Form Ion Channels Research Articles

Non-selective ion channel activity of polymorphic human islet amyloid polypeptide (amylin) double channels

Fundamental understanding of ion channel formation by amyloid peptides, which is strongly linked to cell toxicity, is very critical for (pre)clinical treatment of neurodegenerative diseases. Here, we combine atomistic simulations and experiments to demonstrate a broad range of conformational states of hIAPP double channels in lipid membranes. All individual channels display high selectivity for Cl(-) ions over cations, but the co-existence of polymorphic double channels of different conformations and orientations with different populations determines the non-ionic selectivity nature of the channels, which is different from the typical amyloid-β channels that exhibit Ca(2+) selective ion-permeable characteristics. This work provides a more complete physicochemical mechanism of amyloid-channel-induced toxicity.

CLIC4 regulates cell adhesion and β1 integrin trafficking.

Chloride intracellular channel protein 4 (CLIC4) exists in both soluble and membrane-associated forms, and is implicated in diverse cellular processes, ranging from ion channel formation to intracellular membrane remodeling. CLIC4 is rapidly recruited to the plasma membrane by lysophosphatidic acid (LPA) and serum, suggesting a possible role for CLIC4 in exocytic-endocytic trafficking. However, the function and subcellular target(s) of CLIC4 remain elusive. Here, we show that in HeLa and MDA-MB-231 cells, CLIC4 knockdown decreases cell-matrix adhesion, cell spreading and integrin signaling, whereas it increases cell motility. LPA stimulates the recruitment of CLIC4 to β1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both the internalization and the serum- or LPA-induced recycling of β1 integrin, but not for EGF receptor trafficking. Furthermore, we show that CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of β1 integrin trafficking. Our results define CLIC4 as a regulator of Rab35 activity and serum- and LPA-dependent integrin trafficking.

Dermcidin, an anionic antimicrobial peptide: influence of lipid charge, pH and Zn2+on its interaction with a biomimetic membrane

The mechanism of membrane permeabilization by dermcidin (DCD-1L), an antimicrobial peptide present in human sweat, was investigated at a mercury-supported monolayer of dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylserine (DOPS) and at a mercury-supported tethered bilayer lipid membrane (tBLM) consisting of a thiolipid (DPTL) with a DOPC or DOPS monolayer self-assembled on top of it. In an unbuffered solution of pH 5.4, DCD-1L is almost neutral and permeabilizes a DPTL/DOPS tBLM at transmembrane potentials, ϕtrans, which are physiological. In a pH 7 buffer solution DCD-1L bears two negative charges and has no effect on a DPTL/DOPC tBLM, whereas it permeabilizes a DPTL/DOPS tBLM only outside the physiological ϕtrans range; however, the presence of zinc ion induces DCD-1L to permeabilize the DPTL/DOPS tBLM at physiological ϕtrans values. The effect of zinc ions suggests a DCD-1L conformation with its positive N-terminus embedded in the lipid bilayer and the negative C terminus floating on the membrane surface. This conformation can be stabilized by a zinc ion bridge between the His(38) residue of the C terminus and the carboxyl group of DOPS. Chronocoulometric potential jumps from ϕtrans ≅ +160 mV to sufficiently negative values yield charge transients exhibiting a sigmoidal shape preceded by a relatively long "foot". This behavior is indicative of ion-channel formation characterized by disruption of DCD-1L clusters adsorbed on top of the lipid bilayer, incorporation of the resulting monomers and their aggregation into hydrophilic pores by a mechanism of nucleation and growth.

Direct recording and molecular identification of the calcium channel of primary cilia.

