Macroscopic Conductance Research Articles

Functional and clinical characterization of a novel homozygous KCNH2 missense variant in the pore region of Kv11.1 leading to a viable but severe long-QT syndrome

BackgroundAmong KCNH2 missense loss of function (LOF) variants, homozygosity –at any position in the Kv11.1/hERG channel – is very rare and generally leads to intrauterine death, while heterozygous variants in the pore are responsible for severe Type 2 long-QT syndrome (LQTS). We report a novel homozygous p.Gly603Ser missense variant in the pore of Kv11.1/hERG (KCNH2 c.1807G > A) discovered in the context of a severe LQTS. MethodsWe carried out a phenotypic family study combined with a functional analysis of mutated and wild-type (WT) Kv11.1 by two-electrode voltage-clamp using the Xenopus laevis oocyte heterologous expression system. ResultsThe variant resulted in a severe LQTS phenotype (very prolonged corrected QT interval, T-wave alternans, multiple Torsades de pointes) with a delayed clinical expression in later childhood in the homozygous state, and in a Type 2 LQTS phenotype in the heterozygous state. Expression of KCNH2 p.Gly603Ser cRNA alone elicited detectable current in Xenopus oocytes. Inactivation kinetics and voltage dependence of activation were not significantly affected by the variant. The macroscopic slope conductance of the variant was three-fold less compared to the WT (18.5 ± 9.01 vs 54.7 ± 17.2 μS, p < 0.001). ConclusionsWe characterized the novel p.Gly603Ser KCNH2 missense LOF variant in the pore region of Kv11.1/hERG leading to a severe but viable LQTS in the homozygous state and an attenuated Type 2 LQTS in heterozygous carriers. To our knowledge we provide the first description of a homozygous variant in the pore-forming region of Kv11.1 with a functional impact but a delayed clinical expression.

Migration of Electroconductive Carbon Nanoparticles in Polymer Composites Melts

AbstractProcess of nanoscale carbon filler migration in polymer composite melts is studied. Monotonous decrease of electrical resistance is observed for composite materials during their stay in a melted state. This is especially pronounced for composites with concentration of filler much lower than percolation threshold determined for a specific matrix/filler/mixing method combination. This phenomenon can be observed for composites based on different polymer matrices. Also, it is demonstrated that the type and geometry of the electrically conductive filler does not affect presence of the phenomena. A setup for measurement of conductance for volume and surface layer components is used. The results of electrical conductance measurements of the polymer composite melt using this setup allows to attribute the increase of composite material conductance to formation of filler enriched surface layer. To obtain information on the processes occurring at the microscopic level, molecular dynamics simulations are conducted. Variation of several simulation parameters with comparison of the results to experimental data allows to understand the macroscopic composite material conductance behavior.

Direct visualization of percolating metal-insulator transition in V2O3 using scanning microwave impedance microscopy

Using the extensively studied V2O3 as a prototype system, we investigate the role of percolation in metal-insulator transition (MIT). We apply scanning microwave impedance microscopy to directly determine the metallic phase fraction p and relate it to the macroscopic conductance G, which shows a sudden jump when p reaches the percolation threshold. Interestingly, the conductance G exhibits a hysteretic behavior against suggesting two different percolating processes upon cooling and warming. Based on our image analysis and model simulation, we ascribe such hysteretic behavior to different domain nucleation and growth processes between cooling and warming, which is likely caused by the decoupled structural and electronic transitions in V2O3 during MIT. Our work provides a microscopic view of how the interplay of structural and electronic degrees of freedom affects MIT in strongly correlated systems.

The Functional Role of Hyperpolarization Activated Current (I f) on Cardiac Pacemaking in Human vs. in the Rabbit Sinoatrial Node: A Simulation and Theoretical Study.

