Welcome to FICN2021

The Palatine Hill. From the King to the Emperor at the origin of Rome.

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We have been investigating the Palatine, aiming at recovering physical lay-out, extent and environment of disappeared ancient landscapes and moving towards historical interpretation and narrative. The series of changing in monuments and places testified by material remains, connected or re-connected to other information, reveal the history of Rome from the foundation of the City to the creation of the Empire as a “tale of the landscape”.

Scientific Introduction

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Opening Lecture: Overcoming the timescale problem: Merging molecular simulation with machine learning

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Molecular simulations of complex systems like ion channels or nanopores are a computational challenge, especially regarding the vastly differing timescales between the fastest and slowest processes under consideration. Moreover, the timescale of the simulations themselves is, in practice, orders of magnitudes smaller than that of the mechanisms of interest, which further adds to the complexity of observing these mechanisms, and of drawing meaningful and significant biological insights from the simulation. We will review recent progress regarding this challenging timescale problem across several application contexts and will discuss in particular how emerging machine learning methods may help to overcome it.

Molecular Dynamics Simulations of a Tight-Junction Paracellular Channel

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Claudins are transmembrane proteins found in tight-junction complexes between adjacent cells of biological barriers, where they regulate transport through the paracellular space. A structural model of a claudin channel has been proposed recently, boosting detailed studies of paracellular transport. Using Molecular Dynamics simulations and free energy calculations, we have refined the model and investigated its structural properties, verifying the experimentally known selectivity for cations.

Voltage sensors and channel opening

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Action potential depends on voltage-dependent Na channels opening before voltage-dependent K channels. Voltage dependence is conferred by charge moving in the electric field generating gating currents. Voltage clamp with site-directed fluorescence describes molecular details of the voltage sensor operation: paths followed by the arginine residues within the protein core. Core nature determines Na channels faster than K channels. Two different pathways couple voltage sensor to channel operation.

Ball-and-chain inactivation in potassium channels

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We used cryo-EM and MD simulations to determine the molecular gating mechanism in Ca2+-activated K+ channels. Channels are closed without Ca2+, while Ca2+-bound conditions display open channels where the N-terminus of one subunit plugs the pore, a strong interaction as shown by simulations. Deletion of this N-terminus leads to non-inactivating channels and structures of open states without pore-plug, indicating that the N-terminal peptide is responsible for ball-and-chain inactivation.

A permeation rate model inspired by molecular dynamics simulations of the KcsA channel

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Molecular dynamics provides key information on ion channel permeation, including energy diagrams and trajectories of moving ions. To gain insight into the basic mechanism of ion permeation, it would be useful to connect this output to rate models of ion permeation, that provide a simple view of the process and can be used to test the prediction of available experimental data. In this talk such as multi-scale approach is proposed to understand K permeation through KcsA channels.

Water and Ions in Membrane Nanopores and Channels: Insights from Molecular Simulations

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Ion channels are proteins which form gated nanopores in biological membranes. Many channels exhibit hydrophobic gating, whereby functional closure of a pore occurs by local de-wetting. Molecular dynamics simulations can be used to explore the dynamic behaviour of water within nanopores and biological channels. Simulation studies of the behaviour of water in idealised models of nanopores have revealed aspects of the organization and dynamics of nano-confined water, including wetting/de-wetting in narrow hydrophobic nanopores. The pentameric ligand gated ion channels (pLGICs) provide a biologically important example of hydrophobic gating. Molecular simulation studies comparing additive vs. polarisable models indicate predictions of hydrophobic gating are robust to the model employed. However, polarisable models suggest favourable interactions of hydrophobic pore-lining regions with chloride ions, of relevance to both synthetic carriers and to channel proteins. Electrowetting of a closed pLGIC hydrophobic gate requires too high a voltage to occur physiologically, but may inform designs of for switchable nanopores. Global analysis of ~200 channels yields a simple heuristic for structure-based prediction of (closed) hydrophobic gates. An overall picture emerges whereby the behaviour of water in a nanopore or channel may be predicted as a function of its hydrophobicity and radius. Simulation-based analysis is shown to provide an aid to interpretation of functional states of new channel structures.

