion channel complexes from the field of pain sensors and voltage-gated potassium channels using fluorescence and electrophysiological techniques. In the first project, we studied the stoichiometry of heteromeric Kv2.1/6.4 channel complexes (chapter three). Single subunit counting (SSC) allows to directly count the number of fluorescently labeled subunits by determining the number of irreversible, step-wise photobleaching events. To determine the most probable stoichiometry, we used weighted likelihood calculations and found that Kv2.1/6.4 channels express in a 2:2 arrangement. More precisely, functional studies of concatemeric channels (performed by our collaborators) illustrate that Kv6.4 and 2.1 subunits need to be arranged in an alternating fashion. The second project was also based on SSC experiments and aimed at determining the oligomeric state of the novel ion channel TACAN (chapter four). We found a significant amount of channels in the intracellular which caused background fluorescence in traditional SSC experiments performed in cells. To improve the signal to background ratio, we performed SSC experiments on purified channels that were immobilized on Ni-NTA functionalized glass coverslips. Using the model selection method described in the first project, we found different oligomeric states and propose that native TACAN channels assemble as tetramers which are unstable when solubilized in detergent. In the last project, we investigated the structure-function relation of the auxiliary DPP6 subunit in Kv4.2 channel complexes (chapter five). Here, we progressively truncated DPP6’s 700 amino acids long extracellular domain and studied its effect on macroscopic currents using the cut-open voltage clamp technique. We found that DPP6 subunits with a short extracellular domain fail to modulate the channel properties as efficiently as the full length DPP6. More precisely, the second half of the extracellular b-propeller domain of DPP6 is responsible for drastically accelerated channel inactivation. Based on the crystal structure of the extracellular domain, we proposed that a stable b-propeller domain and possibly DPP6 dimer formation is responsible for destabilizing the open channel state efficiently.

Nonequilibrium Response Spectroscopy (NRS), the technique of using rapidly fluctuating voltage pulses in the study of ion channels is applied here. NRS is known to drive an ensemble of ion channels far from equilibrium where it has been argued that new details of ion channel kinetics can be studied under nonequilibrium conditions. In this study, single pulse NRS technique with custom designed waveforms built from wavelets are used. Two series of experiments were implemented using custom designed pulses, which were obtained using a pulse search algorithm with suitable 'cost functionals' as constraints in the design of the pulse. The use of NRS pulses in model selection was investigated, as well as an in the analysis of model topology through parametric optimization. Electrophysiological recordings (whole cell ionic current measurements) were made from mammalian cells (HEK 293) which stably expressed the sodium ion channel. Wavelet analysis of the model response and the experimental data is introduced. Its effectiveness in the data analysis is demonstrated in the unique plots of the wavelet transforms. The results show how these new NRS pulses can be applied in a systematic way to study ion channels gating kinetics
acase@tulane.edu

The nicotinic acetylcholine receptors are pentameric ligand-gated ion channels that mediate fast synaptic transmission in the brain and peripheral nervous system. After an introduction (Chapter 1), Chapter 2 describes my development of techniques to combine single-channel and whole-cell recording with nonsense suppression. Having established the feasibility of the combined use of single-channel and whole-cell recording, in Chapter 3 we developed a method to identify the functional interactions of amino acids that are physically far apart in a protein. This is fundamentally a whole-cell recording method to find allosteric interactions in ion channels. The significance of this method is strongly supported by single-channel measurements. Additionally, the relationship between the single-channel gating equilibrium constant, theta, and the whole-cell measurement of EC50 is considered.

In Chapter 4, I describe my progress towards measuring the channel opening rate of the fetal and adult muscle-type nicotinic acetylcholine receptors. Multiple different agonists are used, including acetylcholine, choline, and tetramethylammonium. Single-channel data are reported for the wild-type receptors as well as for receptors with the unnatural amino acid 5-F-Trp (monofluoro-Trp). Data are reported for multiple concentrations for a mutated fetal nAChR, and QuB is used to fit various possible models and estimate theta for this mutant.

A major aim of this dissertation was to use single-molecule studies of ion channels expressing unnatural amino acids to provide even more convincing evidence for cation-pi interactions at the binding sites of ligand-gated ion channels, specifically the neuronal nicotinic acetylcholine receptor. Chapter 5 describes the combined application of single-channel, whole-cell, and unnatural amino acid mutagenesis to the specific question of how two molecules—nicotine and Chantix® (varenicline)—bind to the alpha4beta2 brain receptor. In Chapter 6, I describe single-channel experiments that establish a method for distinguishing between the two known stoichiometries of the wild type alpha4beta2 brain receptor. Specifically, I identify a difference in the rectification properties of the high and low affinity receptors.

