RESUMO
Changes in the peptide chain of amphiphilic heptapeptides known to form ion-conducting pores in bilayers dramatically alter transport efficacy and the aggregation number of pore formation.
Assuntos
Glicina/química , Bicamadas Lipídicas/química , Oligopeptídeos/química , Transporte Biológico , Di-Hidroxifenilalanina/química , Fluoresceínas/química , Íons , Lipossomos/química , Fosfatidilcolinas/química , Porosidade , Tensoativos/química , Fatores de TempoRESUMO
An Ussing chamber was used to demonstrate that synthetic amphiphilic anion transporters function as chloride transporters in mammalian airway epithelial cells.
Assuntos
Canais de Cloreto/farmacologia , Cloretos/metabolismo , Células Epiteliais/efeitos dos fármacos , Sistema Respiratório/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Canais de Cloreto/síntese química , Canais de Cloreto/metabolismo , Células Epiteliais/metabolismo , Mamíferos , Sistema Respiratório/metabolismo , Fatores de TempoRESUMO
The channel-forming diglycolylated heptapeptides containing the amino acid sequence Gly-Gly-Gly-Pro-Gly-Gly-Gly have been found to complex chloride in CDCl(3). The strength of the interaction depends on the terminal alkyl groups and on chloride's countercation.
Assuntos
Cloretos/química , Oligopeptídeos/química , Sequência de Aminoácidos , Clorofórmio/química , Deutério , Glicina/química , Fragmentos de Peptídeos/análise , Prolina/química , Ligação ProteicaRESUMO
Five heptapeptide derivatives, [CH3(CH2)17]2NCOCH2OCH2CO-Gly-Gly-Gly-Pro-Gly-Gly-Gly-OR, in which R = ethyl, 2-propyl, heptyl, benzyl, and cyclohexylmethyl, were found to transport chloride anion through a phospholipid bilayer to varying extents dependent on the identity of R. It was concluded that the R group is a membrane anchor for the synthetic chloride channels.
Assuntos
Ânions/metabolismo , Membrana Celular/metabolismo , Ésteres/química , Canais Iônicos/química , Canais Iônicos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Cloretos/metabolismo , Transporte de Íons , Lipossomos/metabolismo , Estrutura Molecular , Fosfolipídeos/metabolismoRESUMO
Two membrane-anchored heptapeptides have been prepared and their pore-formation behavior in phospholipid bilayer membranes has been found to differ profoundly as a result only of alkyl chain length.
Assuntos
Canais de Cloreto/síntese química , Membranas Artificiais , Aminas/química , Canais de Cloreto/química , Canais de Cloreto/fisiologia , Condutividade Elétrica , Fluoresceínas , Lipossomos , Proteínas de Membrana/síntese química , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Modelos Moleculares , Porosidade , Relação Estrutura-AtividadeRESUMO
The heptapeptide sequence Gly-Gly-Gly-Pro-Gly-Gly-Gly, when anchored to diglycolic acid derived (C18H37)2NCOCH2OCH2COOH, forms chloride-selective ion channels in phospholipid liposomes but the related heptapeptide Gly-Gly-Gly-Leu-Gly-Gly-Gly, and tripeptide Gly-Gly-Gly do not.
Assuntos
Canais de Cloreto/química , Canais de Cloreto/metabolismo , Glicolatos/química , Hidrocarbonetos/química , Lipossomos/química , Oligopeptídeos/química , Hidrocarbonetos/metabolismo , Lipossomos/metabolismo , Oligopeptídeos/análise , Fosfolipídeos/químicaRESUMO
Seven synthetic anion transporters (SAT) of the general form R(2)N-COCH(2)OCH(2)CO-(Gly)(3)-Pro-(Gly)(3)-OR' were prepared. Three pairs of compounds each contained twin n-hexyl, n-decyl, and n-octadecyl (R) groups at the N-terminus and one contained twin n-tetradecyl groups. Three of the compounds were C-terminated by benzyl and three by heptyl (R') residues. The ability of these compounds to mediate ion release from phospholipid vesicles was assessed. Chloride release was measured by ion selective electrode measurements and by chloride quenching of the fluorescent dye lucigenin. Transport of the anion carboxyfluorescein (CF) was measured by fluorescence dequenching. Differences in both the C- (R') and N-terminal (R) residues within the ionophores affected anion transport. The chloride release data acquired by ion selective electrode and fluorescence methods were similar but not identical. A possible carrier mechanism for Cl(-) transport was discredited. Both Cl(-) and CF anions were released from vesicles by these compounds. The results of CF and Cl(-) transport showed good consistency when the ionophore's N-terminal chains were either decyl or octadecyl but not when they were hexyl. The transport of CF and Cl(-) appears to be fundamentally different when R is C(6) compared to C(10) or C(18). Differences between the behavior of SATs with Cl(-) and CF were also reflected in negative ion mass spectrometric studies.
