Control of Cation Permeation through the Nicotinic Receptor Channel

We used molecular dynamics (MD) simulations to explore the transport of single cations through the channel of the muscle nicotinic acetylcholine receptor (nAChR). Four MD simulations of 16 ns were performed at physiological and hyperpolarized membrane potentials, with and without restraints of the structure, but all without bound agonist. With the structure unrestrained and a potential of −100 mV, one cation traversed the channel during a transient period of channel hydration; at −200 mV, the channel was continuously hydrated and two cations traversed the channel. With the structure restrained, however, cations did not traverse the channel at either membrane potential, even though the channel was continuously hydrated. The overall results show that cation selective transport through the nAChR channel is governed by electrostatic interactions to achieve charge selectivity, but ion translocation relies on channel hydration, facilitated by a trans-membrane field, coupled with dynamic fluctuations of the channel structure

Testing the Applicability of Nernst-Planck Theory in Ion Channels: Comparisons with Brownian Dynamics Simulations

The macroscopic Nernst-Planck (NP) theory has often been used for predicting ion channel currents in recent years, but the validity of this theory at the microscopic scale has not been tested. In this study we systematically tested the ability of the NP theory to accurately predict channel currents by combining and comparing the results with those of Brownian dynamics (BD) simulations. To thoroughly test the theory in a range of situations, calculations were made in a series of simplified cylindrical channels with radii ranging from 3 to 15 Å, in a more complex ‘catenary’ channel, and in a realistic model of the mechanosensitive channel MscS. The extensive tests indicate that the NP equation is applicable in narrow ion channels provided that accurate concentrations and potentials can be input as the currents obtained from the combination of BD and NP match well with those obtained directly from BD simulations, although some discrepancies are seen when the ion concentrations are not radially uniform. This finding opens a door to utilising the results of microscopic simulations in continuum theory, something that is likely to be useful in the investigation of a range of biophysical and nano-scale applications and should stimulate further studies in this direction

The Dissolution of Cellulose in Ionic Liquids - A Molecular Dynamics Study

The use of ionic liquids for the dissolution of cellulose promises an alternative method for the thermochemical pretreatment of biomass that may be more efficient and environmentally acceptable than conventional techniques in aqueous solution. Understanding how ionic liquids act on cellulose is essential for improving pretreatment conditions and thus detailed knowledge of the interactions between solute and solvent molecules is necessary. Here, results from the first all-atom molecular dynamics simulation of an entire cellulose microfibril in 1-butyl-3-methylimidazolium chloride (BmimCl) are presented and the interactions and orientations of solvent ions with respect to glucose units on the hydrophobic and hydrophilic surfaces of the fiber are analyzed in detail, shedding light on the initiation stages of cellulose dissolution. Moreover, replica-exchange simulations of a single cellulose chain fully solvated in BmimCl and in water are performed for a total of around 13 μs in order to study the dynamics and thermodynamics of the end state of the dissolution. The results indicate that chloride anions predominantly interact with cellulose hydroxyl groups and disrupt the intrachain O3H’···O5 hydrogen bonds, which are essential for the integrity of cellulose fibers. The cations stack preferentially on the hydrophobic cellulose surface, governed by non-polar interactions with cellulose, which can stabilize detached cellulose chains by compensating the interaction between stacked layers. Moreover, a frequently occurring intercalation of cations on the hydrophilic surface is observed, which by separating cellulose layers can also potentially facilitate the initiation of fiber disintegration. The single-chain simulations indicate that differences in cellulose solvation mechanisms between the two solvents exist. Although global size-related properties of the cellulose chain are comparable in the two solvents, local conformational properties of cellulose differ significantly between the BmimCl and water solutions. In general, the results indicate that solute-solvent interaction energies are more favorable and that the cellulose chain is more flexible in BmimCl than in water. Taken together, the simulations explain how ionic liquids can facilitate cellulose dissolution: the synergistic action of anions and cations helps to initiate fiber deconstruction through specific interactions on the fiber surface and to solvate single cellulose chains through favorable solvent interactions and conformational flexibility

Untersuchung der Affinität und Funktionalität von Bispyridiniumverbindungen an nikotinischen Acetylcholinrezeptoren der Subtypen αβδγ (Torpedo californica) und α7 (Homo sapiens)