Molecular dynamics simulations of membrane transport

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Nature has evolved elaborate machineries for the transport of protons, ions and molecules across lipid bilayers. Molecular dynamics simulations open up a window into their inner workings. I will present results of molecular dynamics simulations of proteins involved in a variety of membrane transport processes, from the exchange of ions to lipid flipping and the extrusion of misfolded proteins. Key questions are how these machines achieve substrate specificity and transport coupling.

Competition among electrophoresis, electroosmosis, and dielectrophoresis in particle capture into nanopores.

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The interaction between nanoparticles dispersed in a fluid and nanopores is governed by the interplay of hydrodynamical, electrical, and chemical effects. We will present a theory for particle capture in nanopores providing analytical expressions for the capture rate under the concurrent action of electrical forces, fluid advection, and Brownian motion. Our approach naturally splits the average capture time in two terms, an "approaching time" due to the migration of particles from the bulk to the pore mouth, and an "entrance time" associated with a free-energy barrier at the pore entrance. Within this theoretical framework, we describe the standard experimental condition where a particle concentration is driven into the pore by an applied voltage, with specific focus on different capture mechanisms: under electrophoresis, in the presence of a competition between electrophoresis and electroosmosis, and finally under dielectrophoretic reorientation of dipolar particles. Our theory predicts that dielectrophoresis is able to induce capture for both positive and negative voltages. We performed a dedicated experiment involving a biological nanopore and a rigid dipolar molecule that confirms the theoretically proposed capture mechanism.

Mechanical understanding and reverse engineering of ion channels

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With the current advances in science and technology, we can understand ion channels' working mechanisms and engineer new functions on them. In this talk, I will present our molecular tools to study and manipulate ion channels in detergent, liposomes, cells, and vacuum environment, findings on the working mechanism of a mechanosensitive ion channel, and understanding the malfunctioning of a human ion channel, involved in both spinocerebellar ataxia and sudden cardiac arrest.

Single Particle Characterization of Protein Amyloid Oligomers

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This talk will highlight recent advances from our group in the context of characterizing single amyloid oligomer particles in solid state nanopores. Specifically, by translocating individual amyloid oligomers through a cylindrical pore with a diameter and length that are at least twice that of longest dimension of the oligomers, this approach makes it possible to determine the volume of the oligomer, the number of amyloid monomer molecules in the oligomer, and the approximate shape of the oligomer. We demonstrate this approach with various amyloid proteins including Alzheimer’s disease-related amyloid-beta and tau protein as well as with Parkinson’s disease-related alpha-synuclein protein. Since the size and shape of amyloid oligomers determine their neurotoxicity, we are currently developing methods to apply this approach to samples of biofluids such as cerebrospinal fluid and blood serum. We hope that single particle characterization and quantification of amyloid oligomers in biofluids will provide useful biomarkers for early disease diagnosis, disease prognosis, correlation with disease progression and therapeutic monitoring of disease.

Inducing electroosmotic flow in uncharged nanopores via confinement and/or induced charge

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Ionic selectivity in nanopores is usually described as a consequence of surface charges at the pore wall. In this talk, two purely geometrical mechanisms to induce ionic selectivity and electroosmotic flow in uncharged nanopores are discussed. The first relies on confinement. The second exploits the charge accumulation in a groove that decorates the membrane. In this case, the selectivity results to be inverted when reverting the voltage leading to a rectification of the electroosmotic flow.

Machine Learning and Computational Chemistry.

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The recent success of machine learning (ML) suggests that neural networks may be capable of approximating high-dimensional functions with controllably small errors. As a result, they could outperform standard function interpolation methods that have been the workhorses of scientific computing but do not scale well with dimension. In this talk, I will discuss the prospects that ML offers in the context of the numerical solution of the numerical solution of partial differential equations in high dimension, a problem of interest in computational chemistry (think of the Schroedinger or the Fokker Planck equations) that was once thought intractable. I will focus on problems with a variational formulation, and discuss three main ingredients of their solution by ML: (i) approximation quality, i.e. how accurate the representation of the solution by a neural network can in principle be; (ii) optimization, i.e. how effective are the methods to train the parameter of the network; and (iii) generalization error, i.e. how much data is necessary to obtain a solution accurate also outside this data set. In particular I will show that this third aspect typically requires using importance sampling methods for data acquisition. These results will be illustrated on eigenvalue problems in high dimension. This is joint work with Grant Rotskoff.