The cardiac hyperpolarization-activated “funny” current (If), which contributes to sinoatrial node (SAN) pacemaking, has a more negative half-maximal activation voltage and smaller fully-activated macroscopic conductance in human than in rabbit SAN cells. The consequences of these differences for the relative roles of If in the two species, and for their responses to the specific bradycardic agent ivabradine at clinical doses have not been systematically explored. This study aims to address these issues, through incorporating rabbit and human If formulations developed by Fabbri et al. into the Severi et al. model of rabbit SAN cells. A theory was developed to correlate the effect of If reduction with the total inward depolarising current (Itotal) during diastolic depolarization. Replacing the rabbit If formulation with the human one increased the pacemaking cycle length (CL) from 355 to 1,139 ms. With up to 20% If reduction (a level close to the inhibition of If by ivabradine at clinical concentrations), a modest increase (~5%) in the pacemaking CL was observed with the rabbit If formulation; however, the effect was doubled (~12.4%) for the human If formulation, even though the latter has smaller If density. When the action of acetylcholine (ACh, 0.1 nM) was considered, a 20% If reduction markedly increased the pacemaking CL by 37.5% (~27.3% reduction in the pacing rate), which is similar to the ivabradine effect at clinical concentrations. Theoretical analysis showed that the resultant increase of the pacemaking CL is inversely proportional to the magnitude of Itotal during diastolic depolarization phase: a smaller If in the model resulted in a smaller Itotal amplitude, resulting in a slower pacemaking rate; and the same reduction in If resulted in a more significant change of CL in the cell model with a smaller Itotal. This explained the mechanism by which a low dose of ivabradine slows pacemaking rate more in humans than in the rabbit. Similar results were seen in the Fabbri et al. model of human SAN cells, suggesting our observations are model-independent. Collectively, the results of study explain why low dose ivabradine at clinically relevant concentrations acts as an effective bradycardic agent in modulating human SAN pacemaking.

An Epilepsy-Associated Mutation at the KV1.2 (KCNA2) Charge Transfer Center Strongly Suppresses Channel Surface Trafficking

We report on a new variant of KCNA2, which encodes the subunits of tetrameric, voltage-gated, potassium-selective channels KV1.2. The de novo, heterozygous mutation was discovered in an infant patient with epilepsy and causes amino-acid substitution F233S. F233 is highly-conserved and constitutes the charge transfer center at the KV1.2 voltage-sensing domain. Our immunocytochemistry (confocal microscopy) investigations in transiently-transfected COS-7 cells revealed that KCNA2(F233S) subunits do not traffic to the cell surface. In flow cytometry experiments, surface-labeled KCNA2(F233S) was detected in 1.60±0.08% of cells, relative to cells transfected with KCNA2(WT). In medium-throughput electrophysiological experiments using the OpusXpress TEVC robot, no currents were detectable in Xenopus oocytes injected with KCNA2(F233S) cRNA (p=0.17 compared with sham-injected oocytes; n≥10). In COVG experiments (n=5), no significant KV1.2(F233S) current was detected in oocytes during depolarizing pulses up to 180mV. We then investigated the interaction of KCNA2(WT) and KCNA2(F233S) subunits on the OpusXpress by comparing oocytes injected with KCNA2 cRNA to emulate homozygous WT, haploinsufficiency (half-dose WT) or heterozygous WT/F233S conditions. All oocytes exhibited currents with similar V0.5, indicating that they were produced by wild-type KV1.2 channels. However, heterozygous WT/F233S cells exhibited a mean macroscopic conductance of 11±1μS, 16% that of homozygous WT (70±12μS) and 28% that of the haploinsufficiency condition (40±4μS). KCNA2(F233S)-mediated suppression of KV1.2 channels was dependent on WT:F233S ratio. These results show that F233S has a dominant loss-of-function effect via a severe trafficking defect, which affects subunits from both alleles and potently suppresses KV1.2 expression. Preliminary experiments show that KCNA4 subunits, which can form heterotetrameric channels with KCNA2, are not sequestered by KCNA2(F233S). We are actively studying the interaction of KCNA2(F233S) with KCNA4 and KCNAB2 subunits, both known to facilitate KCNA2 expression.

Functional analysis of the F337C mutation in the CLCN1 gene associated with dominant myotonia congenita reveals an alteration of the macroscopic conductance and voltage dependence.

