Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pos-Graduação em Farmacologia.; O presente estudo avaliou através do emprego de abordagens farmacológicas in vitro e in vivo, alguns dos mecanismos envolvidos na ativação do receptor TRPA1 (receptor de potencial transitório com domínios anquirina 1), um membro da família de canais catiônicos TRP (receptor de potencial transitório). De forma semelhante ao agonista do receptor TRPV1 (receptor de potencial transitório vanilóide 1), a capsaicina, os agonistas do receptor TRPA1, o isotiocianato de alila e o cinamaldeído, causaram contração da bexiga isolada de rato dependente de cálcio externo. Esta resposta foi susceptível a dessensibilização. O vermelho de rutênio, um antagonista não-seletivo dos receptores TRP, reduziu de forma significativa a resposta contrátil induzida pela capsaicina, pelo isotiocianato de alila ou pelo cinamaldeído. O antagonista seletivo do receptor TRPV1, o SB 366791, foi capaz de inibir completamente a resposta contrátil induzida pela capsaicina, enquanto reduziu parcialmente o efeito produzido pelos agonistas do receptor TRPA1. Ademais, a contração induzida pelo isotiocianato de alila foi significativamente reduzida pelos antagonistas dos receptores taquicinérgicos NK1...
O trabalho apresentado nesta dissertação consiste no
desenvolvimento de uma nova geração de compósitos para
membranas de separação de CO2 a alta temperatura (500-600°C), a
partir de um fase cerâmica (Ce0,9Gd0,1O2-δ - CGO) e uma segunda fase
constituída por mistura de carbonatos alcalinos (Li2CO3 e Na2CO3). O
papel da fase cerâmica é assegurar o transporte de iões óxido
enquanto o papel dos carbonatos fundidos é assegurar o transporte
dos iões carbonato. Quando estes iões se movem em sentidos
opostos, o resultado é um fluxo seletivo de CO2.
Para este fim preparou-se um conjunto de eletrólitos compósitos por
moagem, prensagem e sinterização, variando a fração volúmica, a
granulometria da fase cerâmica e também modificando a composição
da própria fase cerâmica (compósitos baseados em LiAlO2). Os vários
eletrólitos compósitos foram posteriormente estudados por
espetroscopia de impedância em ar e microscopia eletrónica para
avaliar o efeito da fração cerâmica e da sua microestrutura na
condução mista. De modo a quantificar a importância relativa do
transporte iónico nos compósitos por iões óxido e/ou carbonato
utilizou-se o método de eficiência faradaica.
Os principais resultados obtidos mostram que o compósito com 85%
em volume de CGO apresenta uma condutividade total próxima da do
The present work proposes to analyse the results obtained under in vitro conditions where cellulose artificial membranes were incubated with biological fluids
from the freshwater bivalve Anodonta cygnea. The membranes were mounted between two half ‘Ussing chambers’ with different composition solutions in order
to simulate epithelial surfaces separating organic fluid compartments. The membrane surfaces were submitted to two synthetic calcium and phosphate
solutions on opposite sides, at pH 6.0, 7.0 or 9.0 during a period of 6 hours. Additional assays were accomplished mixing these solutions with haemolymph or
extrapallial fluid from A. cygnea, only on the calcium side. A selective ion movement, mainly dependent on the membrane pore size and/or cationic affinity,
occurred with higher permeability for calcium ions to the opposite phosphate chamber supported by calcium diffusion forces across the cellulose membrane. In
general, this promoted a more intense mineral precipitation on the phosphate membrane surface. A strong deposition of calcium phosphate mineral was
observed at pH 9.0 as a primary layer with a homogeneous microstructure, being totally absent at pH 6.0. The membrane showed an additional crystal phase
at pH 7.0 exhibiting a very particular hexagonal or cuttlebone shape...
