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Abdulkader F Arcisio-Miranda M Curi R Procopio J 《Journal of biochemical and biophysical methods》2007,70(3):515-518
One of the methods available for the measurement of surface potentials of planar lipid bilayers uses the conductance ratio between a charged and a neutral bilayer doped with ionophores to calculate the surface potential of the charged bilayer. We have devised a simplification of that method which does not require the use of an electrically neutral bilayer as control. The conductance of the charged bilayer is measured before and after the addition of divalent cations (Ba(2+)) to the bathing solution. Ba(2+) ions screen fixed surface charges, decreasing the surface potential. If the membrane is negatively charged the screening has the effect of decreasing the membrane conductance to cations. The resulting conductance ratio is used to calculate the surface potential change, which is fed into an iterative computer program. The program generates pairs of surface potential values and calculates the surface charge density for the two conditions. Since the surface charge density remains constant during this procedure, there is only one pair of surface potentials that satisfies the condition of constant charge density. Applying this method to experimental data from McLaughlin et al. [McLaughlin, S.G.A., Szabo, G. and Eisenman, G., Divalent ions and the surface potential of charged phospholipid membranes, J. Gen. Physiol., 58 (1971) 667-687.] we have found very similar results. We have also successfully used this method to determine the effect of palmitic acid on the surface potential of asolectin membranes. 相似文献
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Martins Danubia Batista Pacca Carolina Colombelli da Silva Annielle Mendes Brito de Souza Bibiana Monson de Almeida Margarete Teresa Gottardo Palma Mario Srgio Arcisio-Miranda Manoel dos Santos Cabrera Marcia Perez 《Amino acids》2020,52(5):725-741
Amino Acids - Increasing resistance in antibiotic and chemotherapeutic treatments has been pushing studies of design and evaluation of bioactive peptides. Designing relies on different approaches... 相似文献
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dos Santos Cabrera MP Arcisio-Miranda M Gorjão R Leite NB de Souza BM Curi R Procopio J Ruggiero Neto J Palma MS 《Biochemistry》2012,51(24):4898-4908
This study shows that MP-1, a peptide from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. By using model membranes and electrophysiology measurements to investigate the molecular mechanisms underlying this selective action, the porelike activity of MP-1 was identified with several bilayer compositions. The highest average conductance was found in bilayers formed by phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine (70:30). The presence of cholesterol or cardiolipin substantially decreases the MP-1 pore activity, suggesting that the membrane fluidity influences the mechanism of selective toxicity. The determination of partition coefficients from the anisotropy of Trp indicated higher coefficients for the anionic bilayers. The partition coefficients were found to be 1 order of magnitude smaller when the bilayers contain cholesterol or a mixture of cholesterol and sphingomyelin. The blue shift fluorescence, anisotropy values, and Stern-Volmer constants are indications of a deeper penetration of MP-1 into anionic bilayers than into zwitterionic bilayers. Our results indicate that MP-1 prefers to target leukemic cell membranes, and its toxicity is probably related to the induction of necrosis and not to DNA fragmentation. This mode of action can be interpreted considering a number of bilayer properties like fluidity, lipid charge, and domain formation. Cholesterol-containing bilayers are less fluid and less charged and have a tendency to form domains. In comparison to healthy cells, leukemic T-lymphocyte membranes are deprived of this lipid, resulting in decreased peptide binding and lower conductance. We showed that the higher content of anionic lipids increases the level of binding of the peptide to bilayers. Additionally, the absence of cholesterol resulted in enhanced pore activity. These findings may drive the selective toxicity of MP-1 to Jurkat cells. 相似文献
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Arcisio-Miranda M Muroi Y Chowdhury S Chanda B 《The Journal of general physiology》2010,136(5):541-554
The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4-S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors. 相似文献
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Annielle Mendes Brito da Silva Laíz Costa Silva-Gonçalves Fernando Augusto Oliveira Manoel Arcisio-Miranda 《Molecular neurobiology》2018,55(7):5490-5504
Glioblastoma multiforme is the most common and lethal malignant brain tumor. Because of its complexity and heterogeneity, this tumor has become resistant to conventional therapies and the available treatment produces multiple side effects. Here, using multiple experimental approaches, we demonstrate that three mastoparan peptides—Polybia-MP1, Mastoparan X, and HR1—from solitary wasp venom exhibit potent anticancer activity toward human glioblastoma multiforme cells. Importantly, the antiglioblastoma action of mastoparan peptides occurs by membranolytic activity, leading to necrosis. Our data also suggest a direct relation between mastoparan membranolytic potency and the presence of negatively charged phospholipids like phosphatidylserine. Collectively, these data may warrant additional studies for mastoparan peptides as new agents for the treatment of glioblastoma multiforme brain tumor. 相似文献
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Manoel Arcisio-Miranda Rui Curi Joaquim Procopio 《Archives of biochemistry and biophysics》2009,484(1):63-69
Free fatty acids (FFA) are important mediators of proton transport across membranes. However, information concerning the influence of the structural features of both FFA and the membrane environment on the proton translocation mechanisms across phospholipid membranes is relatively scant. The effects of FFA chain length, unsaturation and membrane composition on proton transport have been addressed in this study by means of electrical measurements in planar lipid bilayers. Proton conductance () was calculated from open-circuit voltage and short-circuit current density measurements. We found that cis-unsaturated FFA caused a more pronounced effect on proton transport as compared to saturated and trans-unsaturated FFA. Cholesterol and cardiolipin decreased membrane leak conductance. Cardiolipin also decreased proton conductance. These effects indicate a dual modulation of protein-independent proton transport by FFA: through a flip-flop mechanism and by modifying a proton diffusional pathway. Moreover the membrane phospholipid composition was shown to importantly affect both processes. 相似文献
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Marcia Perez dos Santos Cabrera Manoel Arcisio-Miranda Bibiana Monson de Souza Mario Sérgio Palma Joaquim Procopio 《Archives of biochemistry and biophysics》2009,486(1):1-11
In the last decade, there has been renewed interest in biologically active peptides in fields like allergy, autoimmune diseases and antibiotic therapy. Mast cell degranulating peptides mimic G-protein receptors, showing different activity levels even among homologous peptides. Another important feature is their ability to interact directly with membrane phospholipids, in a fast and concentration-dependent way. The mechanism of action of peptide HR1 on model membranes was investigated comparatively to other mast cell degranulating peptides (Mastoparan, Eumenitin and Anoplin) to evidence the features that modulate their selectivity. Using vesicle leakage, single-channel recordings and zeta-potential measurements, we demonstrated that HR1 preferentially binds to anionic bilayers, accumulates, folds, and at very low concentrations, is able to insert and create membrane spanning ion-selective pores. We discuss the ion selectivity character of the pores based on the neutralization or screening of the peptides charges by the bilayer head group charges or dipoles. 相似文献
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Deborah L. Capes Marcel P. Goldschen-Ohm Manoel Arcisio-Miranda Francisco Bezanilla Baron Chanda 《The Journal of general physiology》2013,142(2):101-112
Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na+ channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K+ current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation. 相似文献
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