ABSTRACTBackground Myotonia congenita (MC) is a common channelopathy affecting skeletal muscle and which is due to pathogenic variants within the CLCN1 gene. Various alterations in the function of the channel have been reported and we here illustrate a novel one.MethodsA patient presenting the symptoms of myotonia congenita was shown to bear a new heterozygous missense variant in exon 9 of the CLCN1 gene (c.1010 T > G, p.(Phe337Cys)). Confocal imaging and patch clamp recordings of transiently transfected HEK293 cells were used to functionally analyze the effect of this variant on channel properties.ResultsConfocal imaging showed that the F337C mutant incorporated as well as the WT channel into the plasma membrane. However, in patch clamp, we observed a smaller conductance for F337C at −80 mV. We also found a marked reduction of the fast gating component in the mutant channels, as well as an overall reduced voltage dependence.ConclusionTo our knowledge, this is the first report of a mixed alteration in the biophysical properties of hClC‐1 consisting of a reduced conductance at resting potential and an almost abolished voltage dependence.

Role of Lipid Environment in the Pore-Forming Activity of Cecropin A

Cecropin A (CecA) is a well-known antimicrobial peptide produced by Hyalophore cecropia. It was found that cecropin A forms ion channels of different conductance and selectivity in planar lipid bilayers in the presence of negatively charged DOPS. Here the pore-forming activity of CecA in lipid bilayer of different lipid composition formed by the Montall and Muller technique in the absence and in the presence of alkaloids was studied. We found that one-side addition of CecA up to 5 µM led to the destabilization of lipid bilayers composed of POPC:POPG (50:50 mol %) or POPC:POPE:POPG (25:25:50 mol %). At the same time 2 µM of CecA induced the step-like current fluctuations corresponding to the openings and closures of transmembrane pores in POPC:TOCL – membranes (50:50 mol %). Increasing of CecA concentration up to 5 µM enhanced the macroscopic conductance. Introduction of piperine or capsaicin to the membrane bathing solution up to 400 µM caused dramatic reduction in multichannel conductance induced by CecA. Using differential scanning microcalorimetry we showed that piperine and capsaicin decreased the maximum temperature of the main transition of DPPC at molar ratio to lipid of 1:10 on 3.6 and 5.7 °C respectively. Analyzing the data we suggested that regulation of CecA induced macroscopic conductance is due to the changes in the elastic properties of the membrane at alkaloid adsorption. The study was supported by the Russian Foundation of Science (# 19-14-00110) and Russian Foundation of Basic Research (# 18-34-20047)

The mechanism and energetics of a ligand-controlled hydrophobic gate in a mammalian two pore channel.

In the last decade two-pore intracellular channels (TPCs) attracted the interest of researchers, still some key questions remain open. Their importance for vacuolar (plants) and endo-lysosomal (animals) function highlights them as a very attractive system to study, both theoretically and experimentally. Indicated as key players in the trafficking of the cell, today they are considered a new potential target for avoiding virus infections, including those from coronaviruses. A particular boost for theoretical examinations has been made with recent high-resolution X-ray and cryo-EM structures. These findings have opened the way for efficient and precise computational studies at the atomistic level. Here we report a set of multiscale-calculations performed on the mTPC1, a ligand- and voltage-gated sodium selective channel. The molecular dynamics and enhanced molecular dynamics simulations were used for a thorough analysis of the mammalian TPC1 behaviour in the presence and absence of the ligand molecule, with a special accent on the supposed bottleneck, the hydrophobic gate. Moreover, from the reconstructed free energy obtained from enhanced simulations, we have calculated the macroscopic conductance of sodium ions through the mTPC1, which we compared with measured single-channel conductance values. The hydrophobic gate works as a steric barrier and the key parameters are its flexibility and the dimension of the sodium first hydration shell.

Thermal properties of thin films made from MoS2 nanoflakes and probed via statistical optothermal Raman method

A deep understanding of the thermal properties of 2D materials is crucial to their implementation in electronic and optoelectronic devices. In this study, we investigated the macroscopic in-plane thermal conductivity (κ) and thermal interface conductance (g) of large-area (mm2) thin film made from MoS2 nanoflakes via liquid exfoliation and deposited on Si/SiO2 substrate. We found κ and g to be 1.5 W/mK and 0.23 MW/m2K, respectively. These values are much lower than those of single flakes. This difference shows the effects of interconnections between individual flakes on macroscopic thin film parameters. The properties of a Gaussian laser beam and statistical optothermal Raman mapping were used to obtain sample parameters and significantly improve measurement accuracy. This work demonstrates how to address crucial stability issues in light-sensitive materials and can be used to understand heat management in MoS2 and other 2D flake-based thin films.

