Ion Channel Blockade

The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the ␣-subunit of nicotinic acetylcholine receptors and in particular the homomeric ␣7 nicotinic receptor. We report the isolation and characterization of an ␣-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the ␣7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the ␣3␤2 nAChR indicating that ␣-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of ␣3␤2 nAChRs. ␣-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17amino-acid, two-disulfide bridge peptide. The ligand is the first ␣-conotoxin with higher affinity for the closely related receptor subtypes, ␣3␤2 versus ␣6␤2, and selectively blocks these two subtypes when compared with ␣2␤2, ␣4␤2, and ␣1␤1␦⑀ nAChRs.

We have developed a computer model of human cardiac ventricular myocyte (CVM), including t-tubular and cleft spaces with the aim of evaluating the impact of accumulation-depletion of ions in restricted extracellular spaces on transmembrane ion transport and ionic homeostasis in human CVM. The model was based on available data from human CVMs. Under steady state, the effect of ion concentration changes in extracellular spaces on [Ca 2+ ] i -transient was explored as a function of critical fractions of ion transporters in t-tubular membrane (not documented for human CVM). Depletion of Ca 2+ and accumulation of K + occurring in extracellular spaces slightly affected the transmembrane Ca 2+ flux, but not the action potential duration (APD 90 ). The [Ca 2+ ] i -transient was reduced (by 2%-9%), depending on the stimulation frequency, the rate of ion exchange between t-tubules and clefts and fractions of ion-transfer proteins in the t-tubular membrane. Under non-steady state, the responses of the model to changes of stimulation frequency were analyzed. A sudden increase of frequency (1-2.5 Hz) caused a temporal decrease of [Ca 2+ ] in both extracellular spaces, a reduction of [Ca 2+ ] i -transient (by 15%) and APD 90 (by 13 ms). The results reveal different effects of activity-related ion concentration changes in human cardiac t-tubules (steady-state effects) and intercellular clefts (transient effects) in the modulation of membrane ion transport and Ca 2+ turnover.

Using a three dimensional relativistic particle-in-cell code we have performed numerical experiments of plasma shells colliding at relativistic velocities. Such scenarios are found in many astrophysical objects e.g. the relativistic outflow from gamma ray bursts, active galactic nuclei jets and supernova remnants. We show how a Weibel like two-stream instability is capable of generating smallscale magnetic filaments with strength up to percents of equipartition. Such field topology is ideal for the generation of jitter radiation as opposed to synchrotron radiation. We also explain how the field generating mechanism involves acceleration of electrons to power law distributions (N (γ) ∝ γ -p ) through a non-Fermi acceleration mechanism. The results add to our understanding of collisionless shocks.

The selectivity filter of the bacterial porin OmpF carries a small net charge close to ÿ1 e and is therefore only slightly cation-selective. Calcium channels, on the other hand, contain four negatively charged glutamates, the EEEE-locus, and are among the most selective cation channels known. We aimed to turn the essentially nonselective OmpF into a Ca 21 -selective channel. To that end, two additional glutamates (R42E and R132E) were introduced in the OmpF constriction zone that already contains D113 and E117. Mutant OmpF containing this DEEE-locus has a high Ca 21 over Cl ÿ selectivity and a Na 1 current with a strongly increased sensitivity to 1 mM Ca 21 . The charge/space competition model, initially applied to the L-type Ca 21 channel, identifies the fixed charge and filter volume as key determinants of ion selectivity, with the precise atomic arrangement having only second-order effects. By implication, the reproduction of fixed charge and filter volume should transform two channels into channels of similar selectivity, even if the two belong to entirely different ion channel families, as is the case for OmpF and the L-type Ca 21 channel. The results presented here fit quite well in the framework of charge/space competition theory.

The influenza B virus BM2 proton-selective ion channel is essential for virus uncoating, a process that occurs in the acidic environment of the endosome. The BM2 channel causes acidification of the interior of the virus particle, which results in dissociation of the viral membrane protein from the ribonucleoprotein core. The BM2 protein is similar to the A/M2 protein ion channel of influenza A virus (A/M2) in that it contains an HXXXW motif. Unlike the A/M2 protein, the BM2 protein is not inhibited by the antiviral drug amantadine. We used mutagenesis to ascertain the pore-lining residues of the BM2 ion channel. The specific activity (relative to wild type), reversal voltage, and susceptibility to modification by (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide of cysteine mutant proteins were measured in oocytes. It was found that mutation of transmembrane domain residues Ser 9 , Ser 12 , Phe 13 , Ser 16 , His 19 , and Trp 23 to cysteine were most disruptive for ion channel function. These cysteine mutants were also most susceptible to (2aminoethyl)-methane thiosulfonate and N-ethylmaleimide modification. Furthermore, considerable amounts of dimer were formed in the absence of oxidative reagents when cysteine was introduced at positions Ser 9 , Ser 12 , Ser 16 , or Trp 23 . Based on these experimental data, a BM2 transmembrane domain model is proposed. The presence of polar residues in the pore is a probable explanation for the amantadine insensitivity of the BM2 protein and suggests that related but more polar compounds might serve as useful inhibitors of the protein.

