Ion Channel Electrophysiology Research Articles

Solute translocation probability, lifetime, and "rectification" in membrane channels with localized constriction.

We study the translocation probability and lifetime of a solute molecule in a cylindrical membrane channel that contains a localized constriction at an arbitrary location. Using a one-dimensional continuous diffusion description of solute dynamics in the channel, we explore two models. The first one describes a molecule's interaction with the constriction in terms of a narrow rectangular barrier in the potential of mean force. The second novel model proposed here represents this interaction by introducing an infinitely thin permeable partition. It is shown that when the parameters of the two models are chosen to warrant the same translocation probability, both models predict the same mean lifetime of the molecule in the channel. While the translocation probability is independent of the constriction location, the mean lifetime is a function of the location. The benefit of the thin partition model is that it allows one to lump together the height and length of the potential barrier into a single parameter, which is the partition's permeability. It is shown that in the case of an asymmetric location of the localized constriction and strong repulsion between the solutes, the solute flux through the channel is a function of the direction in which it goes, analogous to the phenomenon known in ion channel electrophysiology as rectification.

Unexpected neurotoxicity in chronic toxicity studies with a HBV viral expression inhibitor

The non-clinical safety profile of the small molecule hepatitis B virus viral expression inhibitor RG7834 was studied in a package consisting of safety pharmacology, genotoxicity, repeat dose toxicity and reproductive toxicity studies. The chronic monkey toxicity study identified dose- and time-dependent symptoms of polyneuropathy, with correlating nerve conduction velocity reductions and axonal degeneration in peripheral nerves and spinal cord, in all compound treatment groups with no evidence of reversibility after approximately 3 months of treatment cessation. Similar histopathological findings were observed in the chronic rat toxicity study. Subsequent in vitro neurotoxicity investigations and ion channel electrophysiology did not elucidate a potential mechanism for the late toxicity. However, based on similar findings observed with a structurally different molecule, an inhibition of their common pharmacological targets, PAPD5 & PAPD 7, was considered as a possible mechanism of toxicity. In conclusion, the marked neuropathies, only observed after chronic dosing, did not support further clinical development of RG7834 because of its foreseen clinical treatment duration of up to 48 weeks in chronic HBV patients.

Probing actin coated planar lipid bilayers with light and electricity in a microelectrode cavity array.

Planar lipid bilayers play a central role in nanopore sensing and ion-channel electrophysiology. However, bilayer fragility, especially in the presence of osmotic stress, can limit their application. We have been developing methods to enhance the mechanical properties of planar lipid bilayers with a layered, molecularly thin, minimal actin cortex (MAC). Approaches involve both single- and multiple-layered MAC structures (multi-MACs). In this work, we describe the MAC formation process in the context of different microelectrode cavity array chips (MECA and MECA-opto), as well as on glass-supported bilayers. We explore the response of these bilayers to osmotic stress and report single-molecule level imaging results that reveal the kinetic motion of actin filaments on the bilayer. We also discuss our latest permeability tests at both the single-molecule and macroscopic levels that show that multi-MACs do not significantly block access to the bilayer.

Mechanical properties of ester- and ether-DPhPC bilayers: A molecular dynamics study

In addition to its biological importance, DPhPC lipid bilayers are widely used in droplet bilayers, study of integral membrane proteins, drug delivery systems as well as patch-clamp electrophysiology of ion channels, yet their mechanical properties are not fully measured. Herein, we examined the effect of the ether linkage on the mechanical properties of ester- and ether-DPhPC lipid bilayers using all-atom molecular dynamics simulation. The values of area per lipid, thickness, intrinsic lateral pressure profile, order parameter, and elasticity moduli were estimated using various computational frameworks and were compared with available experimental values. Overall, a good agreement was observed between the two. The global properties of the two lipid bilayers are vastly different, with ether bilayer being stiffer, less ordered, and thicker than ester bilayer. Moreover, ether linkage decreased the area per lipid in the ether lipid bilayer. Our computational framework and output demonstrate how ether modification changes the mechano-chemical properties of DPhPC bilayers.