Transverse conductance in a three-dimensional Weyl semimetal and Weyl superconductor hybrid under a strong magnetic field

There are two competing pairing mechanisms for the superconductivity of doped Weyl semimetals, i.e., the internode Bardeen-Cooper-Schrieffer (BCS) pairing and the intranode Fulde-Ferrell-Larkin-Ovchinnikov (FFLO) pairing. To understand the edge excitations at the interface between the Weyl semimetal and the superconducting Weyl semimetal (WSM/SWSM) mediated by two different pairings, we study the energy dispersions and the density of states under a strong magnetic field. It is found that only the chiral zeroth Landau level exhibits a significant difference for the two pairings; the excitation spectra of higher Landau levels are insensitive to the way of pairings. In the vicinity of interface in the hybrid of WSM/SWSM, the spatial distributions of transverse current and the transverse conductance are independent of pairing mechanism. The pairing independence in the macroscopic conductance can be understood with the quantum effect of phase-coherent electron-hole states at the WSM/SWSM interface, which is responsible for the magnetically induced edge states supported by the Weyl Landau levels.

Control of Membrane Permeability via Voltage Regulated Lysenin Channels

Lysenin channels inserted into planar lipid membranes containing asolectin, cholesterol, and sphingomyelin present voltage-induced gating which manifests as conformational changes that abrogate the channel's conductance at positive bias potentials. This remarkable feature enables controlled transmembrane transport by modulation of the transmembrane voltage with diffusion potentials created by using selective transporters. In this work we show that the macroscopic conductance of lysenin channels, thus the membrane permeability, can be adjusted with electrochemical gradients produced by the action of specific transporters such as valinomycin. The lysenin channels close and the macroscopic conductance of the membrane approaches zero as the transmembrane potential becomes greater than 20 mV (membrane depolarization). After channel closure, the membrane conductance is reinstated by adjusting the ionic concentrations such that the diffusion potentials drop under 20 mV (membrane hyperpolarization). In addition, upon investigating the selectivity of lysenin channels to monovalent ions we found that their moderate selectivity may be used to produce diffusion potentials sufficient to elicit conformational changes and modulate the channel conductance. Accordingly, we succeeded to adjust the membrane permeability by sole adjustments of the ionic concentrations and in the absence of any other selective transporter. Our experiments suggest that lysenin channels may be used to achieve controlled transport across artificial and natural lipid membranes, with direct applications to drug delivery or the design of microbioreactors with controlled permeability.

ZnO nanoparticles modulate the ionic transport and voltage regulation of lysenin nanochannels

BackgroundThe insufficient understanding of unintended biological impacts from nanomaterials (NMs) represents a serious impediment to their use for scientific, technological, and medical applications. While previous studies have focused on understanding nanotoxicity effects mostly resulting from cellular internalization, recent work indicates that NMs may interfere with transmembrane transport mechanisms, hence enabling contributions to nanotoxicity by affecting key biological activities dependent on transmembrane transport. In this line of inquiry, we investigated the effects of charged nanoparticles (NPs) on the transport properties of lysenin, a pore-forming toxin that shares fundamental features with ion channels such as regulation and high transport rate.ResultsThe macroscopic conductance of lysenin channels greatly diminished in the presence of cationic ZnO NPs. The inhibitory effects were asymmetrical relative to the direction of the electric field and addition site, suggesting electrostatic interactions between ZnO NPs and a binding site. Similar changes in the macroscopic conductance were observed when lysenin channels were reconstituted in neutral lipid membranes, implicating protein-NP interactions as the major contributor to the reduced transport capabilities. In contrast, no inhibitory effects were observed in the presence of anionic SnO2 NPs. Additionally, we demonstrate that inhibition of ion transport is not due to the dissolution of ZnO NPs and subsequent interactions of zinc ions with lysenin channels.ConclusionWe conclude that electrostatic interactions between positively charged ZnO NPs and negative charges within the lysenin channels are responsible for the inhibitory effects on the transport of ions. These interactions point to a potential mechanism of cytotoxicity, which may not require NP internalization.