Background-Recent genome-wide association studies have identified a large number of genetic risk factors for schizophrenia (SCZ) featuring ion channels and calcium transporters. For some of these risk factors, independent prior investigations have examined the effects of genetic alterations on the cellular electrical excitability and calcium homeostasis. In the present proof-ofconcept study, we harnessed these experimental results for modeling of computational properties on layer V cortical pyramidal cells and identified possible common alterations in behavior across SCZ-related genes. Methods-We applied a biophysically detailed multicompartmental model to study the excitability of a layer V pyramidal cell. We reviewed the literature on functional genomics for variants of genes associated with SCZ and used changes in neuron model parameters to represent the effects of these variants. Results-We present and apply a framework for examining the effects of subtle single nucleotide polymorphisms in ion channel and calcium transporter-encoding genes on neuron excitability. Our analysis indicates that most of the considered SCZ-related genetic variants affect the spiking behavior and intracellular calcium dynamics resulting from summation of inputs across the dendritic tree.

Cancer has recently been proposed as a type of channelopathy due to the aberrant expression of various ion channels. Voltage-gated potassium (K +) channels (VGKCs) are notably upregulated during tumor proliferation, while voltage-gated sodium (Na +) channels are predominantly associated with the invasive stage of cancer progression. Among these, the Kv10.1 channel has been found to be overexpressed in breast cancer, making it a promising therapeutic target. 4-Aminopyridine (4-AP), a non-selective voltage-gated potassium channel blocker, has emerged as a potential novel agent for breast cancer treatment. In this study, we aimed to elucidate the mechanism of action of 4-aminopyridine in breast cancer cells. To investigate the involvement of various cell death pathways, cycloheximide (CHX) (a paraptosis inhibitor), Z-VAD-FMK (a pan-caspase inhibitor), and 2-Aminoethoxydiphenyl borate (2-APB) (a phosphoinositide 3-kinase [PI3K] inhibitor) were employed. Experiments were conducted using the MCF-7 human breast cancer cell line and the L929 mouse fibroblast cell line as a healthy control. Assessments included cell viability assays, intracellular calcium (Ca 2+) and K + concentration measurements, and plasma membrane potential analysis. Our findings aim to contribute to the understanding of the therapeutic potential and cellular effects of VGKC blockers, particularly 4-aminopyridine, in breast cancer treatment strategies.

Fragment-based lead discovery (FBLD) Chemogenomics Serotonin 5-HT 3A receptor Histamine H 4 receptor Dual activity ligand G-protein coupled receptor (GPCR) Ligand gated ion channel (LGIC) a b s t r a c t A fragment library was screened against the G protein-coupled histamine H 4 receptor (H 4 R) and the ligand-gated ion channel serotonin 5-HT 3A (5-HT 3A R). Interestingly, significant overlap was found between H 4 R and 5-HT 3A R hit sets. The data indicates that dual active H 4 R and 5 HT 3A R fragments have a higher complexity than the selective compounds which has important implications for chemical genomics approaches. The results of our fragment-based library screening study illustrate similarities in ligand recognition between H 4 R and 5-HT 3A R and have important consequences for selectivity profiling in ongoing drug discovery efforts on H 4 R and 5-HT 3A R. The affinity profiles of our fragment screening studies furthermore match the chemical properties of the H 4 R and 5-HT 3A R binding sites and can be used to define molecular interaction fingerprints to guide the in silico prediction of protein-ligand interactions and structure.

We explore stochastic models for the study of ion transport in biological cells. Analysis of these models explains and explores an interesting feature of ion transport observed by biophysicists. Namely, the average time it takes ions to cross certain ion channels is the same in either direction, even if there is an electric potential difference across the channels. It is shown for simple single ion models that the distribution of a path ͑i.e., the history of location versus time͒ of an ion crossing the channel in one direction has the same distribution as the time-reversed path of an ion crossing the channel in the reverse direction. Therefore, not only is the mean duration of these paths equal, but other measures, such as the variance of passage time or the mean time a path spends within a specified section of the channel, are also the same for both directions of traversal. The feature is also explored for channels with interacting ions. If a system of interacting ions is in reversible equilibrium ͑net flux is zero͒, then the equivalence of the left-to-right trans paths with the time-reversed right-to-left trans paths still holds. However, if the system is in equilibrium, but not reversible equilibrium, then such equivalence need not hold.