Ruggedized Chip-Based Lipid Bilayer Arrays with Permeable multi-MAC Support

Planar lipid bilayers play a central role in nanopore sensing and ion-channel electrophysiology. However, bilayer fragility often limits their application. Previously, we enhanced the mechanical properties of planar lipid bilayers with a single-layered, molecularly thin, minimal actin cortex (MAC). To further increase bilayer robustness, we are developing multiple layered MAC structures (multi-MACs) that can be chemically cross-linked to arbitrary thicknesses, without blocking diffusive access to the outer leaflet of the bilayer. In this work, we describe the multi-MAC formation process in the context of a bilayer array on a fluidic chip, as well as on glass supported bilayers. We report on permeability tests at both the single-molecule and macroscopic levels that show multi-MACs do not significantly block access to the bilayer. We discuss how permeability is achieved by controlling the openness of the F-actin network, which can be tailored using the density of linkage sites, the electrostatic interactions between linkers and filaments, and specific F-actin growth and deposition techniques. We also present results from single-molecule electrical and optical measurements that characterize the multi-MAC structure, permeability, and the approximate pore size of the multi-layered network.

Solving the Gating Mechanism of the Mitochondrial β-Barrel Metabolite Channel VDAC

The Voltage-Dependent Anion Channel (VDAC) is a β-barrel protein at the mitochondrial outer membrane (MOM) serving as the major pathway for ions and respiratory metabolites between mitochondria and the cytosol. Under applied voltage, VDAC transitions from an anionic “open” state, allowing the passive diffusion of neutral and anionic metabolites, to multiple low-conducting “closed” states with reduced anion selectivity. Importantly, VDAC closed states are virtually impermeable to ATP. A potential implication of these closed states in regulating metabolite traffic across the MOM has triggered vigorous investigations on their structural nature. Still, the mechanism behind VDAC closure remains enigmatic. Here, we study VDAC1 voltage-gating by using ion-channel electrophysiology guided by ANTON2 multi-microsecond Molecular Dynamics (MD) simulations. MD simulations highlight the particular role played by conformational dynamics of a “trigger” region, which is especially impacted by voltage polarity or solution acidification. We found that a basic residue in this region is key for initiating gating transitions at neutral pH, and its mutation to the oppositely charged residue hinders channel closure and reverses selectivity. We further explore VDAC voltage-gating by combining selectivity and pH-titration experiments with Markov State Model analysis on the motions of charged residues obtained from MD simulations. We propose a VDAC gating mechanism in which all charged residues are part of an ensemble where the motion of the single basic residue at the trigger region acts as a seed coordinating distinct dynamic rearrangements of the salt-bridge complex network. Thus, VDAC closure does not imply significant conformational changes of the β-barrel. This is strikingly different from the gating mechanism of most highly ion-selective channels and thus establishes a new paradigm of β-barrel channel gating that does not involve the translocation of a voltage sensor or a major change in pore geometry.

The Durability of Lipid Bilayers Modified with a Minimal Actin Cortex (MAC) for Nanopore Sensing and Ion Channel Electrophysiology

A minimal actin cortex (MACs) employs a thin layer of polymerized actin that is chemically coupled to a lipid membrane. Recently, we have shown that MACs can enhance the mechanical resilience of black lipid membranes containing biological nanopores, without blocking access to bulk solution. Our ongoing work explores the creation of actin multilayers for enhanced support using cross-linked filaments. We observe a density and thickness increase following layering and cross-linking procedures. We also investigate the durability of MAC-coated lipid bilayers formed on chips after exposure to non-ambient conditions of temperature, hydration, and mechanical stress. The actin network and filaments dynamics are imaged using total internal reflection fluorescence microscopy and the fluidity of the lipid bilayers is probed using fluorescence recovery after photobleaching. Results from these studies impact the development of biological nanopore sensors and ion channel electrophysiology.

Electrical recordings of the mitochondrial calcium uniporter in Xenopus oocytes.

