Computational analyses on the structure-function relation in ion channels

Ion channels are protein molecules, embedded in the lipid bilayer of the cell membranes. They act as powerful sensing elements switching chemicalphysical stimuli into ion-fluxes. At a glance, ion channels are water-filled pores, which can open and close in response to different stimuli (gating), and one once open select the permeating ion species (selectivity). They play a crucial role in several physiological functions, like nerve transmission, muscular contraction, and secretion. Besides, ion channels can be used in technological applications for different purpose (sensing of organic molecules, DNA sequencing). As a result, there is remarkable interest in understanding the molecular determinants of the channel functioning. Nowadays, both the functional and the structural characteristics of ion channels can be experimentally solved. The purpose of this thesis was to investigate the structure-function relation in ion channels, by computational techniques. Most of the analyses focused on the mechanisms of ion conduction, and the numerical methodologies to compute the channel conductance. The standard techniques for atomistic simulation of complex molecular systems (Molecular Dynamics) cannot be routinely used to calculate ion fluxes in membrane channels, because of the high computational resources needed. The main step forward of the PhD research activity was the development of a computational algorithm for the calculation of ion fluxes in protein channels. The algorithm - based on the electrodiffusion theory - is computational inexpensive, and was used for an extensive analysis on the molecular determinants of the channel conductance. The first record of ion-fluxes through a single protein channel dates back to 1976, and since then measuring the single channel conductance has become a standard experimental procedure. Chapter 1 introduces ion channels, and the experimental techniques used to measure the channel currents. The abundance of functional data (channel currents) does not match with an equal abundance of structural data. The bacterial potassium channel KcsA was the first selective ion channels to be experimentally solved (1998), and after KcsA the structures of four different potassium channels were revealed. These experimental data inspired a new era in ion channel modeling. Once the atomic structures of channels are known, it is possible to define mathematical models based on physical descriptions of the molecular systems. These physically based models can provide an atomic description of ion channel functioning, and predict the effect of structural changes. Chapter 2 introduces the computation methods used throughout the thesis to model ion channels functioning at the atomic level. In Chapter 3 and Chapter 4 the ion conduction through potassium channels is analyzed, by an approach based on the Poisson-Nernst-Planck electrodiffusion theory. In the electrodiffusion theory ion conduction is modeled by the drift-diffusion equations, thus describing the ion distributions by continuum functions. The numerical solver of the Poisson- Nernst-Planck equations was tested in the KcsA potassium channel (Chapter 3), and then used to analyze how the atomic structure of the intracellular vestibule of potassium channels affects the conductance (Chapter 4). As a major result, a correlation between the channel conductance and the potassium concentration in the intracellular vestibule emerged. The atomic structure of the channel modulates the potassium concentration in the vestibule, thus its conductance. This mechanism explains the phenotype of the BK potassium channels, a sub-family of potassium channels with high single channel conductance. The functional role of the intracellular vestibule is also the subject of Chapter 5, where the affinity of the potassium channels hEag1 (involved in tumour-cell proliferation) and hErg (important in the cardiac cycle) for several pharmaceutical drugs was compared. Both experimental measurements and molecular modeling were used in order to identify differences in the blocking mechanism of the two channels, which could be exploited in the synthesis of selective blockers. The experimental data pointed out the different role of residue mutations in the blockage of hEag1 and hErg, and the molecular modeling provided a possible explanation based on different binding sites in the intracellular vestibule. Modeling ion channels at the molecular levels relates the functioning of a channel to its atomic structure (Chapters 3-5), and can also be useful to predict the structure of ion channels (Chapter 6-7). In Chapter 6 the structure of the KcsA potassium channel depleted from potassium ions is analyzed by molecular dynamics simulations. Recently, a surprisingly high osmotic permeability of the KcsA channel was experimentally measured. All the available crystallographic structure of KcsA refers to a channel occupied by potassium ions. To conduct water molecules potassium ions must be expelled from KcsA. The structure of the potassium-depleted KcsA channel and the mechanism of water permeation are still unknown, and have been investigated by numerical simulations. Molecular dynamics of KcsA identified a possible atomic structure of the potassium-depleted KcsA channel, and a mechanism for water permeation. The depletion from potassium ions is an extreme situation for potassium channels, unlikely in physiological conditions. However, the simulation of such an extreme condition could help to identify the structural conformations, so the functional states, accessible to potassium ion channels. The last chapter of the thesis deals with the atomic structure of the !- Hemolysin channel. !-Hemolysin is the major determinant of the Staphylococcus Aureus toxicity, and is also the prototype channel for a possible usage in technological applications. The atomic structure of !- Hemolysin was revealed by X-Ray crystallography, but several experimental evidences suggest the presence of an alternative atomic structure. This alternative structure was predicted, combining experimental measurements of single channel currents and numerical simulations. This thesis is organized in two parts, in the first part an overview on ion channels and on the numerical methods adopted throughout the thesis is provided, while the second part describes the research projects tackled in the course of the PhD programme. The aim of the research activity was to relate the functional characteristics of ion channels to their atomic structure. In presenting the different research projects, the role of numerical simulations to analyze the structure-function relation in ion channels is highlighted.