The small HIV-1 accessory protein Vpr (virus protein R) is a multifunctional protein that is present in the serum and cerebrospinal fluid of AIDS patients. We previously showed that Vpr can form cation-selective ion channels across planar lipid bilayers, introducing the possibility that, if incorporated into the membranes of living cells, Vpr might form ion channels and consequently perturb the maintained ionic gradient. In this study, we demonstrate, by a variety of approaches, that Vpr added extracellularly to intact cells does indeed form ion channels. We use confocal laser scanning microscopy to examine the subcellular localization of fluorescently labeled Vpr. Plasmalemma depolarization and damage are examined using the anionic potential-sensitive dye bis(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI), respectively, and the effect of Vpr on whole-cell current is demonstrated directly by using the patch-clamp technique. We show that recombinant purified extracellular Vpr associates with the plasmalemma of hippocampal neurons to cause a large inward cation current and depolarization of the plasmalemma, eventually resulting in cell death. Thus, we demonstrate a physiological action of extracellular Vpr and present its mechanistic basis. These findings may have important implications for neuropathologies in AIDS patients who possess significant amounts of Vpr in the cerebrospinal fluid.
The determinants of the Actinobacillus pleuropneumoniae RTX toxins ApxI, ApxII, and ApxIII were expressed in an Escherichia coli strain. The toxins were concentrated from the supernatants of cell cultures. The addition of the toxins to the aqueous-phase-bathing lipid bilayer membranes resulted in an increase in the membrane conductance when membranes made of asolectin or phosphatidylethanolamine were used. The toxins were relatively inactive in membranes made of other lipids. The membrane activity (i.e., the number of channels formed at a given Apx concentration) was different for each of the three Apx toxins. That of ApxI, which has the strongest cytotoxic activity, was highest, followed by that of ApxIII and ApxII, which is the least cytotoxic. The conductance increases of ApxIII and ApxII were smaller by factors of 10 and 50, respectively, than that of ApxI under otherwise identical conditions. Single-channel experiments demonstrated that all three Apx toxins formed ion-permeable channels of different conductances. The major open state was approximately the same for the two hemolytic toxins ApxI and ApxII (540 and 620 pS in 0.15 M KCI), whereas the single-channel conductance of the nonhemolytic ApxIII was approximately one-fifth of that of the other two toxins (95 pS). Experiments with different salts suggested that the Apx channels of A. pleuropneumoniae were exclusively cation selective because of negative charges localized at the channel mouth. Analysis of the single-channel data using the Renkin correction factor suggested that the Apx toxins formed aqueous channels with different diameters for the three toxins. Pore-forming properties of the Apx toxins were compared with those of other RTX toxins. All of these toxins have common features and form channels that are transient but have different sizes as judged from the different single-channel conductances.
Lipid bilayer experiments were performed in the presence of hemolysin of Escherichia coli. The toxin had a rather low activity in membranes formed of pure lipids, such as phosphatidylcholine or phosphatidylserine. In membranes from asolectin, a crude lipid mixture from soybean, hemolysin was able to increase the conductance by many orders of magnitude in a steep concentration-dependent fashion, which suggested that several hemolysin molecules could be involved in the conductive unit. Furthermore, the much higher toxin activity in asolectin membranes would be consistent with the assumption that this lipid contains a receptor needed for membrane activity of the toxin. The results of single-channel records showed that the membrane activity of hemolysin is due to the formation of ion-permeable channels with a single-channel conductance of about 500 pS in 0.15 M KCl. The hemolysin channel seemed to be formed by a toxin oligomer which showed an association-dissociation reaction and had a mean lifetime of about 2 s at small transmembrane voltages. The conductance of the hemolysin channels was only moderately dependent on the salt concentration in the aqueous phase. Zero-current membrane potential experiments showed that the hemolysin channel is cation selective. The mobility sequence of the cations in the channel was similar to their mobility sequence in the aqueous phase...