Modeling drying of nanoconfined liquids

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I will present recent work using simulation and theory to investigate drying in nanoconfinement. I will first discuss water in hydrophobic confinement, with a focus on density fluctuations. I will then discuss extensions to concentrated electrolytes, which exhibit structural changes and non-trivial concentration dependence. I will end with a discussion of a physically intuitive and computationally useful theoretical approach for modeling hydrophobic assembly of large and small solutes.

Gas-Induced Drying of Nanopores

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We investigated the role of a dilute dissolved gas in the microscopic intrusion and extrusion of water inside cylindrical hydrophobic pores with diameter 14 Å. By means of restrained molecular dynamics simulations, we obtained the two dimensional free-energy landscape as a function of the number of water molecules inside the nanopore and the penetration of an argon atom inside the nanopore. Different transition paths connecting the pure wetting state of the pore to dry state of the pore only containing the argon atom were investigated. This study shows that it is possible to decrease and, for the highest hydrophobicity, suppress the drying free energy barrier of the nanopore, thus achieving bubble formation with a single argon atom entering the pore. The discovered physical mechanism may provide a general explanation for the action of volatile anesthetics on some class of ion channels by drying of the pore domain – hydrophobic gating – induced by the hydrophobic gas. Important implications for technological applications, such as nanopore sensing and energy-storage and release nano-devices, also derive from this study since the presence of hydrophobic gases in the liquid can significantly affect the wetting state of these devices.

The physical chemistry of K+ channel gates

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Ion channels fluctuate in a stochastic manner between open and closed states. We attempt to understand the physicochemical basis of this so-called channel gating by analyzing structure function/correlates in very small K+ channels from viral origin. From the diversity of single channel gating between natural channel variants we find that simple chemical interactions in a protein are sufficient for distinct gating phenomena.

MscS is a smart bacterial osmolyte release valve

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Ion permeation in wild-type and mutated human α7 nicotinic receptor: a thermodynamics and kinetics study via advanced sampling molecular dynamics

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Physical mechanisms of selective ion transport in biological channels and nanopores

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The ability to discriminate between ions is essential to the function of many biological channels; and similar principles can be used to design selective synthetic pores. Many factors have been suggested to dictate ion selective transport, each of which can be important in different situations. In this talk I will go back to basics, examining the simple principles that can be used to discriminate between ions, giving examples where each factor may be important.

Molecular basis of modulation of voltage-gated ion channels

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Voltage gated ion channels are involved in propagating action potentials, and, as such, are the target of anti hyperexcitability drugs. Changes in the membrane potential lead the domain responsible for voltage-sensing to reorganize, and the signal therefrom is propagated to the pore domain in an allosteric fashion, regulating the ion permeation. Drugs, lipids, and auxiliary proteins can modulate this electromechanical signal propagation. In this talk we describe how we use molecular dynamics simulations to inform electrophysiology recordings of mutant channels, to understand this phenomenon and its regulation by lipid-like drugs and the protein calmodulin in the Kv1 Shaker and the KCNQ (also called Kv7) subfamilies.

Characterizing gating in ion channels through MD simulations: hERG and CRAC channels

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Gating is the property of ion channels to open and close in response to specific stimuli. Here gating was analyzed in two channels: hERG and CRAC. In CRAC channel our simulations highlighted a hydrophobic gating mechanism whereby the formation of a vapour bubble occludes the pore in the absence of steric block. In hERG channel Molecular Dynamics simulations combined with a network analysis revealed for the first time the elusive gating mechanism of non domain swapped K+ channels.

Liquid intrusion/extrusion from metal-organic frameworks for technological applications

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Metal-organic frameworks (MOFs) are organic-inorganic hybrid crystalline porous materials. The tunability of their porous structure via the modification of organic linkers and metal nodes makes MOFs suitable for many technological applications via gas and liquid intrusion/extrusion. In this talk I will present our recent results on water intrusion in ZIF8, the zeolitic imidazolate framework whose peculiar characteristics allow to violate consolidated intrusion/extrusion theories.