We explore stochastic models for the study of ion transport in biological cells. Analysis of these models explains and explores an interesting feature of ion transport observed by biophysicists. Namely, the average time it takes ions to cross certain ion channels is the same in either direction, even if there is an electric potential difference across the channels. It is shown for simple single ion models that the distribution of a path ͑i.e., the history of location versus time͒ of an ion crossing the channel in one direction has the same distribution as the time-reversed path of an ion crossing the channel in the reverse direction. Therefore, not only is the mean duration of these paths equal, but other measures, such as the variance of passage time or the mean time a path spends within a specified section of the channel, are also the same for both directions of traversal. The feature is also explored for channels with interacting ions. If a system of interacting ions is in reversible equilibrium ͑net flux is zero͒, then the equivalence of the left-to-right trans paths with the time-reversed right-to-left trans paths still holds. However, if the system is in equilibrium, but not reversible equilibrium, then such equivalence need not hold.

In the E-157 experiment now being conducted at the Stanford Linear Accelerator Center, a 30 GeV electron beam of 2×1010 electrons in a 0.65-mm-long bunch is propagated through a 1.4-m-long lithium plasma of density up to 2×1014 e−/cm3. The initial beam density is greater than the plasma density, and the head of the bunch expels the plasma electrons leaving behind a uniform ion channel with transverse focusing fields of up to several thousand tesla per meter. The initial transverse beam size with σ=50–100 μm is larger than the matched size of 5 μm resulting in up to three beam envelope oscillations within the plasma. Time integrated optical transition radiation is used to study the transverse beam profile immediately before and after the plasma and to characterize the transverse beam dynamics as a function of plasma density. The head of the bunch deposits energy into plasma wakes, resulting in longitudinal accelerating fields which are witnessed by the tail of the same bunch. A time-...

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via...

Membrane ion channels and synapses are among the most important computational elements of nerve cells. Both have stochastic components that are reflected in random fluctuations of the membrane potential. We measured the spectral characteristics of membrane voltage noise in vitro at the soma and the apical dendrite of layer 4/5 (L4/5) neocortical neurons of rats near the resting potential. We found a remarkable similarity between the voltage noise power spectra at the soma and the dendrites, despite a marked difference in their respective input impedances. At both sites, the noise levels and the input impedance are voltage dependent; in the soma, the noise level increased from sigma = 0.33 +/- 0.28 mV at 10 mV hyperpolarization from the resting potential to sigma = 0.59 +/- 0.3 at a depolarization of 10 mV. At the dendrite, the noise increased from sigma = 0.34 +/- 0.28 to sigma = 0.56 +/- 0.30 mV, respectively. TTX reduced both the input impedance and the voltage noise, and eliminat...

1) Background: Virtual screening campaigns require target structures in which the pockets are properly arranged for binding. Without these, MD simulations can be used to relax the available target structures, optimizing the fine architecture of their binding sites. Among the generated frames, the best structures can be selected based on available experimental data. Without experimental templates, the MD trajectories can be filtered by energy-based criteria or sampled by systematic analyses. (2) Methods: A blind and methodical analysis was performed on the already reported MD run of the hTRPM8 tetrameric structures; a total of 50 frames underwent docking simulations by using a set of 1000 ligands including 20 known hTRPM8 modulators. Docking runs were performed by LiGen program and involved the frames as they are and after optimization by SCRWL4.0. For each frame, all four monomers were considered. Predictive models were developed by the EFO algorithm based on the sole primary LiGen scores. (3) Results: On average, the MD simulation progressively enhances the performance of the extracted frames, and the optimized structures perform better than the non-optimized frames (EF1% mean: 21.38 vs. 23.29). There is an overall correlation between performances and volumes of the explored pockets and the combination of the best performing frames allows to develop highly performing consensus models (EF1% = 49.83). ( ) Conclusions: The systematic sampling of the entire MD run provides performances roughly comparable with those previously reached by using rationally selected frames. The proposed strategy appears to be helpful when the lack of experimental data does not allow an easy selection of the optimal structures for docking simulations. Overall, the reported docking results confirm the relevance of simulating all the monomers of an oligomer structure and emphasize the efficacy of the SCRWL4.0 method to optimize the protein structures for docking calculations.

We have isolated a rat cDNA clone that displays 75% amino acid homology with the mouse 6 and rat K opioid receptors. The cDNA (designated pRMuR-12) encodes a protein of 398 amino acids comprising, in part, seven hydrophobic domains similar to those described for other C protein-linked receptors. Data from binding assays conducted with COS-1 cells transiently transfected with a CMV mammalian expression vector containing the full coding region of pRMuR-12 demonstrated p receptor selectivity. In situ hybridization mRNA analysis revealed an mRNA distribution in rat brain that corresponds well to the distribution of binding sites labeled with p-selective ligands. Based upon these observations, we conclude that pRMuR-12 encodes a p opioid receptor.