La fonction des canaux ioniques est finement régulée par des changements structuraux de sites clés contrôlant l’ouverture du pore. Ces modulations structurales découlent de l’interaction du canal avec l’environnement local, puisque certains domaines peuvent être suffisamment sensibles à des propriétés physico-chimiques spécifiques. Les mouvements engendrés dans la structure sont notamment perceptibles fonctionnellement lorsque le canal ouvre un passage à certains ions, générant ainsi un courant ionique mesurable selon le potentiel électrochimique. Une description détaillée de ces relations structure-fonction est cependant difficile à obtenir à partir de mesures sur des ensembles de canaux identiques, puisque les fluctuations et les distributions de différentes propriétés individuelles demeurent cachées dans une moyenne. Pour distinguer ces propriétés, des mesures à l’échelle de la molécule unique sont nécessaires. Le but principal de la présente thèse est d’étudier la structure et les mécanismes moléculaires de canaux ioniques par mesures de spectroscopie de fluorescence à l’échelle de la molécule unique. Les études sont particulièrement dirigées vers le développement de nouvelles méthodes ou leur amélioration. Une classe de toxine formeuse de pores a servi de premier modèle d’étude. La fluorescence à l’échelle de la molécule unique a aussi été utilisée pour l’étude d’un récepteur glutamate, d’un récepteur à la glycine et d’un canal potassique procaryote. Le premier volet porte sur l’étude de la stœchiométrie par mesures de photoblanchiment en temps résolu. Cette méthode permet de déterminer directement le nombre de monomères fluorescents dans un complexe isolé par le décompte des sauts discrets de fluorescence suivant les événements de photoblanchiment. Nous présentons ici la première description, à notre connaissance, de l’assemblage dynamique d’une protéine membranaire dans un environnement lipidique. La toxine monomérique purifiée Cry1Aa s’assemble à d’autres monomères selon la concentration et sature en conformation tétramérique. Un programme automatique est ensuite développé pour déterminer la stœchiométrie de protéines membranaires fusionnées à GFP et exprimées à la surface de cellules mammifères. Bien que ce système d’expression soit approprié pour l’étude de protéines d’origine mammifère, le bruit de fluorescence y est particulièrement important et augmente significativement le risque d’erreur dans le décompte manuel des monomères fluorescents. La méthode présentée permet une analyse rapide et automatique basée sur des critères fixes. L’algorithme chargé d’effectuer le décompte des monomères fluorescents a été optimisé à partir de simulations et ajuste ses paramètres de détection automatiquement selon la trace de fluorescence. La composition de deux canaux ioniques a été vérifiée avec succès par ce programme. Finalement, la fluorescence à l’échelle de la molécule unique est mesurée conjointement au courant ionique de canaux potassiques KcsA avec un système de fluorométrie en voltage imposé. Ces enregistrements combinés permettent de décrire la fonction de canaux ioniques simultanément à leur position et densité alors qu’ils diffusent dans une membrane lipidique dont la composition est choisie. Nous avons observé le regroupement de canaux KcsA pour différentes compositions lipidiques. Ce regroupement ne paraît pas être causé par des interactions protéine-protéine, mais plutôt par des microdomaines induits par la forme des canaux reconstitués dans la membrane. Il semble que des canaux regroupés puissent ensuite devenir couplés, se traduisant en ouvertures et fermetures simultanées où les niveaux de conductance sont un multiple de la conductance « normale » d’un canal isolé. De plus, contrairement à ce qui est actuellement suggéré, KcsA ne requiert pas de phospholipide chargé négativement pour sa fonction. Plusieurs mesures indiquent plutôt que des lipides de forme conique dans la phase cristalline liquide sont suffisants pour permettre l’ouverture de canaux KcsA isolés. Des canaux regroupés peuvent quant à eux surmonter la barrière d’énergie pour s’ouvrir de manière coopérative dans des lipides non chargés de forme cylindrique.
The function of ion channels is finely regulated by structural changes of key domains controlling the pore opening. These structural modulations arise from interactions with the local environment, since several domains can be sensitive to specific physico-chemical properties. Movements generated in the structure become notably perceptible when channels open a passage for some ions, thus generating a measurable ionic current according to the electrochemical potential. A detailed description of these structure-function relationships is however difficult to obtain from measurements involving a set of identical channels, since the fluctuations and distributions of different individual properties remain hidden in an average. To differentiate these properties, single-molecule recordings are required. The main purpose of this thesis is to study the structural aspects and molecular mechanisms of ion channels using fluorescence spectroscopy at the single-molecule level. Studies are oriented towards the development or improvement of new methods. A class of pore-forming toxin served as a first study model. Single-molecule fluorescence was also used to study an ionotropic glutamate receptor, a glycine receptor and a prokaryotic potassium channel. The first part focuses on the study of stoichiometry using fluorescent subunit counting. This method allows a direct measure of the number of fluorescent monomers within a single complex by counting the number of step-wise fluorescence intensity decrease following photobleaching events. Here we present the first description, to our knowledge, of the dynamic assembly of a membrane protein in a lipid environment. The purified monomeric Cry1Aa toxin clusters with other monomers depending on the concentration and saturates in a tetrameric conformation. An automated method has been developed to determine the stoichiometry of GFP-tagged membrane proteins expressed on mammalian cell surface. Although this expression system is suitable for the study of proteins of mammalian origin, background fluorescence is particularly important and significantly increases the risk of error in the manual counting process. The presented method allows a fast and automated analysis based on fixed criteria. The algorithm responsible for counting fluorescent monomers was optimized from simulations and adjusts its detection parameters automatically according to the fluorescence trace recording. The composition of two ion channels was successfully verified using this program. Finally, single-molecule fluorescence is measured together with ionic current of KcsA channels using a voltage-clamp fluorometry setup. These combined recordings allowed us to describe the function of ion channels simultaneously to their position and density as they diffuse in a lipid membrane of defined composition. We observed clustering of KcsA channels for various lipid compositions. Clustering does not appear to be caused by protein-protein interaction, but rather by microdomains induced by the shape of reconstructed channels in the lipid bilayer. It seems that clustered KcsA channels could then become coupled, resulting in cooperative gating events with conductance levels multiple to the “normal” unitary channel conductance. Moreover, as opposed to what is currently suggested, KcsA does not require a negatively charged phospholipid for its function. Several of our recordings rather suggest that conically shaped lipids in the lamellar liquid crystalline phase are sufficient to allow single channel opening. Clustered channels can on the other hand overcome the energy barrier to open cooperatively in uncharged cylindrical lipids.

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.