RESUMO
OBJECTIVES: Clonal kidney cells (Vero cells) are extensively utilized in the manufacture of biological preparations for disease diagnostics and therapeutics and also in preparation of vaccines. In all cells, regulation of volume is an essential function coupled to a variety of physiological processes and is a topic of interest. The objective here was to investigate involvement of ion channels in the process of volume regulation of Vero cells. METHODS: Involvement of ion channels in cell volume regulation was studied using video-microscopy and flow cytometry. Pharmacologically unaltered cells of different sizes, which are presumably at different phases of the cell cycle, were used. RESULTS: Ion transport inhibitors altered all phases of regulatory volume decrease (RVD) of Vero cells, rate of initial cell swelling, V(max) and volume recovery. Effects were dependent on type of inhibitor and on cell size (cell cycle phase). Participation of aquaporins in RVD was suggested. Inhibitors decelerated growth, arresting Vero cells at the G(0) /G(1) phase boundary. Electrophysiological study confirmed presence of volume-activated Cl(-) channels and K(+) channels in plasmatic membranes of the cells. CONCLUSION: Vero cells of all sizes maintained the ability to recover from osmotic swelling. Activity of ion channels was one of the key factors that controlled volume regulation and proliferation of the cells.
Assuntos
Tamanho Celular , Canais Iônicos/metabolismo , Rim/citologia , Rim/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Citometria de Fluxo , Glibureto/farmacologia , Canais Iônicos/antagonistas & inibidores , Microscopia , Nitrobenzoatos/farmacologia , Tetraetilamônio/farmacologia , Células VeroRESUMO
Plants used in traditional medicine are rich sources of hemolysins and cytolysins, which are potential bactericidal and anticancer drugs. The present study demonstrates for the first time the presence of a hemolysin in the leaves of Passiflora quadrangularis L. This hemolysin is heat stable, resistant to trypsin treatment, has the capacity to froth, and acts very rapidly. The hemolysin activity is dose-dependent, with a slope greater than 1 in a double-logarithmic plot. Polyethylene glycols of high molecular weight were able to reduce the rate of hemolysis, while liposomes containing cholesterol completely inhibited it. In contrast, liposomes containing phosphatidylcholine were ineffective. The Passiflora hemolysin markedly increased the conductance of planar lipid bilayers containing cholesterol but was ineffective in cholesterol-free bilayers. Successive extraction of the crude hemolysin with n-hexane, chloroform, ethyl acetate, and n-butanol resulted in a 10-fold purification, with the hemolytic activity being recovered in the n-butanol fraction. The data suggest that membrane cholesterol is the primary target for this hemolysin and that several hemolysin molecules form a large transmembrane water pore. The properties of the Passiflora hemolysin, such as its frothing ability, positive color reaction with vanillin, selective extraction with n-butanol, HPLC profile, cholesterol-dependent membrane susceptibility, formation of a stable complex with cholesterol, and rapid erythrocyte lysis kinetics indicate that it is probably a saponin.
Assuntos
Colesterol/metabolismo , Eritrócitos/metabolismo , Proteínas Hemolisinas/farmacologia , Passiflora/química , Saponinas/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Eritrócitos/efeitos dos fármacos , Proteínas Hemolisinas/isolamento & purificação , Hemólise , Bicamadas Lipídicas/metabolismo , Extratos Vegetais/farmacologia , Folhas de Planta/química , Coelhos , Saponinas/isolamento & purificaçãoRESUMO
Plants used in traditional medicine are rich sources of hemolysins and cytolysins, which are potential bactericidal and anticancer drugs. The present study demonstrates for the first time the presence of a hemolysin in the leaves of Passiflora quadrangularis L. This hemolysin is heat stable, resistant to trypsin treatment, has the capacity to froth, and acts very rapidly. The hemolysin activity is dose-dependent, with a slope greater than 1 in a double-logarithmic plot. Polyethylene glycols of high molecular weight were able to reduce the rate of hemolysis, while liposomes containing cholesterol completely inhibited it. In contrast, liposomes containing phosphatidylcholine were ineffective. The Passiflora hemolysin markedly increased the conductance of planar lipid bilayers containing cholesterol but was ineffective in cholesterol-free bilayers. Successive extraction of the crude hemolysin with n-hexane, chloroform, ethyl acetate, and n-butanol resulted in a 10-fold purification, with the hemolytic activity being recovered in the n-butanol fraction. The data suggest that membrane cholesterol is the primary target for this hemolysin and that several hemolysin molecules form a large transmembrane water pore. The properties of the Passiflora hemolysin, such as its frothing ability, positive color reaction with vanillin, selective extraction with n-butanol, HPLC profile, cholesterol-dependent membrane susceptibility, formation of a stable complex with cholesterol, and rapid erythrocyte lysis kinetics indicate that it is probably a saponin.