The cytotoxic effect of microcin E492, a low-molecular-mass channel-forming bacteriocin (7,887 Da) produced by a strain of Klebsiella pneumoniae , was characterized in HeLa cells. At low (5 μg/ml) and intermediate (10 μg/ml) concentrations, microcin E492 induced biochemical and morphological changes typical of apoptosis, such as cell shrinkage, DNA fragmentation, extracellular exposure of phosphatidylserine, caspase activation, and loss of mitochondrial membrane potential. Treatment with zVAD-fmk, a general caspase inhibitor, completely blocked the cytotoxic effect of this bacteriocin. At higher microcin concentrations (>20 μg/ml) a necrotic phenotype was observed. Induction of apoptosis by microcin E492 was associated with the release of calcium from intracellular stores, probably after microcin-triggered ion channel formation. Microcin E492 also presented a cytotoxic effect on Jurkat and RJ2.25 cells, but had no effect on KG-1 cells nor on a primary culture of human tonsil endo...

Desensitization is a common feature of ligand-gated ion channels, although the molecular cause varies widely between channel types. Mutations that greatly reduce or nearly abolish desensitization have been described for many ligand-gated ion channels, including glutamate, GABA, glycine, and nicotinic receptors, but not for acid-sensing ion channels (ASICs) until recently. Mutating Gln276 to a glycine (Q276G) in human ASIC1a was reported to mostly abolish desensitization at both the macroscopic and the single channel levels, potentially providing a valuable tool for subsequent studies. However, we find that in both human and chicken ASIC1, the effect of Q276G is modest. In chicken ASIC1, the equivalent Q277G slightly reduces desensitization when using pH 6.5 as a stimulus but desensitizes, essentially like wild-type, when using more acidic pH values. In addition, steady-state desensitization is intact, albeit right-shifted, and recovery from desensitization is accelerated. Molecular ...

Desensitization is a common feature of ligand-gated ion channels, although the molecular cause varies widely between channel types. Mutations that greatly reduce or nearly abolish desensitization have been described for many ligand-gated ion channels, including glutamate, GABA, glycine, and nicotinic receptors, but not for acid-sensing ion channels (ASICs) until recently. Mutating Gln276 to a glycine (Q276G) in human ASIC1a was reported to mostly abolish desensitization at both the macroscopic and the single channel levels, potentially providing a valuable tool for subsequent studies. However, we find that in both human and chicken ASIC1, the effect of Q276G is modest. In chicken ASIC1, the equivalent Q277G slightly reduces desensitization when using pH 6.5 as a stimulus but desensitizes, essentially like wild-type, when using more acidic pH values. In addition, steady-state desensitization is intact, albeit right-shifted, and recovery from desensitization is accelerated. Molecular ...

The interaction between energetic electrons and a circularly polarized laser pulse inside an ion channel is studied. Laser radiation can be resonantly absorbed by electrons executing betatron oscillations in the ion channel and absorbing angular momentum from the laser. The absorbed angular momentum manifests itself as a strong axial magnetic field (inverse Faraday effect). The magnitude of this magnetic field is calculated and related to the amount of the absorbed energy. Absorbed energy and generated magnetic field are estimated for the small and large energy gain regimes. Qualitative comparisons with recent experiments are also made.

Introduction: Neurofibromatosis Type I (NF1), also known as von Recklinghausen disease, is an autosomal dominant disorder that affects one in every 3000 births. There is no gender or race predilection seen in NF1 patients. Common clinical features in...

Background: Cardiomyocytes located at the ischemic border zone of infarcted ventricle are accompanied by redistribution of gap junctions, which mediate electrical transmission between cardiomyocytes. This ischemic border zone provides an arrhythmogenic substrate. It was also shown that sodium (Na + ) channels are redistributed within myocytes located in the ischemic border zone. However, the roles of the subcellular redistribution of Na + channels in the arrhythmogenicity under ischemia remain unclear. Methods: Computer simulations of excitation conduction were performed in a myofiber model incorporating both subcellular Na + channel redistribution and the electric field mechanism, taking into account the intercellular cleft potentials. We found in the myofiber model that the subcellular redistribution of the Na + channels under myocardial ischemia, decreasing in Na + channel expression of the lateral cell membrane of each myocyte, decreased the tissue excitability, resulting in conduction slowing even without any ischemia-related electrophysiological change. The conventional model (i.e., without the electric field mechanism) did not reproduce the conduction slowing caused by the subcellular Na + channel redistribution. Furthermore, Na + channel blockade with the coexistence of a non-ischemic zone with an ischemic border zone expanded the vulnerable period for reentrant tachyarrhythmias compared to the model without the ischemic border zone. Na + channel blockade tended to cause unidirectional conduction block at sites near the ischemic border zone. Thus, such a unidirectional conduction block induced by a premature stimulus at sites near the ischemic border zone is associated with the initiation of reentrant tachyarrhythmias. Conclusions: Proarrhythmia of Na + channel blockade in patients with old myocardial infarction might be partly attributable to the ischemia-related subcellular Na + channel redistribution.