SummaryA primary cilium is a solitary slender non-motile protuberance of structured microtubules (9+0) enclosed by plasma membrane1. Housing components of the cell division apparatus between cell divisions, they also serve as specialized compartments for calcium signaling2 and Hedgehog (Hh) signaling pathways3. Specialized sensory cilia such as retinal photoreceptors and olfactory cilia employ diverse ion channels4-7. An ion current has been measured from primary cilia of kidney cells8 but the responsible genes have not been identified. The polycystin proteins (PC, PKD), identified in linkage studies of polycystic kidney disease9, are candidate channels divided into two structural classes: 11-transmembrane (TM) proteins (PKD1, PKD1-L1 and PKD1-L2) remarkable for a large extracellular N-terminus of putative cell adhesion domains and a GPCR proteolytic site, and the 6-TM channel proteins (PKD2, PKD2-L1, PKD2-L2; TRPPs). Evidence suggests that the PKD1s associate with the PKD2s via coiled-coil domains10-12. Here, we employ a transgenic mouse in which only cilia express a fluorophore and employ it to directly record from primary cilia and demonstrate that PKD1-L1 and PKD2-L1 form ion channels at high densities in several cell types. In conjunction with the companion manuscript2, we show that the PKD1-L1/PKD2-L1 heteromeric channel establishes the cilia as a unique calcium compartment within cells that modulates established Hedgehog pathways.

NMR localization of the O-mycoloylation on PorH, a channel forming peptide from Corynebacterium glutamicum

PorH and PorA are two small peptides that, in complex, form a voltage-dependent ion channel in the outer membrane of Corynebacterium glutamicum. Specific post-translational modifications on PorA and PorH are required for the formation of a functional ion channel. The assignment of PorH proton NMR chemical shifts in DMSO, allowed identifying unambiguously the exact position of the PorH O-mycoloylation on Ser 56 side chain. This was further confirmed by site directed mutagenesis and mass spectrometry. Together with the previously published localization of PorA mycoloylation, this provides the complete primary structure characterization of this outer membrane porin.

Molecular Dynamics Simulations of Homo-oligomeric Bundles Embedded Within a Lipid Bilayer

Using molecular dynamics simulations, we studied the structure, interhelix interactions, and dynamics of transmembrane proteins. Specifically, we investigated homooligomeric helical bundle systems consisting of synthetic α-helices with either the sequence Ac-(LSLLLSL)3-NH2 (LS2) or Ac-(LSSLLSL)3-NH2 (LS3). The LS2 and LS3 helical peptides are designed to have amphipathic characteristics that form ion channels in membrane. We simulated bundles containing one to six peptides that were embedded in palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayer and placed between two lamellae of water. We aim to provide a fundamental understanding of how amphipathic helical peptides interact with each other and their dynamical behaviors in different homooligomeric states. To understand structural properties, we examined the helix lengths, tilt angles of individual helices and the entire bundle, interhelix distances, interhelix cross-angles, helix hydrophobic-to-hydrophilic vector projections, and the average number of interhelix hydrophilic (serine–serine) contacts lining the pore of the transmembrane channel. To analyze dynamical properties, we calculated the rotational autocorrelation function of each helix and the cross-correlation of the rotational velocity between adjacent helices. The observed structural and dynamical characteristics show that higher order bundles containing four to six peptides are composed of multiple lower order bundles of one to three peptides. For example, the LS2 channel was found to be stable in a tetrameric bundle composed of a “dimer of dimers.” In addition, we observed that there is a minimum of two strong hydrophilic contacts between a pair of adjacent helices in the dimer to tetramer systems and only one strong hydrophilic interhelix contact in helix pairs of the pentamer and hexamer systems. We believe these results are general and can be applied to more complex ion channels, providing insight into ion channel stability and assembly.

Permeabilization of mercury-supported biomimetic membranes by amphotericin B and the role of calcium ions

The mechanism of membrane permeabilization by the antifungal heptaene macrolide amphotericin B (AmB) was investigated by using two different mercury-supported biomimetic membranes, namely a lipid self-assembled monolayer and a lipid bilayer tethered to the mercury surface through a hydrophilic spacer (tethered bilayer lipid membrane: tBLM). The disruptive permeabilization of a sterol-free mercury-supported lipid monolayer by low AmB concentrations, after a sufficiently long exposure to the macrolide, suggests a similar effect on the distal lipid monolayer of vesicular or bilayer lipid membranes. The notable permeabilization of a tBLM to Cd(II), with amalgam formation, by 0.6μM AmB complexed with sterols in a 1:3 molar ratio, as compared with the absence of any effect in the absence of sterols, suggests a direct participation of sterols in ion channel formation. The fact that the tBLM permeabilization requires the presence in solution of a freshly added AmB–sterol mixture and of Cd(II) or Ca2+ ions points out the notable role played by these divalent inorganic ions in the formation of AmB ion channels. Ion channels formed in a tBLM by AmB–ergosterol complexes are stable at physiological transmembrane potentials, whereas those formed by AmB–cholesterol complexes require application of non-physiological transmembrane potentials for their stability.