Stochastic sensing of Angiotensin II with lysenin channels

The ability of pore-forming proteins to interact with various analytes has found vast applicability in single molecule sensing and characterization. In spite of their abundance in organisms from all kingdoms of life, only a few pore-forming proteins have been successfully reconstituted in artificial membrane systems for sensing purposes. Lysenin, a pore-forming toxin extracted from the earthworm E. fetida, inserts large conductance nanopores in lipid membranes containing sphingomyelin. Here we show that single lysenin channels may function as stochastic nanosensors by allowing the short cationic peptide angiotensin II to be electrophoretically driven through the conducting pathway. Long-term translocation experiments performed using large populations of lysenin channels allowed unequivocal identification of the unmodified analyte by Liquid Chromatography-Mass Spectrometry. However, application of reverse voltages or irreversible blockage of the macroscopic conductance of lysenin channels by chitosan addition prevented analyte translocation. This investigation demonstrates that lysenin channels have the potential to function as nano-sensing devices capable of single peptide molecule identification and characterization, which may be further extended to other macromolecular analytes.

Growth hormone secretagogues prevent dysregulation of skeletal muscle calcium homeostasis in a rat model of cisplatin-induced cachexia.

BackgroundCachexia is a wasting condition associated with cancer types and, at the same time, is a serious and dose‐limiting side effect of cancer chemotherapy. Skeletal muscle loss is one of the main characteristics of cachexia that significantly contributes to the functional muscle impairment. Calcium‐dependent signaling pathways are believed to play an important role in skeletal muscle decline observed in cachexia, but whether intracellular calcium homeostasis is affected in this situation remains uncertain. Growth hormone secretagogues (GHS), a family of synthetic agonists of ghrelin receptor (GHS‐R1a), are being developed as a therapeutic option for cancer cachexia syndrome; however, the exact mechanism by which GHS interfere with skeletal muscle is not fully understood.MethodsBy a multidisciplinary approach ranging from cytofluorometry and electrophysiology to gene expression and histology, we characterized the calcium homeostasis in fast‐twitch extensor digitorum longus (EDL) muscle of adult rats with cisplatin‐induced cachexia and established the potential beneficial effects of two GHS (hexarelin and JMV2894) at this level. Additionally, in vivo measures of grip strength and of ultrasonography recordings allowed us to evaluate the functional impact of GHS therapeutic intervention.ResultsCisplatin‐treated EDL muscle fibres were characterized by a ~18% significant reduction of the muscle weight and fibre diameter together with an up‐regulation of atrogin1/Murf‐1 genes and a down‐regulation of Pgc1‐a gene, all indexes of muscle atrophy, and by a two‐fold increase in resting intracellular calcium, [Ca2+]i, compared with control rats. Moreover, the amplitude of the calcium transient induced by caffeine or depolarizing high potassium solution as well as the store‐operated calcium entry were ~50% significantly reduced in cisplatin‐treated rats. Calcium homeostasis dysregulation parallels with changes of functional ex vivo (excitability and resting macroscopic conductance) and in vivo (forelimb force and muscle volume) outcomes in cachectic animals. Administration of hexarelin or JMV2894 markedly reduced the cisplatin‐induced alteration of calcium homeostasis by both common as well as drug‐specific mechanisms of action. This effect correlated with muscle function preservation as well as amelioration of various atrophic indexes, thus supporting the functional impact of GHS activity on calcium homeostasis.ConclusionsOur findings provide a direct evidence that a dysregulation of calcium homeostasis plays a key role in cisplatin‐induced model of cachexia gaining insight into the etiopathogenesis of this form of muscle wasting. Furthermore, our demonstration that GHS administration efficaciously prevents cisplatin‐induced calcium homeostasis alteration contributes to elucidate the mechanism of action through which GHS could potentially ameliorate chemotherapy‐associated cachexia.

Purinergic control of lysenin's transport and voltage-gating properties.