1. The ryanodine receptor protein of sheep cardiac muscle sarcoplasmic reticulum membranes functions as a ligand-regulated ion channel following solubilization with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1- propane sulphonate); purification by density gradient centrifugation, reconstitution into proteo-liposomes and incorporation into planar phospholipid bilayers. 2. In the absence of divalent cations, measurable conductance is observed with the group 1a cations and with some larger organic cations. In symmetric 210 mM solutions the following conductance sequence was determined: K+ greater than Rb+ = NH4+ greater than Na+ = Cs+ greater than Li+ much greater than Tris+. 3. Other organic cations, e.g. TEA+, do not produce measurable current under these conditions. 4. Single-channel conductance saturates with increasing ionic activities of K+, Na+ and Li+. Saturation curves are described by Michaelis-Menten kinetic schemes with the following values of maximal conductance and apparent dissociation constant: K+ 900 pS, 19.9 mM; Na+ 516 pS, 17.8 mM; Li+ 248 pS, 9.1 mM. 5. The channel displays only minor differences in permeability amongst the group 1a cations. Relative permeability, monitored under bi-ionic conditions...
Gramicidin A (gA) molecules were covalently linked with a dioxolane ring. Dioxolane-linked gA dimers formed ion channels, selective for monovalent cations, in planar lipid bilayers. The main goal of this study was to compare the functional single ion channel properties of natural gA and its covalently linked dimer in two different lipid bilayers and HCl concentrations (10-8000 mM). Two ion channels with different gating and conductance properties were identified in bilayers from the product of dimerization reaction. The most commonly observed and most stable gramicidin A dimer is the main object of this study. This gramicidin dimer remained in the open state most of the time, with brief closing flickers (tau(closed) approximately 30 micros). The frequency of closing flickers increased with transmembrane potential, making the mean open time moderately voltage dependent (tau(open) changed approximately 1.43-fold/100 mV). Such gating behavior is markedly different from what is seen in natural gA channels. In PEPC (phosphatidylethanolamine-phosphatidylcholine) bilayers, single-channel current-voltage relationships had an ohmic behavior at low voltages, and a marked sublinearity at relatively higher voltages. This behavior contrasts with what was previously described in GMO (glycerylmonooleate) bilayers. In PEPC bilayers...
Ionic conduction in the axolemmal and septal membranes of the medial giant fiber (MGF) of the earthworm (EW) Lumbricus terrestris was assessed by impedance spectroscopy in the frequency range 2.5-1000 Hz. Impedance loci in the complex plane were described by two semi-circular arcs, one at a lower characteristic frequency (100 Hz) and the other at a higher frequency (500 Hz). The lower frequency arc had a chord resistance of 53 k omega and was not affected by membrane potential changes or ion channel blockers [tetrodotoxin (TTX), 3,4-diaminopyridine (3,4-DAP), 4-aminopyridine (4-AP), and tetraethylammonium (TEA)]. The higher frequency arc had a chord resistance of 274 k omega at resting potential, was voltage-dependent, and was affected by the addition of TTX, 3,4-DAP, 4-AP, and TEA to the physiological EW salines. When all four blockers were added to the bathing solution, the impedance locus was described by two voltage-independent arcs. Considering the effects of these and other (i.e., Cd and Ni) ion channel blockers, we conclude that: 1) the higher frequency locus reflects conduction by voltage-sensitive ion channels in the axolemmal membrane, which contains at least four ion channels selective for sodium, calcium, and potassium (delayed rectifier and calcium-dependent)...
We examined ion channels derived from a chloroform extract of isolated, dehydrated rat liver mitochondria. The extraction method was previously used to isolate a channel-forming complex containing poly-3-hydroxybutyrate and calcium polyphosphate from Escherichia coli. This complex is also present in eukaryotic membranes, and is located primarily in mitochondria. Reconstituted channels showed multiple subconductance levels and were voltage-dependent, showing an increased probability of higher conductance states at voltages near zero. In symmetric 150 mM KCl, the maximal conductance of the channel ranged from 350 pS to 750 pS. For voltages >±60 mV, conductance fluctuated in the range of ∼50–∼200 pS. In the presence of a 1:3 gradient of KCl, at pH = 7.4, selectivity periodically switched between different states ranging from weakly anion-selective (Vrev ∼ −15 mV) to ideally cation-selective (Vrev ∼ +29 mV), without a significant change in its conductance. Overall, the diverse, but highly reproducible, channel activity most closely resembled the behavior of the permeability transition pore channel seen in patch-clamp experiments on native mitoplasts. We suggest that the isolated complex may represent the ion-conducting module from the permeability transition pore.