Membrane Partitioning of the Pore-Forming Domain of Colicin A. Role of the Hydrophobic Helical Hairpin

The colicins are bacteriocins that target Escherichia coli and kill bacterial cells through different mechanisms. Colicin A forms ion channels in the inner membranes of nonimmune bacteria. This activity resides exclusively in its C-terminal fragment (residues 387–592). The soluble free form of this domain is a 10 α-helix bundle. The hydrophobic helical hairpin, H8–H9, is buried inside the structure and shielded by eight amphipathic surface helices. The interaction of the C-terminal colicin A domain and several chimeric variants with lipidic vesicles was examined here by isothermal titration calorimetry. In the mutant constructions, natural sequences of the hydrophobic helices H8 and H9 were either removed or substituted by polyalanine or polyleucine. All the constructions fully associated with DOPG liposomes including the mutant that lacked helices H8 and H9, indicating that amphipathic rather than hydrophobic helices were the major determinants of the exothermic binding reactions. Alanine is not specially favored in the lipid-bound form; the chimeric construct with polyalanine produced lower enthalpy gain. On the other hand, the large negative heat capacities associated with partitioning, a characteristic feature of the hydrophobic effect, were found to be dependent on the sequence hydrophobicity of helices H8 and H9.

The ORF4a protein of human coronavirus 229E functions as a viroporin that regulates viral production

In addition to a set of canonical genes, coronaviruses encode additional accessory proteins. A locus located between the spike and envelope genes is conserved in all coronaviruses and contains a complete or truncated open reading frame (ORF). Previously, we demonstrated that this locus, which contains the gene for accessory protein 3a from severe acute respiratory syndrome coronavirus (SARS-CoV), encodes a protein that forms ion channels and regulates virus release. In the current study, we explored whether the ORF4a protein of HCoV-229E has similar functions. Our findings revealed that the ORF4a proteins were expressed in infected cells and localized at the endoplasmic reticulum/Golgi intermediate compartment (ERGIC). The ORF4a proteins formed homo-oligomers through disulfide bridges and possessed ion channel activity in both Xenopus oocytes and yeast. Based on the measurement of conductance to different monovalent cations, the ORF4a was suggested to form a non-selective channel for monovalent cations, although Li+ partially reduced the inward current. Furthermore, viral production decreased when the ORF4a protein expression was suppressed by siRNA in infected cells. Collectively, this evidence indicates that the HCoV-229E ORF4a protein is functionally analogous to the SARS-CoV 3a protein, which also acts as a viroporin that regulates virus production. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.

Peptide-induced membrane leakage by lysine derivatives of gramicidin A in liposomes, planar bilayers, and erythrocytes

Introducing a charged group near the N-terminus of gramicidin A (gA) is supposed to suppress its ability to form ion channels by restricting its head-to-head dimerization. The present study dealt with the activity of [Lys1]gA, [Lys3]gA, [Glu1]gA, [Glu3]gA, [Lys2]gA, and [Lys5]gA in model membrane systems (planar lipid bilayers and liposomes) and erythrocytes. In contrast to the Glu-substituted peptides, the lysine derivatives of gA caused non-specific liposomal leakage monitored by fluorescence dequenching of lipid vesicles loaded with carboxyfluorescein or other fluorescent dyes. Measurements of electrical current through a planar lipid membrane revealed formation of giant pores by Lys-substituted analogs, which depended on the presence of solvent in the bilayer lipid membrane. The efficacy of unselective pore formation in liposomes depended on the position of the lysine residue in the amino acid sequence, increasing in the row: [Lys2]gA<[Lys5]gA<[Lys1]gA<[Lys3]gA. The similar series of potency was exhibited by the Lys-substituted gA analogs in facilitating erythrocyte hemolysis, whereas the Glu-substituted analogs showed negligible hemolytic activity. Oligomerization of the Lys-substituted peptides is suggested to be involved in the process of nonselective pore formation.