Bei der Behandlung von Vergiftungen mit phosphororganischen Verbindungen gibt es immer noch therapeutische Lücken. Insbesondere bei Vergiftungen mit Soman oder Tabun kann die gehemmte AChE nicht mehr adäquat reaktiviert werden. Die Hydrolyse des Neurotransmitters Acetylcholin wird nicht mehr katalysiert, so dass es zur Akkumulation dieses Agonisten im synaptischen Spalt und zur Desensitisierung der mAChR und nAChR kommt. Die muskarinisch innervierten Systeme können mit Atropin kompetitiv antagonisiert werden, aber für die direkte Intervention an den nikotinischen Effektorsystemen sind keine Wirkstoffe verfügbar. Bispyridiniumverbindungen zeigten in vitro eine Wiederherstellung der Muskelfunktion und in vivo eine Erhöhung der Überlebensrate, ohne dabei die AChE zu reaktivieren. Um aufzuklären, ob der Effekt Rezeptor vermittelt ist und was genau am Rezeptor bewirkt wird, wurden hierfür aussagekräftige Screeningmethoden entwickelt, etabliert und eingesetzt: - Radioligand-Rezeptor-Bindungsassay - Bilayer-basierte Elektrophysiologie - Muskelkraftuntersuchungen am Rattendiaphragma Für die Untersuchung der Affinität von Liganden an nAChR und mAChR wurden Ligand-Rezeptor-Bindungsassays auf Basis der Filtrationstechnik und Radioisotopen entwickelt. Hierfür wurden Plasmamembranpräparationen eingesetzt, die im Fall der humanen mAChR kommerziell erworben, bei humanen α7 nAChR und Muskeltyp-nAChR (Torpedo californica) selbst hergestellt wurden. Um zu erkennen, welchen Effekt die zu testenden Substanzen auf den nAChR ausüben, wurde eine Bilayer-basierte elektrophysiologische Methode entwickelt, die im Gegensatz zu der Patch Clamp-Technik nicht auf ganze Zellen angewiesen ist. Diese SSM-basierte Elektrophysiologie wurde so konzipiert, dass der desensitisierte, pathophysiologische nAChR gezielt herbeigeführt wurde und die Testsubstanzen auf ihre resensitisierende Wirkung, d.h. Eigenschaft als Typ II PAM untersucht wurden. Auch die Muskelkraftmessungen mittels indirekter elektrischer Feldstimulation stellen ein Modell dar, das die Gegebenheiten einer Nervenkampfstoffvergiftung wiedergeben soll. Bei Soman vergifteten Ratten-Zwerchfellhemisphären wurde der pharmakologische Effekt der drei tert-Butyl-Bispyridinium-Regioisomeren MB327, PTM0001 und PTM0002 untersucht. Es stellte sich heraus, dass es klare Korrelationen zwischen den Effekten auf Rezeptor- und Gewebeebene gibt. Die Kombination dieser drei verschiedenen Screeningmethoden brachte zu Tage, dass positiv allosterische Modulatoren des nAChR therapeutisch von Interesse sind. Hierbei werden nicht orthosterische, sondern allosterische Bindungsstellen adressiert. Insbesondere Typ II PAM, die in der Lage sind, den desensitisierten in einen funktionalen Rezeptorzustand zu überführen (sogenannte „Resensitizer“), könnten zur Therapie von Nervenkampfstoffen eingesetzt werden. Auch ohne Reaktivierung inhibierter AChE (z.B. bei einer Soman-Vergiftung) ist die Wiederherstellung der Muskelkraft möglich; der pharmakologische Effekt ist rezeptorvermittelt. Erste Struktur-Wirkungsbeziehung deuten darauf hin, dass die Alkylkette zwischen den beiden Heteroaromaten kurz, idealerweise ein Propyl-Linker sein soll. Auch das Substitutionsmuster wirkt sich auf die Aktivität aus, wenngleich in etwas geringerem Ausmaß. Da die Leitsubstanz MB327 und seine Strukturanaloga noch nicht für einen Medikament tauglichen Wirkstoff geeignet sind, muss noch nach weiteren, besseren Wirkstoffen gesucht werden. In einem iterativen Prozess aus virtuellen (Molecular Modeling), präparativen (zielgerichtete Synthesen) und pharmakologischen Methoden werden diese hier vorgestellten Screeningmethoden einen wichtigen Platz einnehmen.Treatment of nerve agent poisoning still comprises therapeutic gaps. Especially in the case of soman or tabun poisoning, the inhibited AChE cannot be reactivated adequately. The hydrolysis of the neurotransmitter acetylcholine is reduced, resulting in accumulation of this agonist in the synaptic cleft and desensitization of mAChRs and nAChRs. Musca-rinic based effector systems allow a competitive antagonism, but drugs for direct intervention at nicotinic acetylcholine receptors are missing. Bispyridinium compounds showed in vitro a recovery of muscle force and in vivo increased survival without reactivation of the AChE. However, it was unknown if and which effect was receptor-mediated. For enlightenment of the mechanism and search of more effective substances, screening methods were developed, established and used: - Radioligand-receptor-binding assay - Bilayer-based electrophysiology - Muscle force measurements using rat diaphragm hemispheres Ligand-receptor binding assays basing on filtration technique and radioisotopes allow the estimation of the affinity of chemical substances toward the mAChRs and nAChRs. The plasma membrane preparations used for this purpose were purchased (in the case of human mAChR) or prepared (in the case of human α7 nAChRs and muscle-type nAChRs (Torpedo californica)). For testing functional effects on the nAChRs, a bilayer-based electrophysiological method (so-called SSM-based electrophysiology) was developed. In contrast to patch clamp techniques, this method depends not on whole cells (“cell-free electrophysiology”). Using SSM-based electrophysiology, the desensitized, pathophysiological receptor state was intentionally induced and type II PAM properties (“resensitizing” effects) of the testing substances evaluated. Additionally, muscle force measurements using indirect electric field stimulation represent a model for OPC poisoning. The application of substances to soman-poisoned rat diaphragm hemispheres detects restoring effects without reactivation of AChE. Combining the results obtained from binding assays, functional receptor assays and diaphragm tests, clear correlations between receptor- and tissue-based screening methods were observed. The results obtained from these different screening methods showed that positive allosteric modulators are therapeutically of interest. Allosteric ligands address binding sites distinct from orthosteric binding sites. Especially type II PAM, which are able to recover the activity of desensitized nAChRs, may be beneficial in treatment of OPC poisoning. When nAChRs are resensitized, restoring of muscle force is possible – even without reactivation of inhibited AChE (e.g. soman poisoning). Initial structure-activity-relations (SAR) implicate that short alkyl spacers between the pyridinium moieties, ideally a propyl chain is advantageous for activity. Additionally, the substitution pattern of the pyridinium moieties influences the manner of activity, albeit to a lesser extent. The leading structure MB327 and its analogous structures lack drug like properties and are not suitable as antidotes. Consequently, more effective drugs have to be identified. As part of an iterative process of virtual (Molecular Modeling), preparative (target-based synthesis) and pharmacological methods, the described screenings methods will take an essential place