The mitochondrial calcium uniporter is a multisubunit Ca2+ channel that mediates mitochondrial Ca2+ uptake, a cellular process crucial for the regulation of oxidative phosphorylation, intracellular Ca2+ signaling, and apoptosis. In the last few years, genes encoding uniporter proteins have been identified, but a lack of efficient tools for electrophysiological recordings has hindered quantitative analysis required to determine functional mechanisms of this channel complex. Here, we redirected Ca2+-conducting subunits (MCU and EMRE) of the human uniporter to the plasma membrane of Xenopus oocytes. Two-electrode voltage clamp reveals inwardly rectifying Ca2+ currents blocked by a potent inhibitor, Ru360 (half maximal inhibitory concentration, ~4 nM), with a divalent cation conductivity of Ca2+ > Sr2+ > Ba2+, Mn2+, and Mg2+ Patch clamp recordings further reveal macroscopic and single-channel Ca2+ currents sensitive to Ru360. These electrical phenomena were abolished by mutations that perturb MCU-EMRE interactions or disrupt a Ca2+-binding site in the pore. Altogether, this work establishes a robust method that enables deep mechanistic scrutiny of the uniporter using classical strategies in ion channel electrophysiology.

Directional K+ channel insertion in a single phospholipid bilayer: Neutron reflectometry and electrophysiology in the joint exploration of a model membrane functional platform

We investigated the insertion of small potassium (K+) channel proteins (KcvMA-1D and KcvNTS) into model membranes and the lipid-protein structural interference, combining neutron reflectometry and electrophysiology. Neutron reflectometry experiments showed how the transverse structure and mechanical properties of the bilayer were modified, upon insertion of the proteins in single model-membranes, either supported on solid substrate or floating. Parallel electrophysiology experiments were performed on the same channels reconstituted in free-standing planar lipid bilayers, of both typical composition and matched to the neutron reflectometry experiment, assessing their electrical features. Functional and structural results converge in detecting that the proteins, conical in shape, insert with a directionality, cytosolic side first. Our work addresses the powerful combination of the two experimental approaches. We show here that membrane structure spectroscopy and ion channel electrophysiology can become synergistic tools in the analysis of structural-functional properties of biomimetic complex environment.

Modeling pathogenesis and treatment response in childhood absence epilepsy.

Childhood absence epilepsy (CAE) is a genetic generalized epilepsy syndrome with polygenic inheritance, with genes for γ-aminobutyric acid (GABA) receptors and T-type calcium channels implicated in the disorder. Previous studies of T-type calcium channel electrophysiology have shown genetic changes and medications have multiple effects. The aim of this study was to use an established thalamocortical computer model to determine how T-type calcium channels work in concert with cortical excitability to contribute to pathogenesis and treatment response in CAE. The model is comprised of cortical pyramidal, cortical inhibitory, thalamocortical relay, and thalamic reticular single-compartment neurons, implemented with Hodgkin-Huxley model ion channels and connected by AMPA, GABAA , and GABAB synapses. Network behavior was simulated for different combinations of T-type calcium channel conductance, inactivation time, steady state activation/inactivation shift, and cortical GABAA conductance. Decreasing cortical GABAA conductance and increasing T-type calcium channel conductance converted spindle to spike and wave oscillations; smaller changes were required if both were changed in concert. In contrast, left shift of steady state voltage activation/inactivation did not lead to spike and wave oscillations, whereas right shift reduced network propensity for oscillations of any type. These results provide a window into mechanisms underlying polygenic inheritance in CAE, as well as a mechanism for treatment effects and failures mediated by these channels. Although the model is a simplification of the human thalamocortical network, it serves as a useful starting point for predicting the implications of ion channel electrophysiology in polygenic epilepsy such as CAE.

Electrophysiology of Epithelial Sodium Channel (ENaC) Embedded in Supported Lipid Bilayer Using a Single Nanopore Chip.

Nanopore-based technologies are highly adaptable supports for developing label-free sensor chips to characterize lipid bilayers, membrane proteins, and nucleotides. We utilized a single nanopore chip to study the electrophysiology of the epithelial Na+ channel (ENaC) incorporated in supported lipid membrane (SLM). An isolated nanopore was developed inside the silicon cavity followed by fusing large unilamellar vesicles (LUVs) of DPPS (1,2-dipalmitoyl-sn-glycero-3-phosphoserine) and DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) to produce a solvent-free SLM with giga-ohm (GΩ) sealed impedance. The presence and thickness of SLM on the nanopore chip were confirmed using atomic force spectroscopy. The functionality of SLM with and without ENaC was verified in terms of electrical impedance and capacitance by sweeping the frequency from 0.01 Hz to 100 kHz using electrochemical impedance spectroscopy. The nanopore chip exhibits long-term stability for the lipid bilayer before (144 h) and after (16 h) incorporation of ENaC. Amiloride, an inhibitor of ENaC, was utilized at different concentrations to test the integrity of fused ENaC in the lipid bilayer supported on a single nanopore chip. The developed model presents excellent electrical properties and improved mechanical stability of SLM, making this technology a reliable platform to study ion channel electrophysiology.