Analysis of ion channel stochastic signals for biosensing

In biological world, life of cells is guaranteed by their ability to sense and to respond to a large variety of internal and external stimuli. In particular, excitable cells, like muscle or nerve cells, produce quick depolarizations in response to electrical, mechanical or chemical stimuli: this means that they can change their internal potential through a quick exchange of ions between cytoplasm and the external environment. This can be done thanks to the presence of ion channels, proteins that span the lipid bilayer and act like switches, allowing ionic current to flow opening and shutting in a stochastic way. For a particular class of ion channels, ligand-gated ion channels, the gating processes is strongly influenced by binding between receptive sites located on the channel surface and specific target molecules. These channels, inserted in biomimetic membranes and in presence of a proper electronic system for acquiring and elaborating the electrical signal, could give us the possibility of detecting and quantifying concentrations of specific molecules in complex mixtures from ionic currents across the membrane; in this thesis work, this possibility is investigated. In particular, it reports a description of experiments focused on the creation and the characterization of artificial lipid membranes, the reconstitution of ion channels and the analysis of their electrical and statistical properties. Moreover, after a chapter about the basis of the modelling of the kinetic behaviour of ligand gated ion channels, a possible approach for the estimation of the target molecule concentration, based on a statistical analysis of the ion channel open probability, is proposed. The fifth chapter contains a description of the kinetic characterisation of a ligand gated ion channel: the homomeric α2 isoform of the glycine receptor. It involved both experimental acquisitions and signal analysis. The last chapter represents the conclusions of this thesis, with some remark on the effective performance that may be achieved using ligand gated ion channels as sensing elements.

Studio elettrofisiologico di modificazioni a lungo termine della forza della trasmissione sinaptica nel sistema nervoso centrale

Il nucleo accumbens (NAc), il maggior componente del sistema mesocorticolimbico, è coinvolto nella mediazione delle proprietà di rinforzo e nella dipendenza da diverse sostanze d’abuso. Le sinapsi glutammatergiche del NAc possono esprimere plasticità, tra cui una forma di depressione a lungo termine (LTD) dipendente dagli endocannabinoidi (eCB). Recenti studi hanno dimostrato un’interazione tra le vie di segnalazione del sistema eCB e quelle di altri sistemi recettoriali, compreso quello serotoninergico (5-HT); la vasta colocalizzazione di recettori serotoninergici e CB1 nel NAc suggerisce la possibilità di un’interazione tra questi due sistemi. In questo studio abbiamo riscontrato che una stimolazione a 4 Hz per 20 minuti (LFS-4Hz) delle afferenze glutammatergiche in fettine cerebrali di ratto, induce una nuova forma di eCB-LTD nel core del NAc, che richiede l’attivazione dei recettori CB1 e 5-HT2 e l’apertura dei canali del Ca2+ voltaggio-dipendenti di tipo L. Inoltre abbiamo valutato che l’applicazione esogena di 5-HT (5 M, 20 min) induce una LTD analoga (5-HT-LTD) a livello delle stesse sinapsi, che richiede l’attivazione dei medesimi recettori e l’apertura degli stessi canali del Ca2+; LFS-4Hz-LTD e 5-HT-LTD sono reciprocamente saturanti. Questi risultati suggeriscono che la LFS-4Hz induce il rilascio di 5-HT, che si lega ai recettori 5-HT2 a livello postsinaptico incrementando l’influsso di Ca2+ attraverso i canali voltaggio-dipendenti di tipo L e la produzione e il rilascio di 2-arachidonoilglicerolo; l’eCB viaggia a ritroso e si lega al recettore CB1 a livello presinaptico, causando una diminuzione duratura del rilascio di glutammato, che risulta in una LTD. Queste osservazioni possono essere utili per comprendere i meccanismi neurofisiologici che sono alla base della dipendenza da sostanze d’abuso, della depressione maggiore e di altre malattie psichiatriche caratterizzate dalla disfunzione della neurotrasmissione di 5-HT nel NAc.