Extramembrane Control of Ion Channel Peptide Assemblies, Using Alamethicin as an Example

Ion channels allow the influx and efflux of specific ions through a plasma membrane. Many ion channels can sense, for example, the membrane potential (the voltage gaps between the inside and the outside of the membrane), specific ligands such as neurotransmitters, and mechanical tension within the membrane. They modulate cell function in response to these stimuli. Researchers have focused on developing peptide- and non-peptide-based model systems to elucidate ion-channel protein functions and to create artificial sensing systems. In this Account, we employed a typical peptide that forms ion channels,alamethicin, as a model to evaluate our methodologies for controlling the assembly states of channel-forming molecules in membranes. As alamethicin self-assembles in membranes, it prompts channel formation, but number of peptide molecules in these channels is not constant. Using planar-lipid bilayer methods, we monitored the association states of alamethicin in real time. Many ligand-gated, natural-ion channel proteins have large extramembrane domains. As these proteins interact with specific ligands, those conformational alterations in the extramembrane domains are transmitted to the transmembrane, pore-forming domains to open and close the channels. We hypothesized that if we conjugated suitable extramembrane segments to alamethicin, ligand binding to the extramembrane segments could alter the structure of the extramembrane domains and influence the association states or association numbers of alamethicin in the membranes. We could then assess those changes by using single-channel current recording. We found that we could modulate channel assembly and eventual ion flux with attached leucine-zipper extramembrane peptide segments. Using conformationally switchable leucine-zipper extramembrane segments that respond to Fe(3+), we fabricated an artificial Fe(3+)-sensitive ion channel; a decrease in the helical content of the extramembrane segment led to an increase in the channel current. When we added a calmodulin C-terminus segment, we formed a channel that was sensitive to Ca(2+). This result demonstrated that we could prepare artificial channels that were sensitive to specific ligands by adding appropriate extramembrane segments from natural protein motifs that respond to external stimuli. In conclusion, our research points to the possibility of creating tailored sensor or signal transduction systems through the conjugation of a conformationally switchable extramembrane peptide/protein segment to a suitable transmembrane peptide segment.

Abstract B05: Targeting nonapoptotic cell death in chemoresistant patient-derived breast cancer cells

Abstract The development of anti-cancer therapeutics that target non-apoptotic pathways has received increasing attention from the scientific community in recent years. The prevalence of chemoresistance and the characterization of defective apoptotic pathways in cancer has promoted interest in the expansion of current therapeutic regimens to include drugs that induce non-apoptotic cell death. From a phenotypic screen we have identified a small molecule named C6 that induces caspase-independent, non-apoptotic cell death in chemoresistant patient-derived breast cancer cells. Additionally, C6 is also selectively cytotoxic against cancer cells compared to normal mammary epithelial cells. In an effort to characterize this small molecule's mechanism of action and identify relevant biological pathways that might be used as therapeutic drug targets, we have utilized a photoaffinity pull-down strategy to identify biological binding partners of C6. Our photoaffinity pull-down studies have revealed Mitsugumin 23 (MG23), an endoplasmic reticulum-bound transmembrane protein capable of ion channel formation, as a binding partner for C6. Additionally, we have identified a metabolic component of C6-induced cell death through the use of mitochondrial respiration measurements, metabolomic analyses, and mitochondrial transmission electron microscopy (TEM) imaging. Metabolic studies have identified mitochondrial respiration defects, excess lactic acid production, and gross changes in mitochondrial morphology as a result of C6 treatment. Collectively, our findings suggest a role for ionic imbalance and subsequent metabolic disruption in this form of caspase-independent cell death. Our ongoing work is focused on further mechanistic characterization of this non-apoptotic pathway as a potential target for breast cancer drug development. Citation Format: Rachel M. Vaden, Keith M. Gligorich, Dawne N. Shelton, Guoying Wang, Cindy B. Matsen, Ryan E. Looper, Matthew S. Sigman, Bryan E. Welm. Targeting nonapoptotic cell death in chemoresistant patient-derived breast cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B05.