Annotation of functional impact of voltage-gated sodium channel mutations.

Voltage‐gated sodium channels are pore‐forming transmembrane proteins that selectively allow sodium ions to flow across the plasma membrane according to the electro‐chemical gradient thus mediating the rising phase of action potentials in excitable cells and playing key roles in physiological processes such as neurotransmission, skeletal muscle contraction, heart rhythm, and pain sensation. Genetic variations in the nine human genes encoding these channels are known to cause a large range of diseases affecting the nervous and cardiac systems. Understanding the molecular effect of genetic variations is critical for elucidating the pathologic mechanisms of known variations and in predicting the effect of newly discovered ones. To this end, we have created a Web‐based tool, the Ion Channels Variants Portal, which compiles all variants characterized functionally in the human sodium channel genes. This portal describes 672 variants each associated with at least one molecular or clinical phenotypic impact, for a total of 4,658 observations extracted from 264 different research articles. These data were captured as structured annotations using standardized vocabularies and ontologies, such as the Gene Ontology and the Ion Channel ElectroPhysiology Ontology. All these data are available to the scientific community via neXtProt at https://www.nextprot.org/portals/navmut.

ICEPO: the ion channel electrophysiology ontology.

Ion channels are transmembrane proteins that selectively allow ions to flow across the plasma membrane and play key roles in diverse biological processes. A multitude of diseases, called channelopathies, such as epilepsies, muscle paralysis, pain syndromes, cardiac arrhythmias or hypoglycemia are due to ion channel mutations. A wide corpus of literature is available on ion channels, covering both their functions and their roles in disease. The research community needs to access this data in a user-friendly, yet systematic manner. However, extraction and integration of this increasing amount of data have been proven to be difficult because of the lack of a standardized vocabulary that describes the properties of ion channels at the molecular level. To address this, we have developed Ion Channel ElectroPhysiology Ontology (ICEPO), an ontology that allows one to annotate the electrophysiological parameters of the voltage-gated class of ion channels. This ontology is based on a three-state model of ion channel gating describing the three conformations/states that an ion channel can adopt: closed, open and inactivated. This ontology supports the capture of voltage-gated ion channel electrophysiological data from the literature in a structured manner and thus enables other applications such as querying and reasoning tools. Here, we present ICEPO (ICEPO ftp site: ftp://ftp.nextprot.org/pub/current_release/controlled_vocabularies/), as well as examples of its use.

C3-OH of Amphotericin B Plays an Important Role in Ion Conductance.

Amphotericin B (AmB) is the archetype for small molecules that form ion channels in living systems and has recently been shown to replace a missing protein ion transporter and thereby restore physiology in yeast. Molecular modeling studies predict that AmB self-assembles in lipid membranes with the polyol region lining a channel interior that funnels to its narrowest region at the C3-hydroxyl group. This model predicts that modification of this functional group would alter conductance of the AmB ion channel. To test this hypothesis, the C3-hydroxyl group was synthetically deleted, and the resulting derivative, C3deoxyAmB (C3deOAmB), was characterized using multidimensional NMR experiments and single ion channel electrophysiology recordings. C3deOAmB possesses the same macrocycle conformation as AmB and retains the capacity to form transmembrane ion channels, yet the conductance of the C3deOAmB channels is 3-fold lower than that of AmB channels. Thus, the C3-hydroxyl group plays an important role in AmB ion channel conductance, and synthetic modifications at this position may provide an opportunity for further tuning of channel functions.