in the COMPASS Tokamak J. Mlynar, O. Ficker, M. Vlainic, V. Weinzettl, M. Imrisek, R. Paprok, M. Rabinski, M. Jakubowski , M. Tomes, M. Peterka, R. Panek and the COMPASS Team Institute of Plasma Physics AS CR, Prague, Czech Republic Czech Technical University in Prague, Faculty of Nuclear Sciences and Physical Engineering, Czech Republic Department of Applied Physics, Ghent University, Ghent, Belgium Charles University in Prague, Faculty of Mathematics and Physics, Prague, Czech Republic 5 National Centre for Nuclear Research (NCBJ), Otwock-Swierk, Poland

We report on a simple and high‐yield manufacturing process for silicon planar patch‐clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high‐quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high‐impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high‐fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole‐cell current recordings obtained from a voltage‐clamp stimulation protocol, and in current‐clamp mode we report action potentials stimulated by membrane depola...

Human brain development is a continuum governed by differential gene expression. Therefore, we proceeded to identify genes selectively expressed in the developing brain. Using differential display and library screening, a novel rat cDNA, neuronatin, was identified and used to screen a human fetal brain cDNA library. Human neuronatin cDNA was isolated and sequenced. The cDNA was 1159 bp long and corresponded in size to the 1.25 kb message detected on Northern analysis. Neuronatin mRNA was selectively expressed in human brain during fetal development, but became repressed in adulthood. When studied in the rat, neuronatin mRNA first appeared at mid-gestation in association with the onset of neurogenesis, becoming most pronounced later in development when neuroepithelial proliferation and neuroblast commitment are manifest, and declined postnatally coinciding with the completion of neurogenesis. The deduced protein has two distinct domains, a hydrophobic N-terminal and basic C-terminal rich in arginine residues. Both the amino acid sequence and secondary structure of this amphipathic polypeptide exhibited homology to PMP1 and phospholamban, members of the "proteolipid' class of proteins which function as regulatory subunits of membrane channels. The neuronatin gene, 3973 bases long, contains in its 5'-flanking region a neural restrictive silencer element which may govern neuronspecific expression. Based on screening a somatic cell hybrid panel, neuronatin gene was assigned to chromosome-20. And, using deletion constructs of chromosome-20 and fluorescence in situ hybridization, neuronatin was localized to chromosome-20q11.2-12. In conclusion, neuronatin is a novel human gene that is developmentally regulated and expressed in the brain. The deduced protein is a proteolipid that may function as a unique regulator of ion channels during brain development. The definitive localization of neuronatin to human chromosome 20q11.2-12 provides the basis to investigate this gene as a candidate in neuro-developmental diseases that may also map to this region.

In the animal world, the regulation of ion channels by phosphoinositides (PIs) has been investigated extensively, demonstrating a wide range of channels controlled by phosphatidylinositol (4,5)bisphosphate (PtdInsP2). To understand PI regulation of plant ion channels, we examined the in planta effect of PtdInsP2 on the K+-efflux channel of tobacco (Nicotiana tabacum), NtORK (outward-rectifying K channel). We applied a patch clamp in the whole-cell configuration (with fixed “cytosolic” Ca2+ concentration and pH) to protoplasts isolated from cultured tobacco cells with genetically manipulated plasma membrane levels of PtdInsP2 and cellular inositol (1,4,5)trisphosphate: “Low PIs” had depressed levels of these PIs, and “High PIs” had elevated levels relative to controls. In all of these cells, K channel activity, reflected in the net, steady-state outward K+ currents (IK), was inversely related to the plasma membrane PtdInsP2 level. Consistent with this, short-term manipulations decrea...

SummaryWe present near-atomic-resolution cryo-EM structures of the mammalian voltage-gated potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and sodium-bound states at 3.2 Å, 2.5 Å, 2.8 Å, and 2.9Å. These structures, all obtained at nominally zero membrane potential in detergent micelles, reveal distinct ion-occupancy patterns in the selectivity filter. The first two structures are very similar to those reported in the related Shaker channel and the much-studied Kv1.2-2.1 chimeric channel. On the other hand, two new structures show unexpected patterns of ion occupancy. First, in the toxin-blocked channel α-Dendrotoxin, like Charybdotoxin, is seen to attach to the negatively-charged channel outer mouth, and a lysine residue penetrates into the selectivity filter. Penetration by α-Dendrotoxin is however deeper than with Charybdotoxin, occupying two of the four ion-binding sites. Second, a structure of Kv1.2 in Na+solution does not show collapse of the selectivity filt...

Temperature sensing is one of the most ancient capabilities of living organisms and is essential to sustain life, because failures in avoiding extreme noxious temperatures can result in tissue damage or death. A subset of members of the transient receptor potential (TRP) ion channel family is finely tuned to detect temperatures ranging from extreme cold to noxious heat, giving rise to thermoTRP channels. Structural and functional experiments have shown that thermoTRP channels are allosteric proteins, containing different domains that sense changes in temperature, among other stimuli, triggering pore opening. Although temperature-dependence is well characterized in thermoTRP channels, the molecular nature of the temperature-sensing elements remains unknown. Importantly, thermoTRP channels are involved in pain sensation, related to pathological conditions. Here we provide an overview of thermoTRP channels activation. We also discuss the structural and functional evidence supporting th...