Assuntos
Animais , Coelhos , Colesterol/metabolismo , Eritrócitos/metabolismo , Proteínas Hemolisinas/farmacologia , Passiflora/química , Saponinas/farmacologia , Cromatografia Líquida de Alta Pressão , Eritrócitos/efeitos dos fármacos , Hemólise , Proteínas Hemolisinas/isolamento & purificação , Bicamadas Lipídicas/metabolismo , Extratos Vegetais/farmacologia , Folhas de Planta/química , Saponinas/isolamento & purificaçãoRESUMO
Vibrio cholerae EL Tor cytolysin, a water-soluble protein with a molecular mass of 63 kDa, forms small pores in target cell membranes. In this communication, planar lipid bilayers under voltage clamp conditions were used to investigate the geometric properties of the pores. It was established that all cytolysin channels were inserted into membranes with the same orientation. Sharp asymmetry in the I-V curve of fully open cytolysin channels persisting at high electrolyte concentrations indicated asymmetry in the geometry of the channel lumen. Using the nonelectrolyte exclusion method, evidence was obtained that the cis opening of the channel had a larger diameter (< or = 1.9 nm) than the trans opening (< or = 1.6 nm). The channel lumen appeared constricted, with a diameter of < or = 1.2 nm. Cup-shaped lumen geometry was deduced for both channel openings, which appeared to be connected to each other via a central narrow part. The latter contributed significantly to the total electrical resistance and determined the discontinuous character of channel filling with nonelectrolytes. Comparisons of the properties of pores formed by cytolysins of two V. cholerae biotypes (EL Tor and non-O1) indicated that the two ion channels possessed a similar geometry.
Assuntos
Citotoxinas/química , Canais Iônicos/química , Vibrio cholerae/química , Eletroquímica , Eletrólitos , Modelos Teóricos , Vibrio cholerae/genéticaRESUMO
Staphylococcal alpha-toxin forms homo-oligomeric channels in lipid bilayers and cell membranes. Here, we report that electrophysiological monitoring of single-channel function using a derivatized cysteine substitution mutant allows accurate determination of the subunit stoichiometry of the oligomer in situ. The electrophysiological phenotype of channels formed in planar lipid bilayers with the cysteine replacement mutant I7C is equal to that of the wild type. When pores were formed with I7C, alterations of several channel properties were observed upon modification with SH reagents. Decreases in conductance then occurred that were seen only as negative voltage was applied. At the level of single channels, these were manifest as stepwise changes in conductance, each step most probably reflecting modification of a single SH group within the oligomer. Because seven steps were observed, the functional channel formed by alpha-toxin in planar lipid membranes is a heptamer.
Assuntos
Toxinas Bacterianas/química , Eletrofisiologia/métodos , Proteínas Hemolisinas/química , Canais Iônicos/química , Bicamadas Lipídicas , Toxinas Bacterianas/genética , Cisteína , Proteínas Hemolisinas/genética , Mutação , Relação Estrutura-AtividadeRESUMO
Asymmetrical (one-sided) application of penetrating water-soluble polymers, polyethylene glycols (PEGs), to a well-defined channel formed by Staphylococcus aureus alpha-toxin is shown to probe channel pore geometry in more detail than their symmetrical (two-sided) application. Polymers added to the cis side of the planar lipid membrane (the side of protein addition) affect channel conductance differently than polymers added to the trans side. Because a satisfactory theory quantitatively describing PEG partitioning into a channel pore does not exist, we apply the simple empirical rules proposed previously (, J. Membr. Biol. 161:83-92) to gauge the size of pore openings as well as the size and position of constrictions along the pore axis. We estimate the radii of the two openings of the channel to be practically identical and equal to 1. 2-1.3 nm. Two apparent constrictions with radii of approximately 0. 9 nm and approximately 0.6-0.7 nm are inferred to be present in the channel lumen, the larger one being closer to the cis side. These structural findings agree well with crystallographic data on the channel structure (, Science. 274:1859-1866) and verify the practicality of polymer probing. The general features of PEG partitioning are examined using available theoretical considerations, assuming there is no attraction between PEG and the channel lumen. It is shown that the sharp dependence of the partition coefficient on polymer molecular weight found under both symmetrical and asymmetrical polymer application can be rationalized within a "hard sphere nonideal solution model." This finding is rather surprising because PEG forms highly flexible coils in water with a Kuhn length of only several Angstroms.