A DFT study of bubble gating

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Classical density functional theory (DFT) allows one to study the structure and thermodynamics of strongly confined fluids such as water inside of a gate of an ion channel. If the hydrophobic gate is sufficiently narrow a vapor bubble can form, by capillary evaporation, and close the gate. In this talk DFT is employed to study the formation of bubbles in the gate and to discuss some of its consequences.

Transport of electrolyte across varying section channels

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We characterize the dynamics of a z − z electrolyte embedded in a varying-section channel. In the linear response regime, by means of suitable approximations, we derive the Onsager matrix associated with externally enforced gradients in electrostatic potential, chemical potential, and pressure, for both dielectric and conducting channel walls. We show here that the linear transport coefficients are particularly sensitive to the geometry and the conductive properties of the channel walls when the Debye length is comparable to the channel width. In this regime, we found that one pair of off-diagonal Onsager matrix elements increases with the corrugation of the channel transport, in contrast to all other elements which are either unaffected by or decrease with increasing corrugation. Our results have a possible impact on the design of blue-energy devices as well as on the understanding of biological ion channels.

Posters - flash presentations

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Allosteric Modulation of Membrane Proteins by Small Ligands: Applications to Ion Channels and General Anesthetics

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Understanding how lipophilic small ligands impact membrane proteins requires knowledge on the molecular structure of ligand binding, a reasoning that has driven relentless efforts in drug discovery and translational research. Looking for new developments in the field, we will present and discuss a statistical mechanical formulation of the equilibrium properties of ligand binding to membrane proteins, clarifying the impact of multiple binding events on the energetic of two-state membrane proteins intrinsically driven by a variety of thermodynamic conjugates. The theory in combination with docking or flooding MD may be of special interest by establishing a microscopic framework to be linked with macroscopic measurements. Illustration of the approach will be presented in the context of anesthetic binding to ion channels, highlighting how such calculations in combination with measurements have been applied to investigate anesthetic action.

How mitochondrial uncouplers induce proton leak

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I will discuss on going work in collaboration with Dr. Yuriy Kirchok’s lab attempting to understand the mechanism of action of common mitochondrial uncouplers. Using both simulation and electrophysiological recordings directly from mitochondria, we show that fatty acids and well-known chemical uncouplers primarily induce proton leak across the inner-mitochondrial membrane via membrane proteins – not by shuttling directly across the membrane. Implications for energy metabolism will be discussed.

From sequence to function: design principles of ion channels and other molecular machines

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Ion channels are nature’s nanodevices that control transmembrane potential and propagate electrical signals. Natural selection has led to the occurrence of different types of ion channels, each activated by a different environmental stimulus, each associated to a different thermodynamic generalized force such as temperature, external electric fields and surface tension. I will discuss our recent efforts to connect this macroscopic behavior to the microscopic interactions responsible for it, using molecular simulations and data-science.

Best Poster Prizes

Diffusive Transport Phenomena through constricted channels & pores

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In this talk I’ll review our effort to combine molecular simulations and electrophysiology with a multi-scale approach for quantifying the passive transport of ions/molecules through channels and pores. Recently we explored a family of cationic intracellular channels, the two-pore channels. In humans they are involved in the exocytosis and have been reported as key for the entry of diverse viruses, thus of primary interest for the discovery of inhibitors to combat the actual COVID-19 pandemy.

{Heat + work}-to-electricity conversion via reversible water intrusion into hydrophobic nanopore

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Reversible water intrusion into highly hydrophobic nanoporous Metal−Organic Frameworks and grafted silica is explored via high-pressure calorimetry, dielectric spectroscopy and in operando small-angle neutron scattering. It is shown that triboelectrification during the intrusion-extrusion process can be used to efficiently convert ambient heat and undesired vibrations to electricity. This allows a new type of regenerative shock-absorbers, extending the autonomy of electric vehicles.