Interdroplet bilayer arrays in millifluidic droplet traps from 3D-printed moulds

In droplet microfluidics, aqueous droplets are typically separated by an oil phase to ensure containment of molecules in individual droplets of nano-to-picoliter volume. An interesting variation of this method involves bringing two phospholipid-coated droplets into contact to form a lipid bilayer in-between the droplets. These interdroplet bilayers, created by manual pipetting of microliter droplets, have proved advantageous for the study of membrane transport phenomena, including ion channel electrophysiology. In this study, we adapted the droplet microfluidics methodology to achieve automated formation of interdroplet lipid bilayer arrays. We developed a 'millifluidic' chip for microliter droplet generation and droplet packing, which is cast from a 3D-printed mould. Droplets of 0.7-6.0 μL volume were packed as homogeneous or heterogeneous linear arrays of 2-9 droplets that were stable for at least six hours. The interdroplet bilayers had an area of up to 0.56 mm(2), or an equivalent diameter of up to 850 μm, as determined from capacitance measurements. We observed osmotic water transfer over the bilayers as well as sequential bilayer lysis by the pore-forming toxin melittin. These millifluidic interdroplet bilayer arrays combine the ease of electrical and optical access of manually pipetted microdroplets with the automation and reproducibility of microfluidic technologies. Moreover, the 3D-printing based fabrication strategy enables the rapid implementation of alternative channel geometries, e.g. branched arrays, with a design-to-device time of just 24-48 hours.

Variability in high-throughput ion-channel screening data and consequences for cardiac safety assessment

IntroductionUnwanted drug interactions with ionic currents in the heart can lead to an increased pro-arrhythmic risk to patients in the clinic. It is therefore a priority for safety pharmacology teams to detect block of cardiac ion channels, and new technologies have enabled the development of automated and high-throughput screening assays using cell lines. As a result of screening multiple ion-channels there is a need to integrate information, particularly for compounds affecting more than one current, and mathematical electrophysiology in-silico action potential models are beginning to be used for this. MethodsWe quantified the variability associated with concentration-effect curves fitted to recordings from high-throughput Molecular Devices IonWorks® Quattro™ screens when detecting block of IKr (hERG), INa (NaV1.5), ICaL (CaV1.2), IKs (KCNQ1/minK) and Ito (Kv4.3/KChIP2.2), and the Molecular Devices FLIPR® Tetra fluorescence screen for ICaL (CaV1.2), for control compounds used at AstraZeneca and GlaxoSmithKline. We examined how screening variability propagates through in-silico action potential models for whole cell electrical behaviour, and how confidence intervals on model predictions can be estimated with repeated simulations. ResultsThere are significant levels of variability associated with high-throughput ion channel electrophysiology screens. This variability is of a similar magnitude for different cardiac ion currents and different compounds. Uncertainty in the Hill coefficients of reported concentration-effect curves is particularly high. Depending on a compound's ion channel blocking profile, the uncertainty introduced into whole-cell predictions can become significant. DiscussionOur technique allows confidence intervals to be placed on computational model predictions that are based on high-throughput ion channel screens. This allows us to suggest when repeated screens should be performed to reduce uncertainty in a compound's action to acceptable levels, to allow a meaningful interpretation of the data.

Wavelet-based protocols for ion channel electrophysiology

BackgroundFluctuation-induced phenomena caused by both random and deterministic stimuli have been previously studied in a variety of contexts. They are based on the interplay between the spectro-temporal patterns of the signal and the kinetics of the system it is applied to. The aim of this study was to develop a method for designing fluctuating inputs into nonlinear system which would elicit the most desired system output and to implement the method to studies of ion channels.ResultsWe describe an algorithm based on constructing the input as a superposition of wavelets and optimizing it according to a selected cost functional. The algorithm is applied to ion channel electrophysiology where the input is the fluctuating voltage delivered through a patch-clamp experimental apparatus and the output is the whole-cell ionic current. The algorithm is optimized to aid selection of Markov models of the gating kinetics of the voltage-gated Shaker K+ channel and tested by comparison of numerically obtained ionic currents predicted by different models with experimental data obtained from the Shaker K+ channels. Other applications and optimization criteria are also suggested.ConclusionThe method described in this paper can be useful in development and testing of models of ion channel gating kinetics, developing voltage inputs that optimize certain nonequilibrium phenomena in ion channels, such as the kinetic focusing, and potentially has applications to other fields.