Bacillus thuringiensis is a Gram-positive bacterium, widely used in agriculture as a biological pesticide. The biocidal activity mainly resides in a parasporal protein inclusion body, or crystal. The inclusion is composed of one or more types of ␦-endotoxins (Cry and Cyt proteins). Cry proteins are selectively toxic to different species from several invertebrate phyla: arthropods (mainly insects), nematodes, flatworms and protozoa. The mode of action of the insecticidal proteins is still a matter of investigation; generally, the active toxin is supposed to bind specific membrane receptors on the insect midgut brush-border epithelium, leading to intestinal cell lysis and subsequent insect death by starvation or septicemia. The toxin-encoding cry genes have been extensively studied and expressed in a large number of prokaryotic and eukaryotic organisms. The expression of such genes in transgenic plants has provided a powerful alternative for crop protection.

Many encoded gene products responsible for neurodevelopmental disorders (NDs) like autism spectrum disorders (ASD), schizophrenia (SCZ), intellectual disability (ID), and idiopathic generalized epilepsy (IGE) converge on networks controlling synaptic function. An increase in KCC2 (SLC12A5) Cl(-) transporter activity drives the developmental GABA excitatory-inhibitory sequence, but the role of KCC2 in human NDs is essentially unknown. Here, we report two rare, non-synonymous (NS), functionally-impairing variants in the KCC2 C-terminal regulatory domain (CTRD) in human ASD (R952H and R1049C) and SCZ (R952H) previously linked with IGE and familial febrile seizures, and another novel NS KCC2 variant in ASD (R1048W) with highly-predicted pathogenicity. Exome data from 2517 simplex families in the ASD Simon Simplex Collection (SSC) revealed significantly more KCC2 CTRD variants in ASD cases than controls, and interestingly, these were more often synonymous and predicted to disrupt or intr...

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels involved in the modulation of essential brain functions such as memory, learning, and attention. Homomeric α7 nAChR, formed exclusively by five identical α7 subunits, is involved in rapid synaptic transmission, whereas the heteromeric oligomers composed of α7 in combination with β subunits display metabotropic properties and operate in slower time frames. At the cellular level, the activation of nAChRs allows the entry of Na+ and Ca2+; the two cations depolarize the membrane and trigger diverse cellular signals, depending on the type of nAChR pentamer and neurons involved, the location of the intervening cells, and the networks of which these neuronal cells form part. These features make the α7 nAChR a central player in neurotransmission, metabolically associated Ca2+-mediated signaling, and modulation of diverse fundamental processes operated by other neurotransmitters in the brain. Due to its ubiqu...

The topography of nicotinic acetylcholine receptor (AChR) membrane-embedded domains and the relative affinity of lipids for these protein regions were studied using fluorescence methods. Intact Torpedo californica AChR protein and transmembrane peptides were derivatized with N-(1-pyrenyl)maleimide (PM), purified, and reconstituted into asolectin liposomes. Fluorescence mapped to proteolytic fragments consistent with PM labeling of cysteine residues in ␣M1, ␣M4, ␥M1, and ␥M4. The topography of the pyrene-labeled Cys residues with respect to the membrane and the apparent affinity for representative lipids were determined by differential fluorescence quenching with spin-labeled derivatives of fatty acids, phosphatidylcholine, and the steroids cholestane and androstane. Different spin label lipid analogs exhibit different selectivity for the whole AChR protein and its transmembrane domains. In all cases labeled residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear ␣-helical structure, but not so for M1, for which "classical" models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of non-helical structure, and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily.

> Les canaux ioniques dépendants du voltage (VGIC) représentent une large famille de protéines canalaires assurant le passage d'ions de part et d'autre de la membrane plasmique. Les VGIC ont la particularité d'être dotés d'un domaine senseur de voltage (VSD). Cette unité régule l'activité des VGIC en fonction des variations de potentiel membranaire. Récemment, des mutations décrites dans le VSD ont conduit à la découverte d'une nouvelle voie de perméation directement localisée au sein de ce domaine : le pore oméga. Au vu du grand nombre de protéines présentant un VSD et de leur rôle crucial dans diverses fonctions physiologiques, déchiffrer le potentiel pathogénique du pore oméga s'avère désormais essentiel. < Le mouvement du segment S4 possédant de 3 à 7 charges positives (apportées par des arginines et des lysines) induit un changement de conformation du pore aboutissant à son ouverture, et donc au passage des ions. Récemment, plusieurs études ont montré que des modifications du VSD peuvent générer une autre voie pour le passage des ions appelée pore oméga. L'objectif de cette revue est de présenter le concept de pore oméga, ses propriétés biophysiques ainsi que son potentiel pathologique.