Molecular orbital calculations (CNDO/2) are reported for the interaction of Na+ and Mg+2 with the carbonyl of a model peptide moiety (N-methyl acetamide) as a function of the C--O ... Me distance and angle and with variation in the number of ligands for the purpose of determining the steepness of the distance dependence of the binding energy and for the purpose of determining the reduction of charge on the ion with increasing numbers of ligands. The greater energy derived on divalent ion binding and the steeper distance dependence indicate that selective, divalent over monovalent, ion binding will occur whenever the liganding system can provide a coordination shell of appropriate dimension. The calculations indicate that the preferred C--O ... Me angle is not 180 degrees. Of particular note is the decrease of charge on the cation on binding to N-methyl acetamide. One ligand bound to Na+ reduces the charge from 1.0 to 0.7 electron units and four ligands bound to Mg+2 reduces the charge from 2.0 to 0.7 electron units. This is of primary significance in carrier and channel mechanisms for cation permeation of lipid membranes; and although the numerical values are qualitative, the implication is for allowance of multiple occupancy of channels by monovalent cations.
Binding of the novel radioligand 3H-2-(2-dimethylaminomethyl-phenylsulphanyl)-5-methyl-phenylamine (3H-MADAM) to the serotonin transporter (SERT) was used to characterise a range of selective serotonin re-uptake inhibitors (SSRIs) in vitro and in vivo.3H-MADAM bound with high affinity in a saturable manner to both human SERT expressed in CHO cells (Kd=0.20 nM (pKd=9.74±0.12), Bmax=35±4 fmol mg−1 protein) and mouse cerebral cortex membranes (Kd=0.21 nM (pKd=9.66±0.10), Bmax=50±24 fmol mg−1 protein).Binding of 3H-MADAM was highly selective for SERT in vitro as demonstrated by the in vitro profile of MADAM tested at 75 different receptors, ion channels and transporters. This was further substantiated by the pharmacological profile of the binding. Hence, the binding of 3H-MADAM was potently inhibited by SSRIs but not by selective inhibitors of noradrenaline transport and dopamine transport. Likewise, a 5-HT2A/2C receptor antagonist did not inhibit 3H-MADAM binding.3H-MADAM binding in vivo was inhibited only by compounds which also inhibited the binding of 3H-MADAM in vitro (the SSRIs, mixed SERT/noradrenaline transport inhibitors and clomipramine), confirming the selectivity of 3H-MADAM for SERT also in vivo. Moreover, compounds effective in inhibiting 3H-MADAM binding were the only ones found to be active in the mouse 5-HTP potentiation test confirming the model as a behavioural correlate to in vivo 5-HT uptake.Finally...
Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease of the kidney, characterized by cystic enlargement of renal tubules, aberrant epithelial proliferation, and ion and fluid secretion into the lumen. Previous studies have shown abnormalities in polarization of membrane proteins, including mislocalization of the NaK-ATPase to the apical plasma membranes of cystic epithelia. Apically located NaK-ATPase has previously been shown to be fully functional in vivo and in membrane-grown ADPKD epithelial cells in vitro, where basal-to-apical 22Na transport was inhibited by application of ouabain to the apical membrane compartment. Studies were conducted with polymerase chain reaction-generated specific riboprobes and polyclonal peptide antibodies against human sequences of α1, α3, β1, and β2 subunits of NaK-ATPase. High levels of expression of α1 and β1 messenger RNA were detected in ADPKD and age-matched normal adult kidneys in vivo, whereas β2 messenger RNA was detected only in ADPKD kidneys. Western blot analysis and immunocytochemical studies showed that, in normal adult kidneys, peptide subunit-specific antibodies against α1 and β1 localized to the basolateral membranes of normal renal tubules, predominantly thick ascending limbs of Henle’s loop. In ADPKD kidneys...