Electrophysiology and Single Cell Reverse Transcription Quantitative PCR in GnRH Neurons

Release of gonadotrophin-releasing hormone (GnRH) from specialized hypothalamic neurons is the final common pathway in the endocrine reproductive axis and plays an important role in reproductive health. Regular and timely electrical activity of GnRH neurons accompanies the pulsatile release of GnRH that is plays a central role in release of luteinizing hormone and follicle stimulating hormone required for normal fertility in all vertebrates. Understanding the molecular basis of the neuronal electrical activity is critical to understanding excitation-release coupling in GnRH neurons. Despite the fundamental importance of GnRH neurons in the reproductive axis, little is known about the molecular basis of electrical activity in GnRH neurons. GnRH neurons are spontaneously active, and pacemaking potentials have been identified in GnRH neurons. We used transgenic rats expressing GFP driven by a GnRH promoter to identify individual GnRH neurons. We performed electrophysiology and single cell quantitative RT-PCR to determine the electrophysiology and molecular identity of ion channels in isolated GFP-labeled GnRH neurons isolated from brains of regularly cycling female rats. We have identified Kv4.3 transcripts, suggesting the presence of a transient outward-type current (IA). We also identified a hyperpolarization-activated current that is probably encoded by HCN2 and/or HCN3 and may be responsible for the pacemaking activity critical to GnRH neuronal function.

Kv3.3b expression defines the shape of the complex spike in the Purkinje cell

The complex spike (CS) in cerebellar Purkinje Cells (PC) is not an all-or-nothing phenomena as originally proposed, but shows variability depending on the spiking behavior of the Inferior Olive and intrinsic variability in the number and shape of spikelets. The potassium channel Kv3.3b, which has been proposed to undergo developmental changes during the postnatal PC maturation, has been shown to be crucial for the repolarization of the spikelets in the CS. We address here the regulation of the intrinsic CS variability by the expression of inactivating Kv3.3 channels in PCs by combining patch-clamp recordings and single-cell PCR methods on the same neurons, using a technique that we recently optimized to correlate single cell transcription levels with membrane ion channel electrophysiology. We show that while the inactivating TEA sensitive Kv3.3 current peak intensity increases with postnatal age, the channel density does not, arguing against postnatal developmental changes of Kv3.3b expression. Real time PCR of Kv3.3b showed a high variability from cell to cell, correlated with the Kv3.3 current density, and suggesting that there are no mechanisms regulating these currents beyond the mRNA pool. We show a significant correlation between normalized quantity of Kv3.3b mRNA and both the number of CS spikelets and their rate of voltage fluctuation, linking the intrinsic CS shape directly to the Kv3.3b mRNA pool. Comparing the observed cell-to-cell variance with studies on transcriptional noise suggests that fluctuations of the Kv3.3b mRNA pool are possibly not regulated but represent merely transcriptional noise, resulting in intrinsic variability of the CS.

Lipid bilayer coated Al(2)O(3) nanopore sensors: towards a hybrid biological solid-state nanopore.

Solid-state nanopore sensors are highly versatile platforms for the rapid, label-free electrical detection and analysis of single molecules, applicable to next generation DNA sequencing. The versatility of this technology allows for both large scale device integration and interfacing with biological systems. Here we report on the development of a hybrid biological solid-state nanopore platform that incorporates a highly mobile lipid bilayer on a single solid-state Al(2)O(3) nanopore sensor, for the potential reconstitution of ion channels and biological nanopores. Such a system seeks to combine the superior electrical, thermal, and mechanical stability of Al(2)O(3) solid-state nanopores with the chemical specificity of biological nanopores. Bilayers on Al(2)O(3) exhibit higher diffusivity than those formed on TiO(2) and SiO(2) substrates, attributed to the presence of a thick hydration layer on Al(2)O(3), a key requirement to preserving the biological functionality of reconstituted membrane proteins. Molecular dynamics simulations demonstrate that the electrostatic repulsion between the dipole of the DOPC headgroup and the positively charged Al(2)O(3) surface may be responsible for the enhanced thickness of this hydration layer. Lipid bilayer coated Al(2)O(3) nanopore sensors exhibit excellent electrical properties and enhanced mechanical stability (GΩ seals for over 50 h), making this technology ideal for use in ion channel electrophysiology, the screening of ion channel active drugs and future integration with biological nanopores such as α-hemolysin and MspA for rapid single molecule DNA sequencing. This technology can find broad application in bio-nanotechnology.