Epithelia of the cornea, lens and retina contain a vast array of ion channels and pumps. Together they produce a polarized flow of ions in and out of cells, as well as across the epithelia. These naturally occurring ion fluxes are essential to the hydration and metabolism of the ocular tissues, especially for the avascular cornea and lens. The directional transport of ions generates electric fields and currents in those tissues. Applied electric fields affect migration, division and proliferation of ocular cells which are important in homeostasis and healing of the ocular tissues. Abnormalities in any of those aspects may underlie many ocular diseases, for example chronic corneal ulcers, posterior capsule opacity after cataract surgery, and retinopathies. Electric fieldinducing cellular responses, termed electrical signaling here, therefore may be an unexpected yet powerful mechanism in regulating ocular cell behavior. Both endogenous electric fields and applied electric fields could be exploited to regulate ocular cells. We aim to briefly describe the physiology of the naturally occurring electrical activities in the corneal, lens, and retinal epithelia, to provide experimental evidence of the effects of electric fields on ocular cell behaviors, and to suggest possible clinical implications.

In the paper Several integral inequalities published in J. Inequal. Pure Appl. Math. 1 (2000), no. 2, Art. 19 (. 001_00.html) and RGMIA Res. Rep. Coll. 2(7) (1999), Art. 9, 1039-1042 () by F. Qi, an open problem was posed. In this article we give the solution and further generalizations of this problem. Reverse inequalities to the posed one are considered and, finally, the derived results are extended to weighted integral inequalities.

Introduction: Neurofibromatosis Type I (NF1), also known as von Recklinghausen disease, is an autosomal dominant disorder that affects one in every 3000 births. There is no gender or race predilection seen in NF1 patients. Common clinical features in...

This paper deals with the experimental construction, stochastic modeling, and statistical signal processing of a novel, artificially constructed biosensor comprised of biological ion channels. Such nanoscale biosensors have been built by incorporating dimeric gramicidin A (bis-gA) ion channels into bilayer membranes of giant unilamellar liposomes, and then excising small patches of the membrane loaded with ion channels. We present a stochastic model for the response of the biosensor and present statistical model validation tests to verify the adequacy of the model. We show that in the presence of specific target molecules, the statistics of the gating mechanisms of the gA channels are altered. By capturing the change in real time, we devise a maximum-likelihood detector to detect the presence of target molecules. To test the sensitivity of this model, we conducted patch-clamp experiments with two compounds known to inhibit conduction of the gA channels. We found experimentally that the real-time detection algorithm was able to accurately identify the addition of the compounds even when the alterations in the patch-clamp recordings were very small. This algorithm provides the sensitive detection system for ongoing development of lipid-based nanosensors.

VDAC is a voltage-gated anion channel located in the mitochondrial outer membrane, presumably participating in controlling aerobic metabolism. Three distinct wheat vdac cDNAs were expressed in a vdac-minus yeast strain and successfully complemented its defective phenotype. The growth curves of these transformants were different. The wheat channel isoforms were functionally characterized following purification from yeast mitochondria and reconstitution into soybean phospholipid planar membranes. All three isoforms yielded voltagedependent anion channels with electrophysiological parameters comparable to known VDACs. Isoform-related functional features (specific conductance levels, kinetics, and gating behaviors) are reported for the first time in VDACs. The presence (or absence) of protease inhibitors during the purification procedure, and the use of Pronase on reconstituted channels, strongly suggest that some of the unique wheat VDAC properties are due to co-purification of a yeast channel-modulating protein. Its effects, different from the reported functional interactions of the channel with hexo-or creatine kinases, could not be mimicked by the protein termed VDAC modulator, indicating the presence of a novel VDAC modulator. In addition to strengthening VDAC presumed role in metabolism, the functional diversity of the channels (as shown here in two different systems) implies a highly dynamic outer membrane permeability. Our results are consistent with VDAC functioning as a heteromer including one pore protein and other modulating subunits.

We investigated the effects of a 7-day verapamil pretreatment (VPT, 7.5 mg/kg bodyweight subcutaneously every 12 h) on ionic currents and molecular mechanisms underlying tachycardia-induced early electrical remodeling after 24-h rapid atrial pacing (RAP, 600 bpm) in rabbit atrium. Animals were divided into four groups (n=6 each group): control (not paced, no verapamil), paced only, verapamil only and verapamil and paced, respectively. VPT doubled ICa,L [7.0±0.7 pA/pF (control) vs 14.2±0.6 pA/pF (verapamil only)]. RAP reduced ICa,L by 48% to 3.6± 0.7 pA/pF (paced only). RAP did not affect ICa,L in verapamil-treated animals and averaged 15.3±0.2 pA/pF (paced and verapamil). RAP resulted in a significant decrease of the expression of the α1c subunit (-24.7%) and the β2A subunit (-13.3%), respectively. VPT led to a similar alteration of subunit expression as RAP ["control" vs "verapamil only", decrease of α1c subunit (-25.4%), but no significant change in β2A subunit expression]. However, after VPT, further diminishment of α1c and β2A subunit expression after rapid atrial pacing was absent. ("verapamil" vs "verapamil and paced", n=6 both groups). RAP decreased Ito [-45%, 51.5±3.9 pA/pF (control) vs 26.8±1.5 pA/pF (paced only)] and was not influenceable by VPT. IK1 was neither affected by RAP nor verapamil pretreatment. Downregulation of α1c and β2A subunit expression and the resulting decay of ICa,L current densities were prevented by verapamil. However, these effects are abolished by multiple other adverse effects of verapamil on atrial electrophysiology.