Engineering and functional characterisation of pentameric concatenated (alpha 4)2 (beta 2)3 and (alpha 4)3 (beta 2)2 nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that influence neurotransmitter release, hence constituting a key component of the physiological mechanisms of neuronal signalling. This thesis is concerned with the properties of the a4P2 nAChR, the most abundant nAChR in the brain, and the major contributor to the central effects of nicotine. The a4P2 nAChR is made up of five subunits, which in heterologous systems can assemble into at least two different stoichiometries: the high sensitivity (HS) (a4h(P2)3 stoichiometry and the low sensitivity (LS) (a4)3(p2)2 stoichiometry, which might both exist in native tissues. Despite the attractiveness of the a.4P2 nAChR as a target for therapeutic intervention, progress in the development of a4P2 nAChR-selective drugs has been slowed, partly because of the lack of stoichiometricspecific receptor models. This study presents a strategy to express homogenous populations of a4P2 nAChRs with fixed stoichiometry. By using standard molecular biological techniques, pentameric concatenated (a4)2(P2)3 and (a4)3(P2)2 nAChRs were engineered. These receptors were expressed in Xenopus laevis oocytes and functional studies showed that their functional properties resembled those of their non-linked counterparts. Subsequent site-directed mutagenesis in combination with functional analysis allowed the identification of the agonist-binding subunits in both concatamers. Concatenated receptors proved to be suitable for comparative studies of the effects of receptor mutation linked to autosomal dominant nocturnal frontal lobe epilepsy. Studies carried out on non-linked receptors, showed that the properties of the (a4)3(p2)2 stoichiometry were affected more markedly than those of the (a4)2(p2)3 stoichiometry. Insertion of the mutation in concatenated receptors revealed that the mutation not only affected the functional properties of a.4P2 nAChRs but also altered the subunit composition of the receptor. These studies show that pentameric concatenated constructs are a powerful tool to study the function and structure of receptors that assemble in multimeric types in expression systems

Investigation Of The Allosteric Modulators Desformylflustrabromine And 4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid (Hepes) Interactions On Nicotinic Acetylcholine Receptors