Potassium channels are exquisitely selective, allowing K+ to pass across cell membranes while blocking other ion types. Here we demonstrate that the number of carbonyl oxygen atoms that surround permeating ions is the most important factor in determining ion selectivity rather than the size of the pore or the strength of the coordinating dipoles. Although the electrostatic properties of the coordinating ligands can lead to Na+ or K+ selectivity at some values of the dipole moment, no significant selectivity arises at the specific value of the dipole moment for carbonyl groups found in potassium channels when the ligands have complete freedom. Rather, we show that the main contribution to selectivity arises from slight constraints on the conformational freedom of the channel protein that limit the number of carbonyl oxygen atoms to a value better suited to K+ than Na+, despite the pore being flexible. This mechanism provides an example of a general framework for explaining ion discrimination in a range of natural and synthetic macromolecules in which selectivity is controlled by the number of coordinating ligands in addition to their dipole moment.
The results of the present study show that bilaterally vasectomized rabbits with high levels of antibodies to sperm antigens frequently develop an orchitis associated with granular deposits of rabbit IgG and C3 in the basement membranes of seminiferous tubules. The immune deposits correspond in location to electron-opaque deposits seen by electron microscopy. The "membranous orchitis" is characterized by thickening of tubular basement membranes, acc-mulation of macrophages and a few polymorphonuclear leukocytes, and destruction of the basal lamina, of the Sertoli and spermatogenetic cells. The pathogenetic role of the immune deposits and the possibility that they contain antigen- antibody complexes is indicated by: (a) selective accumulation of IgG and C3 granular deposits along the basement membranes of seminiferous tubules in rabbits producing high and persistent levels of antibodies to sperm antigens; (b) the elution of immunoglobulins from tissues with chaotropic ion-containing buffers, acid buffers, or heat; (c) the observation that the immuno-globulins accumulated in the testis contain antibody to sperm antigens; and (d) the demonstration of sperm antigens in a location similar to that of IgG and C3. It is postulated that sperm antigen-antibody complexes are formed in the basement membranes of seminiferous tubules when antigens leaking out of the tubules react with specific antibody coming from the circulation. In two rabbits with higher levels of circulating antisperm antibodies and severe orchitis...
Although it is clear that amyloid beta (Aβ) peptides play a pivotal role in the development of Alzheimer’s disease, the precise molecular model of action remains unclear. Aβ peptides form assemble both in aqueous solution and in lipid membranes. It has been proposed that deleterious effects occur when the peptides interact with membranes, possibly by forming Ca2+ permeant ion channels. In the accompanying manuscript, we propose models in which the C-terminus third of six Aβ42 peptides forms a six-stranded β-barrel in highly toxic soluble oligomers. Here we extend this hypothesis to membrane-bound assemblies. In these Aβ models, the hydrophobic β-barrel of a hexamer may either reside on the surface of the bilayer, or span the bilayer. Transmembrane pores are proposed to form between several hexamers. Once the β-barrels of six hexamers have spanned the bilayer, they may merge to form a more stable 36-stranded β-barrel. We favor models in which parallel β-barrels formed by N-terminus segments comprise the lining of the pores. These types of models explain why the channels are selective for cations and how metal ions, such as Zn2+, synthetic peptides that contain histidines, and some small organic cations may block channels or inhibit formation of channels. Our models were developed to be consistent with microscopy studies of Aβ assemblies in membranes...