Lysenin, a pore-forming protein extracted from the coelomic fluid of the earthworm Eisenia foetida, manifests cytolytic activity by inserting large conductance pores in host membranes containing sphingomyelin. In the present study, we found that adenosine phosphates control the biological activity of lysenin channels inserted into planar lipid membranes with respect to their macroscopic conductance and voltage-induced gating. Addition of ATP, ADP, or AMP decreased the macroscopic conductance of lysenin channels in a concentration-dependent manner, with ATP being the most potent inhibitor and AMP the least. ATP removal from the bulk solutions by buffer exchange quickly reinstated the macroscopic conductance and demonstrated reversibility. Single-channel experiments pointed to an inhibition mechanism that most probably relies on electrostatic binding and partial occlusion of the channel-conducting pathway, rather than ligand gating induced by the highly charged phosphates. The Hill analysis of the changes in macroscopic conduction as a function of the inhibitor concentration suggested cooperative binding as descriptive of the inhibition process. Ionic screening significantly reduced the ATP inhibitory efficacy, in support of the electrostatic binding hypothesis. In addition to conductance modulation, purinergic control over the biological activity of lysenin channels has also been observed to manifest as changes of the voltage-induced gating profile. Our analysis strongly suggests that not only the inhibitor's charge but also its ability to adopt a folded conformation may explain the differences in the observed influence of ATP, ADP, and AMP on lysenin's biological activity.

Gabapentinoids Suppress Early Afterdepolarizations by Uncoupling α2δ-1 Subunits from CAV1.2 Channels: Implications for CAV Channel Gating-Modifiers as a New Class of Antiarrhythmics

Under certain pathological conditions, the repolarization phase of the cardiac action potential (AP) is interrupted by membrane potential oscillations called early afterdepolarizations (EADs), well-recognized triggers of deadly cardiac arrhythmias. The reactivation of L-type calcium current (ICa,L) plays a prominent role in EAD formation. In recent studies using the dynamic clamp technique, we have shown that a small depolarizing shift (<5mV) of the CaV1.2 steady-state activation potently suppresses EAD occurrence. Based on this findings, we hypothesized that pharmacological interventions that induce a depolarizing shift of macroscopic ICa,L activation (gating modifiers) can suppress EADs and their arrhythmogenic consequences. Gabapentinoids (gabapentin and pregabalin), drugs that selectively bind to α2δ-1 CaV subunits and are commonly used to treat epilepsy and neuropathic pain, are good candidates as Cav gating modifiers. We found that gabapentin induced a time-dependent depolarizing shift of the voltage-dependent activation of CaV1.2 channels expressed in Xenopus oocytes (α1C β3 α2δ-1; 1mM gabapentin, 2h incubation). Since α2δ-1 facilitates CaV1.2 voltage-dependent activation, these results suggest a functional uncoupling of α2δ-1 from a fraction of channels. Importantly, the maximal macroscopic conductance was unaltered, implying that CaV1.2 channel trafficking was not perturbed, in contrast to the effects shown in other CaV isoforms. We then studied gabapentinoids effect on EAD susceptibility in freshly-dissociated rabbit ventricular myocytes under EAD-promoting conditions (600μM H2O2). Strikingly, 1 mM pregabalin suppressed EAD occurrence and restored AP duration as predicted based on the depolarizing shift in voltage-dependent activation. Taken together, these results support the view that gabapentinoids, and more generally CaV1.2 gating modifiers, show promise in suppressing EAD-mediated arrhythmias and could constitute a novel class of antiarrhythmics.