Towards Predicting And Controlling Ionic Hydration Patterns In Nanopores

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We propose a method to describe analytically the ionic hydration patterns near sub-nanopores. It agrees well with molecular dynamics simulations, predicts the locations of the trapped water molecules, and captures the intrinsic and extrinsic features of the nanopores. Our method would open the way to designing and optimising controllable nanoionic devices with on-demand selective and conductive properties, finding applications in water desalination, energy harvesting, and DNA sequencing.

Correlative 3D microscopy of single cells using Super-Resolution and Scanning Ion-Conductance Microscopy

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Expérimental investigation of fluid and ion transport in sub-nanometer channels.

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New paradigms for fluid transport are expected to emerge from the confinement of liquids at the nanoscales with potential breakthroughs in ultra-filtration, desalination, and energy conversion. Nevertheless, advancing the fundamental understanding of fluid transport at the smallest scales requires mass and ion dynamics to be ultimately characterized across an individual channel so to avoid averaging over many pores. A major challenge for nanofluidics consists in building distinctive and well-controlled nano-channels, amenable for systematic exploration of their properties. Carbon materials offer an unprecedented opportunity to study flow and ion transport in controlled molecular size channel: thanks to the développement of nanotube and bidimensional material we have been able to investigate the subtle coupling between mass and charge transport at the sub-nanometer scale.

Ions and water transport through µm-long single-walled carbon nanotubes

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Ionic currents measured on nanofluidic devices containing one or several long SWCNTs with diameters between 1.2 to 2 nm will be presented and compared to data already reported on similar systems. Theoretical analysis and all-atom computational simulation will be proposed to explain the experimental data with a focus on surface charge and capacitance, friction and local energy barriers effects.

Engineering ion channels for long term inhibition of cell excitability

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Remote control of potassium channel constitutes a promising way to control firing in excitable cells. In the past, we have engineered a light-gated channel, BLINK2 and demonstrated its potential application in the control of neuropathic pain. More recently, we have exploited other ways to remotely control channels, such as peptide drugs which modulate channel activity under the control of light. Presently, we are investigating the possibility to control K channels by other stimuli such as temperature and magnetic fields.

The molecular anatomy of store-operated Orai channel gating

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Store-operated calcium channels (SOCs) are a major pathway for calcium signaling in virtually all animal cells and serve a wide variety of functions ranging from gene expression, motility, secretion, tissue and organ development, and the immune response. SOCs are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER), triggered physiologically through stimulation of a diverse set of metabotropic surface receptors. The identification of the STIM proteins as ER Ca2+ sensors and the Orai proteins as store-operated channels 10 years ago has enabled rapid progress in understanding the unique and unusual mechanism of SOCE. Depletion of Ca2+ from the ER causes STIM to accumulate at ER-plasma membrane junctions where it traps and activates Orai1 channels diffusing in the closely apposed plasma membrane. The ensuing entry of Ca2+ mediates refills ER Ca2+ stores, and mediated a variety of functions including immune cell activation, muscle function, bone and tooth development, and many others. In this talk, I will discuss our recent work on the molecular and structural basis of SOC gating, focusing on a hydrophobic gating mechanism that leads to opening of the pore following STIM1 binding to, and the role of novel structural motifs in Orai1 that stabilize the channel gate in the open configuration.

Fractional noise in nanopores

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Fluctuations are ubiquitous in bio and artificial nanopores. Their dramatic consequences on transport are subtle and highly intricate Yet bio and artificial pores have to optimize signal to noise to achieve complex tasks. Here we show that even in the simplest nanoporous setting the osmotic pressure exhibits non-trivial fractional noise in time (i.e. grows sublinearly with time). We will rationalize this effect and investigate its consequences for nanoporous transport.

Final Lecture: Unraveling the mechanism of C-type inactivation in potassium channels

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Activation of a K+ channel typically leads to a transient period of ion conduction until the selectivity filter spontaneously undergoes a conformational change toward a constricted non-conductive state (C-type inactivation). Subsequent removal of the stimulus closes the gate and allows the selectivity filter to return back to its conductive conformation (recovery). The recovery process can take up to several seconds, an extraordinarily long time. Yet, the structural differences between the conductive and inactivated filter are very small. Molecular dynamics simulation of large biological macromolecules and free energy computations are used to to explain the origin of the slow recovery process provide meaningful insight on the molecular determinants of inactivation.

Round Table

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