Ion channels lower the energetic barrier for ion passage across cell membranes and enable the generation of bioelectricity. Electrostatic interactions between permeant ions and channel pore helix dipoles have been proposed as a general mechanism for facilitating ion passage. Here, using genetic selections to probe interactions of an exemplar potassium channel blocker, barium, with the inward rectifier Kir2.1, we identify mutants bearing positively charged residues in the potassium channel signature sequence at the pore helix C terminus. We show that these channels are functional, selective, resistant to barium block, and have minimally altered conductance properties. Both the experimental data and model calculations indicate that barium resistance originates from electrostatics. We demonstrate that potassium channel function is remarkably unperturbed when positive charges occur near the permeant ions at a location that should counteract pore helix electrostatic effects. Thus, contrary to accepted models, the pore helix dipole seems to be a minor factor in potassium channel permeation.
Cellular membranes are composed of hundreds of different lipids, ion channels, receptors and scaffolding complexes that act as signalling and trafficking platforms for processes fundamental to life. Cellular signalling and membrane trafficking are often regulated by peripheral proteins, which reversibly interact with lipid molecules in highly regulated spatial and temporal fashions. In most cases, one or more modular lipid-binding domain(s) mediate recruitment of peripheral proteins to specific cellular membranes. These domains, of which more than 10 have been identified since 1989, harbour structurally selective lipid-binding sites. Traditional in vitro and in vivo studies have elucidated how these domains coordinate their cognate lipids and thus how the parent proteins associate with membranes. Cellular activities of peripheral proteins and subsequent physiological processes depend upon lipid binding affinities and selectivity. Thus, the development of novel sensitive and quantitative tools is essential in furthering our understanding of the function and regulation of these proteins. As this field expands into new areas such as computational biology, cellular lipid mapping, single molecule imaging, and lipidomics, there is an urgent need to integrate technologies to detail the molecular architecture and mechanisms of lipid signalling. This review surveys emerging cellular and in vitro approaches for studying protein–lipid interactions and provides perspective on how integration of methodologies directs the future development of the field.
Ion channels modulate ion flux across cell membranes, activate signal transduction pathways, and influence cellular transport—vital biological functions that are inexorably linked to cellular processes that go awry during carcinogenesis. Indeed, deregulation of ion channel function has been implicated in cancer-related phenomena such as unrestrained cell proliferation and apoptotic evasion. As the prototype for ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs) have been extensively studied in the context of neuronal cells but accumulating evidence also indicate a role for nAChRs in carcinogenesis. Recently, variants in the nAChR genes CHRNA3, CHRNA5, and CHRNB4 have been implicated in nicotine dependence and lung cancer susceptibility. Here, we silenced the expression of these three genes to investigate their function in lung cancer. We show that these genes are necessary for the viability of small cell lung carcinomas (SCLC), the most aggressive type of lung cancer. Furthermore, we show that nicotine promotes SCLC cell viability whereas an α3β4-selective antagonist, α-conotoxin AuIB, inhibits it. Our findings posit a mechanism whereby signaling via α3/α5/β4-containing nAChRs promotes lung carcinogenesis.
The purified chloroplast ATP synthase (CF(0)-CF(1)) was reconstituted into azolectin liposomes from which bilayer membranes on the tip of a glass pipette ('dip stick technique')and planar bilayer membranes were form ed. The CF(0)-CF(1) facilitated ion conductance through the bilayer membranes. Our results clearly indicated that the observed single channel currents were carried by H+ through the isolated and reconstituted chloroplast ATPase. We demonstrated that in proteoliposomes it is the whole enzyme complex CF(0)-CF(1) and not the membrane sector CF(0) alone that constitutes a voltagegated, proton-selective channel with a high conductance of 1-5 pS at pH 5.5-8.0. After removal of CF(1) from the liposomes by NaBr treatment the membrane sector CF(0) displayed various kinds of channels also permeable to monovalent cations. The open probability P(0) of the CF(0)-CF(1) channel increased considerable with increasing membrane voltage [from P(0) less than or equal to 1% (V(m) less than or equal to 120 mV) to P(0) less than or equal to 30% (120 mV less than or equal to Vm 200 mV)]. In the presence of ADP (3 microM) and P(i) (5 microM), which specifically bind to CF(1), the open probability decreased and venturicidin (1 microM), a specific inhibitor of H+ flow through CF(0) in thylakoid membranes...