Self-Assembling Organic Nanotubes with Precisely Defined, Sub-nanometer Pores: Formation and Mass Transport Characteristics

The transport of molecules and ions across nanometer-scaled pores, created by natural or artificial molecules, is a phenomenon of both fundamental and practical significance. Biological channels are the most remarkable examples of mass transport across membranes and demonstrate nearly exclusive selectivity and high efficiency with a diverse collection of molecules. These channels are critical for many basic biological functions, such as membrane potential, signal transduction, and osmotic homeostasis. If such highly specific and efficient mass transport or separation could be achieved with artificial nanostructures under controlled conditions, they could create revolutionary technologies in a variety of areas. For this reason, investigators from diverse disciplines have vigorously studied small nondeformable nanopores. The most exciting studies have focused on carbon nanotubes (CNTs), which have exhibited fast mass transport and high ion selectivity despite their very simple structure. However, the limitations of CNTs and the dearth of other small (≤2 nm) nanopores have severely hampered the systematic investigation of nanopore-mediated mass transport, which will be essential for designing artificial nanopores with desired functions en masse. Researchers can overcome the difficulties associated with CNT and other artificial pores by stacking macrocyclic building blocks with persistent shapes to construct tunable, self-assembling organic pores. This effort started when we discovered a highly efficient, one-pot macrocyclization process to efficiently prepare several classes of macrocycles with rigid backbones containing nondeformable cavities. Such macrocycles, if stacked atop one another, should lead to nanotubular assemblies with defined inner pores determined by their constituent macrocycles. One class of macrocycles with aromatic oligoamide backbones had a very high propensity for directional assembly, forming nanotubular structures containing nanometer and sub-nanometer hydrophilic pores. These self-assembling hydrophilic pores can form ion channels in lipid membranes with very large ion conductances. To control the assembly, we have further introduced multiple hydrogen-bonding side chains to enforce the stacking of rigid macrocycles into self-assembling nanotubes. This strategy has produced a self-assembling, sub-nanometer hydrophobic pore that not only acted as a transmembrane channel with surprisingly high ion selectivity, but also mediated a significant transmembrane water flux. The stacking of rigid macrocycles that can be chemically modified in either the lumen or the exterior surface can produce self-assembling organic nanotubes with inner pores of defined sizes. The combination of our approach with the availability and synthetic tunability of various rigid macrocycles should produce a variety of organic nanopores. Such structures would allow researchers to systematically explore mass transport in the sub-nanometer regime. Further advances should lead to novel applications such as biosensing, materials separation, and molecular purifications.

Sodium selective ion channel formation in living cell membranes by polyamidoamine dendrimer

Polyamidoamine (PAMAM) dendrimers are highly charged hyperbranched protein-like polymers that are known to interact with cell membranes. In order to disclose the mechanisms of dendrimer–membrane interaction, we monitored the effect of PAMAM generation five (G5) dendrimer on the membrane permeability of living neuronal cells followed by exploring the underlying structural changes with infrared-visible sum frequency vibrational spectroscopy (SVFS), small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). G5 dendrimers were demonstrated to irreversibly increase the membrane permeability of neurons that could be blocked in low-[Na+], but not in low-[Ca2+] media suggesting the formation of specific Na+ permeable channels. SFVS measurements on silica supported DPPG–DPPC bilayers suggested G5-specific trans-polarization of the membrane. SAXS data and freeze-fracture TEM imaging of self-organized DPPC vesicle systems demonstrated disruption of DPPC vesicle layers by G5 through polar interactions between G5 terminal amino groups and the anionic head groups of DPPC. We propose a nanoscale mechanism by which G5 incorporates into the membrane through multiple polar interactions that disrupt proximate membrane bilayer and shape a unique hydrophilic Na+ ion permeable channel around the dendrimer. In addition, we tested whether these artificial Na+ channels can be exploited as antibiotic tools. We showed that G5 quickly arrest the growth of resistant bacterial strains below 10μg/ml concentration, while they show no detrimental effect on red blood cell viability, offering the chance for the development of new generation anti-resistant antibiotics.