Thesis (Ph.D.) University of Alaska Fairbanks, 2011Neuronal nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop super family of ligand gated ion channels. Dysregulation of nAChRs can lead to pathologies such as Alzheimer's disease, Parkinson's disease, Autism and nicotine addiction. Possible new therapeutic avenues are positive allosteric modulators (PAMs). The natural product desformylflustrabromine (dFBr), a tryptophan metabolite of the marine bryozoan Flustra foliacea, was found to be PAM of alpha4beta2 nAChR. Evaluation of our synthetic water soluble dFBr salt by two-electrode voltage clamp of Xenopus laevis oocytes expressing human nAChR confirmed that synthetic dFBr displayed similar properties as the natural product. Low concentrations of the synthetic dFBr enhanced ACh's efficacy on alpha4beta2 receptors. At higher dFBr concentrations, dFBr inhibited ACh potentiated responses. On alpha7 receptors, dFBr inhibited ACh induced currents. Further pharmacological characterization of dFBr revealed that dFBr was able to enhance partial agonist potencies and efficacies. Evaluation of dFBr on antagonists showed no effect on antagonist inhibition. The mechanisms of biphasic modulation (potentiation and inhibition) of dFBr on alpha4beta2 nAChR were also investigated. Enhanced efficacy of ACh induced currents by dFBr appeared to be accomplished by dFBr stabilization of the open receptor conformation by destabilization of the desensitized state. The inhibition of ACh potentiated currents by dFBr appeared to involve open-channel block. To better understand dFBr mechanisms, its putative binding site was examined. Alanine mutations were made in non-orthosteric clefts on the beta2+ and alpha4- faces. Results revealed residues located on these faces are involved in ACh induced conformational change of the receptor. In addition our data supports our hypothesis that allosteric modulation by dFBr interacts with residues located on the beta2+ and alpha4- faces. The new novel actions of (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) as a alpha4beta2 stoichiometric PAM was discovered and characterized. We showed that HEPES, a common buffering agent, potentiated the high ACh sensitivity alpha4beta2 receptor while only inhibiting the low ACh sensitivity alpha4beta2 receptor. Mutagenesis results suggested that residue beta2D217 is a critical residue in the HEPES binding site. Results from these studies will aid in the development of therapeutic ligands that will assist in the treatment of diseases where nAChRs are dysregulated

Pharmacological characterisation of neuronal nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are the targets for the endogenous neurotransmitter acetylcholine and represent a diverse family of ligand-gated ion channels. They are expressed in the neuromuscular junction, the peripheral nervous system and the central nervous system. In the brain, the most prevalent subtypes are the heteromeric α4β2 and homomeric α7 nAChRs. Neuronal nAChRs are implicated in numerous physiological and pathophysiological functions and are therefore important targets for therapeutic drug discovery for conditions such as Alzheimer’s disease, schizophrenia and tobacco addiction. This thesis aims to further our understanding of the pharmacological and molecular characteristics of neuronal nAChRs. Acetylcholine activates nAChRs by binding at an extracellular orthosteric site. Previous studies have described several ligands that potentiate agonist-evoked responses by binding to an allosteric site of the α7 nAChRs that is distinct from the acetylcholine binding site. These ligands, termed positive allosteric modulators (PAMs) can be described as type I, when they have little or no effect on desensitisation, or type II, when they dramatically slow down the fast desensitisation kinetics of the α7 nAChR subtype. Here, a novel series of α7-selective PAMs with a range of effects on receptor desensitisation is described, using recombinant human receptors. This series consists of PAMs with type I and type II profiles, in addition to PAMs with intermediate properties on desensitisation, therefore increasing the nAChR pharmacological toolbox. Furthermore, the effect of a number of mutations on the pharmacological properties of the receptor is investigated. Three point mutations, two in the transmembrane domain (L247T and M260L) and one in the N-terminal domain (W54A), are shown to have the ability to convert PAMs into agonists. Moreover, the M260L mutation displays this property only with PAMs that have a significant effect on receptor desensitisation. These observations can provide insights into the role of these residues on receptor gating and desensitisation. In addition to the studies on recombinant receptors, the expression and functional properties of nAChRs in neurons derived from human induced pluripotent stem cells (iPSC) is examined. The iPSC-derived neurons represent a potentially valuable tool for the characterisation of neuronal receptors and ion channels in a native environment

Understanding the working mechanism of natural and engineered proteins for cell regulation and biotechnological applications: a molecular dynamics study

This thesis reports on an in-silico investigation of a few proteins whose function takes part in cell regulation. In particular, this work focuses on selected proteins that carry out their tasks by modifying either the concentration or the nature of inorganic ions within the cell. Among them, there are systems so versatile to even allow, upon proper biotechnological modifications, the creation of new macromolecular entities exploitable in contexts not strictly biological. According to the growing interest towards these two classes of systems alike, both natural proteins and engineered protein-based nanodevices have been considered within this work

Control of cation permeation through the nicotinic receptor channel.

We used molecular dynamics (MD) simulations to explore the transport of single cations through the channel of the muscle nicotinic acetylcholine receptor (nAChR). Four MD simulations of 16 ns were performed at physiological and hyperpolarized membrane potentials, with and without restraints of the structure, but all without bound agonist. With the structure unrestrained and a potential of -100 mV, one cation traversed the channel during a transient period of channel hydration; at -200 mV, the channel was continuously hydrated and two cations traversed the channel. With the structure restrained, however, cations did not traverse the channel at either membrane potential, even though the channel was continuously hydrated. The overall results show that cation selective transport through the nAChR channel is governed by electrostatic interactions to achieve charge selectivity, but ion translocation relies on channel hydration, facilitated by a trans-membrane field, coupled with dynamic fluctuations of the channel structure