Phospholamban (PLN), a membrane protein present in the sarcoplasmic reticulum of cardiac myocytes, is a crucial regulator of cardiac function. It is known that PLN appears as a monomer and as a pentamer. However, the role of the PLN pentamer and its ability to generate an ion channel are a matter of debate. To address this issue we employed an experimental approach that combines electrochemical impedance spectroscopy and surface plasmon resonance measurements. In particular, we investigated the channel activity of wildtype PLN reconstituted in a tethered bilayer lipid membrane (tBLM) on a gold surface. Our results indicate that reconstituted PLN can generate ion-conducting channels in a tBLM. Experiments with a PLN monoclonal antibody support an oriented incorporation of PLN in the tBLM. We show that the binding of the antibody to the PLN cytoplasmic domain interferes with PLN channel activity.

Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with aerolysin-type architecture, and has been shown that co-administration of Cry46Ab with other mosquitocidal Cry toxins results in synergistic toxicity against Culex pipiens Coquillett (Diptera: Culicidae) mosquito larvae. Cry46Ab therefore is expected to find use in improving the insecticidal activity of B. thuringiensis-based bioinsecticides. In the present study, the mode of action of Cry46Ab was explored by single-channel measurements of Cry46Ab channel-pores. The single-channel conductances of channel-pores formed in planar lipid bilayers by Cry46Ab were determined to be 31.8 ± 2.7 pS in 150 mM NaCl and 24.2 ± 0.7 pS in 150 mM CaCl2. Ion-selectivity measurements revealed that the channel-pores formed by Cry46Ab were cation selective. The permeability ratio of K + to Cl -was approximately 4, and the preferences for cations were K + > Na + , K + > Ca 2+ , and Ca 2+ > Na + . A calcein release assay using liposomes suggested that Cry46Ab influences the integrity of membrane vesicles. Formation of cation-selective channel-pores has been observed with other insecticidal Cry toxins that have structures distinct from those of Cry46Ab; the capability of forming such pores may be a property required of insecticidal toxins.

TRPV4 is a non-selective cation channel that tunes the function of different tissues including the vascular endothelium, lung, chondrocytes, and neurons. GSK1016790A is the selective and potent agonist of TRPV4 and a pharmacological tool that is used to study the TRPV4 physiological function in vitro and in vivo. It remains unknown how the sensitivity of TRPV4 to this agonist is regulated. The spatial and temporal dynamics of receptors are the major determinants of cellular responses to stimuli. Membrane translocation has been shown to control the response of several members of the transient receptor potential (TRP) family of ion channels to different stimuli. Here, we show that TRPV4 stimulation with GSK1016790A caused an increase in [Ca 2+ ] i that is stable for a few minutes. Single molecule analysis of TRPV4 channels showed that the density of TRPV4 at the plasma membrane is controlled through two modes of membrane trafficking, complete, and partial vesicular fusion. Further, we show that the density of TRPV4 at the plasma membrane decreased within 20 min, as they translocate to the recycling endosomes and that the surface density is dependent on the release of calcium from the intracellular stores and is controlled via a PI3K, PKC, and RhoA signaling pathway.

Highlights d SPECTRUS is a general method for identifying dynamical domains in proteins d It performs spectral clustering of amino acids based on their distance fluctuations d It takes as input conformers from PDB entries or MD trajectories d The SPECTRUS web server and source code are available at spectrus.sissa.it

The dynamic range of ion composition spectrometers is limited by several factors, including saturation of particle counters and spillover of signals from highly dominant species into channels tuned to minor species. Instruments designed for composition measurements of hot plasmas in space can suffer greatly from both of these problems because of the wide energy range required and the wide disparity in fluxes encountered in various regions of interest. In order to detect minor ions in regions of very weak fluxes, geometry factors need to be as large as possible within the mass and volume resources available. As a result, problems with saturation by the dominant fluxes and spillover to minor-ion channels in plasma regions with intense fluxes become especially acute. This article reports on a technique for solving the dynamic-range problem in the few eV to several keV energy/charge range that is of central importance for space physics research where the dominant ion is of low mass/char...