Contribution of rare damaging variants in Autism Spectrum Disorder

Autism Spectrum Disorder (ASD) is a heterogeneous and highly heritable neurodevelopmental disorder with a complex genetic architecture, consisting of a combination of common low-risk and more penetrant rare variants. This PhD project aimed to explore the contribution of rare variants in ASD susceptibility through NGS approaches in a cohort of 106 ASD families including 125 ASD individuals. Firstly, I explored the contribution of inherited rare variants towards the ASD phenotype in a girl with a maternally inherited pathogenic NRXN1 deletion. Whole exome sequencing of the trio family identified an increased burden of deleterious variants in the proband that could modulate the CNV penetrance and determine the disease development. In the second part of the project, I investigated the role of rare variants emerging from whole genome sequencing in ASD aetiology. To properly manage and analyse sequencing data, a robust and efficient variant filtering and prioritization pipeline was developed, and by its application a stringent set of rare recessive-acting and ultra-rare variants was obtained. As a first follow-up, I performed a preliminary analysis on de novo variants, identifying the most likely deleterious variants and highlighting candidate genes for further analyses. In the third part of the project, considering the well-established involvement of calcium signalling in the molecular bases of ASD, I investigated the role of rare variants in voltage-gated calcium channels genes, that mainly regulate intracellular calcium concentration, and whose alterations have been correlated with enhanced ASD risk. Specifically, I functionally tested the effect of rare damaging variants identified in CACNA1H, showing that CACNA1H variation may be involved in ASD development by additively combining with other high risk variants. This project highlights the challenges in the analysis and interpretation of variants from NGS analysis in ASD, and underlines the importance of a comprehensive assessment of the genomic landscape of ASD individuals.

Elf magnetic field influence on ION Channels studied by Patch Clamp Technique: exposure set up and "Whole Cell" measurements on Potassium currents

The aim of this thesis was to study the effects of extremely low frequency (ELF) electromagnetic magnetic fields on potassium currents in neural cell lines ( Neuroblastoma SK-N-BE ), using the whole-cell Patch Clamp technique. Such technique is a sophisticated tool capable to investigate the electrophysiological activity at a single cell, and even at single channel level. The total potassium ion currents through the cell membrane was measured while exposing the cells to a combination of static (DC) and alternate (AC) magnetic fields according to the prediction of the so-called ‘ Ion Resonance Hypothesis ’. For this purpose we have designed and fabricated a magnetic field exposure system reaching a good compromise between magnetic field homogeneity and accessibility to the biological sample under the microscope. The magnetic field exposure system consists of three large orthogonal pairs of square coils surrounding the patch clamp set up and connected to the signal generation unit, able to generate different combinations of static and/or alternate magnetic fields. Such system was characterized in term of field distribution and uniformity through computation and direct field measurements. No statistically significant changes in the potassium ion currents through cell membrane were reveled when the cells were exposed to AC/DC magnetic field combination according to the afore mentioned ‘Ion Resonance Hypothesis’.