Assuntos
Toxinas Bacterianas/química , Proteínas Hemolisinas/química , Canais Iônicos/química , Fenômenos Biofísicos , Biofísica , Cristalografia , Condutividade Elétrica , Concentração de Íons de Hidrogênio , Potenciais da Membrana , Sondas Moleculares , Polietilenoglicóis , Cloreto de Potássio , ÁguaRESUMO
The mammalian porin channel (VDAC, porin-31BM) was reconstituted in planar lipid bilayers under voltage clamp conditions. The radii of both entrances of the channel were examined using a method that consisted in filling the channel with different non-electrolytes through its cis or trans entrances while recording single channel conductances. As a result it was found that the geometry of channels formed by porin-31BM could not be approximated by a perfectly cylindrical pore. In fact there is an asymmetry in the geometry of the channel: the diameters of the cis and trans entrances were estimated to be approximately 2 nm and approximately 4 nm respectively.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Porinas , Animais , Bovinos , Condutividade Elétrica , Eletrólitos , Canais Iônicos/química , Canais Iônicos/fisiologia , Bicamadas Lipídicas , Mamíferos , Proteínas de Membrana/isolamento & purificação , Modelos Estruturais , Músculo Esquelético/fisiologia , Técnicas de Patch-Clamp , Polietilenoglicóis , Conformação Proteica , Canais de Ânion Dependentes de VoltagemRESUMO
Porin isolated from bovine skeletal muscle was reconstitute in planar lipid bilayers under voltage clamp conditions. A set of non-electrolytes were used as molecular probes for determining the pore diameter. The maximal diameter of the open channel was estimated to be 3.02 +/- 0.26 nm. As observed for other porin channels, a large transmembrane potential drove the channel into a "closed" state. The channel transition to the low conductance (closed) state was followed by a decrease in the maximal diameter of the channel to 2.4 +/- 0.08 nm.
Assuntos
Canais Iônicos/fisiologia , Bicamadas Lipídicas , Porinas/ultraestrutura , Animais , Técnicas In Vitro , Mamíferos , Tamanho da Partícula , Técnicas de Patch-ClampRESUMO
Porin isolated from bovine skeletal muscle was reconstituted in planar lipid bilayers under voltage clamp conditions. A set of non-electrolytes were used as molecular probes for determining the pore diameter. The maximal diameter of the open channel was estimated to be 3.02 + 0.26 nm. As observed for other porin channels, a large transmembrane potential drove the channel into a "closed" state. The channel transition to the low conductance (closed) state was followed by a decrease in the maximal diameter of the channel to 2.4 +- 0.08 nm.
Assuntos
Animais , Técnicas In Vitro , Canais Iônicos/fisiologia , Bicamadas Lipídicas , Mamíferos/fisiologia , Porinas , Técnicas de Patch-ClampRESUMO
The effective size of colicin Ia channel was tested by a recently described method (FEMS, Microbiology and Immunology (1992). 105: 93-100) in which the nonelectrolyte molecules with different hydrodynamic diameters (0.52 to 5.0 nm) were used as molecular tools. We have shown that despite low conductance (55-105 pS at 1.5 M KCl, pH 7.0) the ion channels formed by colicin Ia have a fairly large water pore diameter equal to 1.66-1.88 nm. The results are discussed in terms of an energetic barrier for ions passing into the channel lumen.