Mathematical analysis of human red blood cell volume regulation with regard to the elastic effect of the erythrocyte shell on metabolic processes

The mathematical model of the regulation of ion exchange and human erythrocyte volume is extended with a biomechanical model of the erythrocyte shell. This model was used to analyze the influence of elastic properties of the erythrocyte shell on erythrocyte volume in the experiments, where the volume of erythrocytes increased due to the formation of ion channels in the membrane after the treatment with amphotericin B and in case of placing red blood cells in a hypo-osmotic medium. During red blood cell deformation at a constant surface area up to sphericity, the influence of mechanical properties of the shell on volume regulation was shown to be negligible compared to the influence of ion exchange. Further osmotic swelling of red blood cells followed by the increase in their surface area is determined by tensile stiffness of the shell. The high value of tensile stiffness inherent to the erythrocyte shell is constraint for its volume change and also affects ion exchange.

Probing into the interaction of β-amyloid peptides with bilayer lipid membrane by electrochemical techniques

Alzheimer's disease (AD) is the most common degenerative disease in the elderly that is a severe threat to human health. In this paper, we report a simple but efficient electrochemical method to probe into the interaction between β-amyloid peptides and bilayer lipid membrane for revealing the toxic mechanism of AD. For this study, a supported bilayer lipid membrane is firstly modified on an electrode surface as the mimic of cell membrane, which can induce significant steric hindrance to prohibit the electrochemical probe [Fe(CN)6]3−/4− approaching the electrode. However, after incubation of the modified electrode with Aβ peptides, a noticeable electrochemical response of [Fe(CN)6]3−/4− can be obtained on the lipid membrane modified electrode, indicating the formation of ion channels in the lipid membrane. Further experimental results show that the peak current of [Fe(CN)6]3−/4− increases along with Aβ peptides concentration and incubation time. Meanwhile, the electrochemical response can be inhibited by (−)-epigallocatechin gallate, an inhibitor of the aggregation of Aβ peptides. Therefore, electrochemical technique might provide a convenient and powerful approach for in vitro studies of protein-misfolding diseases.

Antihypertensive Antagonists of L-Type Calcium Channel Exert Enhancement of Alzheimer's Aβ Peptides Effect on Cells

The Alzheimer's Aβ peptides are shown to increase the cytosolic calcium concentrations of cells in culture. Such an alteration may cause a variety of secondary effects, leading to cell degeneration and reduced cell culture growth. Cytosolic calcium concentration can be increased by the formation of cation-selective ion channels by the Aβ peptides. This property is well documented and has been extensively studied both in artificial membrane systems as well as in living cells. Here we show that L-type calcium channel antagonists, commonly used in the treatment of hypertension and ischemic heart disease, intensify the capacity of Aβ peptides to increase cytosolic calcium concentrations and consequently enhance the cytotoxicity of the Aβ peptides. Calcium imaging experiments were conducted with the fluorescent indicator Fura-2 to measure at different periods of times the levels of cytosolic calcium during exposure to Aβ peptides and to the L-type calcium channel antagonists. Two different 1,4-dihydropyridines (nifedipine, and nitrendipine), and Diltiazem, a structurally unrelated Ca2+-channel antagonist belonging to the phenylalkylamine class were used and found to exert significant excitatory effects on the toxicity of Aβ. The reduced toxicity observed in solutions of combinations of Aβ40 and Aβ42 is also reversed by L-type calcium channel antagonists. The toxicity of Aβ peptides was weighed by quantifying the growth of cultures of PC12 cells using the colorimetric assay XTT and by measuring the release of LDH to the media. The enhancement of the Aβ peptides toxicity by L-type calcium channel antagonists can be constrained by specific Aβ ion channel blockers suggesting the mediation of the ion channel property of Aβ peptides. The relevance of our finding derives from the widespread clinical use of L-type calcium channel antagonists in the treatment of hypertension and angina pectoris.