Mechanism of Interaction between Adenosine Phosphates and Lysenin Channels

Lysenin, a 297 amino acid pore forming toxin extracted from the earthworm E. foetida, inserts hexameric channels in natural and artificial membranes containing sphingomyelin. A distinct feature of lysenin channels is their asymmetric voltage induced gating which occurs at low positive transmembrane potentials. Although the structure of the channel is not yet solved, previous studies of the lysenin monomeric structure and interaction with membranes indicate the presence of local positive charges within the protein's structure. Consequently, we hypothesized that highly charged organic anions such as ATP, ADP, and AMP may interact with the channel and alter its functionality. Here we show that the macroscopic conductance of the channels was diminished by addition of various adenosine phosphates in a charge and concentration-dependent manner. Single channel explorations have shown a gradual reduction of the ionic currents indicating interaction with absence of gating. Buffer-exchange experiments demonstrated that the current inhibition is reversible, pointing out a potential electrostatic mechanism of interaction between channels and anions which implies binding. This hypothesis was further sustained by analyzing the inhibitory effects of ATP comprising ionic solutions with different ionic strengths. Hill analysis provided further insights into the inhibition process and indicated a positive cooperativity between binding events. In addition, the I-V plots recorded in the presence of increasing inhibitor concentration demonstrate a significant rightward shift of the voltage-induced gating profile. The reduced effects elicited by AMP as compared with ATP and ADP, the reversibility of the inhibition, and the strong dependency of the observed effects on the ionic strength point out an electrostatic mechanism of interaction which may yield partial occlusion of the channels and modulation of the voltage-induced gating. This work sets an example of purinergic signaling that implies modulating the functionality of an exogenous membrane transporter.

Establishment of the Dual Whole Cell Recording Patch Clamp Configuration for the Measurement of Gap Junction Conductance.

The development of the patch clamp technique has enabled investigators to directly measure gap junction conductance between isolated pairs of small cells with resolution to the single channel level. The dual patch clamp recording technique requires specialized equipment and the acquired skill to reliably establish gigaohm seals and the whole cell recording configuration with high efficiency. This chapter describes the equipment needed and methods required to achieve accurate measurement of macroscopic and single gap junction channel conductances. Inherent limitations with the dual whole cell recording technique and methods to correct for series access resistance errors are defined as well as basic procedures to determine the essential electrical parameters necessary to evaluate the accuracy of gap junction conductance measurements using this approach.

Au nanoparticle enhanced thin-film silicon solar cells

Gold nanoparticles (Au NPs) were prepared on the top of the indium tin oxide (ITO) window layer in thin-film silicon solar cells using a magnetron sputtering method. In comparison with the pure ITO film, the samples with the Au NPs show higher optical transmittance. It is found that the photoluminescence (PL) intensities from the ITO films are enhanced significantly by the Au NPs, so do the macroscopic conductance and the microscopic conductive atomic microcopy (C-AFM) current–voltage (I–V) characteristics. It also appears that the surface work function (φs) is changed by the Au NPs, the longer time the Au NP deposition, the more Au NPs on surface, the larger φs. When the sample is illuminated, its φs downshifts due to the local surface plasmon resonance (LSPR) excited by the illumination on the Au NPs. It is found that the short circuit current density (Jsc) of the solar cells is increased by as much as 20.78% for the optimized solar cell configuration.

Positioning of extracellular loop 1 affects pore gating of the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a chloride ion channel, the dysfunction of which directly leads to the life-shortening disease CF. Extracellular loop 1 (ECL1) of CFTR contains several residues involved in stabilizing the open state of the channel; some, including D110, are sites of disease-associated gating mutations. Structures from related proteins suggest that the position of CFTR's extracellular loops may change considerably during gating. To better understand the roles of ECL1 in CFTR function, we utilized functional cysteine cross-linking to determine the effects of modulation of D110C-CFTR and of a double mutant of D110C with K892C in extracellular loop 4 (ECL4). The reducing agent DTT elicited a large potentiation of the macroscopic conductance of D110C/K892C-CFTR, likely due to breakage of a spontaneous disulfide bond between C110 and C892. DTT-reduced D110C/K892C-CFTR was rapidly inhibited by binding cadmium ions with high affinity, suggesting that these residues frequently come in close proximity in actively gating channels. Effects of DTT and cadmium on modulation of pore gating were demonstrated at the single-channel level. Finally, disulfided D110C/K892C-CFTR channels were found to be less sensitive than wild-type or DTT-treated D110C/K892C-CFTR channels to stimulation by IBMX, suggesting an impact of this conformational restriction on channel activation by phosphorylation. The results are best explained in the context of a model of CFTR gating wherein stable channel opening requires correct positioning of functional elements structurally influenced by ECL1.