Chronic Cytosolic Calcium Changes Induced by Alzheimer's Peptides Aβ Allows for Sorting of Aβ-Resistant Cell Subpopulations

The Alzheimer's Aβ peptides interaction with the plasma membrane has been shown to result in a chronic increase in the cytosolic calcium concentrations of cells in culture. Such alteration may cause a variety of secondary effects which may lead to cell degeneration and reduced cell culture growth. We have proposed that the alteration in the cytosolic calcium concentrations induced by Aβ is initiated by early formation of Aβ ion channels in the plasma membrane. To study the Aβ-induced cytosolic calcium change dynamics we monitored the level of cytosolic calcium from hundreds of individual cells in culture exposed to Aβ for prolonged period of time. Because of the continuous cytotoxic effect of Aβ on cells, the number of viable cells in the cultures varied with time of exposure. Histograms based on the individual intracellular calcium levels showed that while most cells in the culture remain unaffected, a defined subpopulation of cells show increasingly higher than normal cytosolic calcium levels. After days of continuous exposure to Aβ cells more sensitive to Aβ died, and consequently the averaged cytosolic calcium for remaining cells in the whole culture approached control values. Aβ ion channel blockers prevented the induced cytosolic calcium changes and preserved cell viability, confirming the participation of Aβ ion channels. Confocal microscopic analysis using fluorescent annexin V on cells loaded with fura 2 showed that those cells that remained after prolonged exposure to Aβ did not display the proposed extracellular Aβ receptor phosphatidyl serine. Further addition of Aβ to this sorted cell subpopulation did not induce cytosolic calcium changes and cells continued to grow and divide. Application of this procedure allows for sorting out Aβ resistant subpopulation of cells.

Characteristics of Transient Receptor Potential Canonical Calcium-Permeable Channels and Their Relevance to Vascular Physiology and Disease

Transient receptor potential canonical (TRPC) proteins assemble to form ion channels that enable influx of calcium and sodium ions into cells. There are 6 TRPC proteins in humans but more TRPC channels may arise through heteromerization among TRPCs and other types of TRP protein. They are widely expressed and have multiple functions throughout the peripheral and central systems of the body. This review summarizes current knowledge of the characteristics of TRPC channels and discusses principles by which the channels operate. Modulators of the channels include lipids, redox factors, and agonists at G-protein and tyrosine kinase receptors. The channels enable coupling between these factors and the calcium ion, which is a master intracellular regulator of multiple cell functions. In the context of this information the review gives specific consideration to TRPC channels in vascular cells, which include endothelial cells, vascular smooth muscle cells, perivascular adipocytes, and cells of the hematopoietic lineage. It is discussed that the channels may have most significance as drivers of change when there is strain or insult in physiology or disease. The TRPC proteins constitute a substantial and important group of calcium-permeable channels. They remain enigmatic but there is increasing understanding of their properties and recognition of their importance in the vasculature as well as in other systems such as the myocardium.

On the Dependence of Initial Dynamics of Nystatin Channel Formation in Phospholipid/Ergosterol Bilayers on Ergosterol Microstructure

Nystatin (NYS) is an antifungal agent that preferentially forms ion channels in membranes containing ergosterol (ERG) or cholesterol (CHOL). In phospholipid bilayers ERG or CHOL segregate into ordered (Lo) and disordered (Ld) domains. We prepared POPC bilayers containing ERG mol fraction 0.18 ≤ χERG ≤ 0.28 separating two chambers containing 150mM KCl solutions. We imposed a potential of 50mV across the bilayer and introduced NYS molecules into the reference chamber, which was stirred at 4Hz. The NYS molecules adhered to the bilayer and formed channels on the boundaries of the Lo domains, as we have shown previously. The resulting bilayer current increased in a series of log-linear sections. A plot of the rate constant for initial NYS channel formation against χERG revealed prominent spikes at χERG = 0.19 and 0.25. These correspond well to the dips in dehydroergosterol fluorescence observed by P. L-G. Chong, which he understood in terms of a superlattice. We conclude that the initial channel formation rate depends strongly on Lo domain structure and propose a semi-empirical theory for the first two log-linear sections of the bilayer current.