Assuntos
Cloretos/química , Fluoresceínas/química , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/química , Fosfolipídeos/química , Água/química , Acridinas/química , Hidrocarbonetos Clorados/química , Íons/química , Lipossomos/química , Estrutura Molecular , Receptor EphA5 , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The synthetic peptide (C(18)H(37))(2)NCOCH(2)OCH(2)CON-(Gly)(3)-Pro-(Gly)(3)-OCH(2)Ph forms chloride-selective channels in liposomes and exhibits voltage-gating properties in planar phospholipid bilayers. The peptide fragment of the channel is based on a conserved motif in naturally occurring chloride transporters. Membrane-anchoring residues at the N- and C-terminal ends augment the peptide. NMR spectra (1D and 2D) of the channel in CDCl(3) showed significant variation in the absence and presence of stoichiometric tetrabutylammonium chloride (Bu(4)NCl). One-dimensional solution-state NMR titration studies combined with computational molecular simulation studies indicate that the peptide interacts with the salt as an ion pair and H-bonds chloride. To our knowledge, this is the first structural analysis of any synthetic anion-channel salt complex.
Assuntos
Canais de Cloreto/química , Oligopeptídeos/química , Canais de Cloreto/metabolismo , Dicroísmo Circular , Simulação por Computador , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Conformação ProteicaRESUMO
Seven heptapeptide derivatives have been prepared. The peptide structure is (Gly)(3)Xxx(Gly)(3) in which Xxx stands for a variable amino acid. The amino acid variations include azetidine carboxylic acid, pipecolic acid, meta-aminobenzoic acid, proline, and leucine. All seven compounds have a C-terminal benzyl group. In all cases, the heptapeptide's N-terminus was linked to diglycolic acid and a dialkylamine. In five cases, the N-terminal group was didecylamine and in two cases, N-ethyl-N-decyl. Chloride and carboxyfluorescein release from phospholipid vesicles was studied with the result that C(10)H(21)N(C(2)H(5)) COCH(2)OCH(2)CO-NH-(Gly)(3)Leu(Gly)(3)-OCH(2)Ph was the most active. Hill analysis showed that this compound involves pore formation by four monomer units rather than two, as previously found for other members of this family.
RESUMO
Heptapeptides having dioctadecyl, N-terminal hydrocarbon chains insert in phospholipid bilayer membranes and form pores through which at least chloride ions pass. Although amphiphilic, these compounds do not typically form vesicles themselves. They insert in the bilayers of phospholipid vesicles and mediate the release of carboxyfluorescein. Hill analysis indicates that at least two molecules of the amphiphile are involved in pore formation. In CD2Cl2, dimer formation is detected by NMR chemical shift changes. The anion release activity of individual anion transporters is increased by linking them covalently at the C-terminus or, even more, by linking them at the N-terminus. Evidence is presented that either linked molecule releases chloride from liposomes more effectively and rapidly than the individual transporter molecule at a comparable concentration.
Assuntos
Cloretos/química , Fluoresceínas/química , Lipossomos/química , Oligopeptídeos/química , Tensoativos/química , Aminas/química , Dimerização , Cinética , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Oligopeptídeos/síntese química , Tensoativos/síntese químicaRESUMO
A family of compounds having twin octadecyl anchor chains and various polar headgroups were designed to be ceramide mimics. The compounds prepared increase the apparent permeability of phospholipid vesicles to chloride and carboxyfluorescein anions. In addition, significantly larger vesicles are observed after exposure to these compounds suggesting the possibility of vesicular fusion.
Assuntos
Ceramidas/química , Lipossomos , Fosfolipídeos/química , Esfingomielinas/química , Cloretos/química , Fluoresceínas/químicaRESUMO
Seventeen peptides, most having the sequence GGGPGGG, but differing in the C- and N-terminal ends, have been studied as anion-complexing agents. These relatively simple, open-chained peptide systems interact with both chloride and the associated cation. Changes in the N- and C-terminal side chains appear to make little difference in the efficacy of binding. NMR studies suggest that the primary interactions involve amide NH contacts with the chloride anion, and CD spectral analyses suggest a concomitant conformational change upon binding. Changes in binding constants, which are expected in different solvents, also suggest selective solvent interactions with the unbound host that helps to preorganize the open-chained peptide system. Significant differences are apparent in complexation strengths when the heptapeptide chain is shortened or lengthened or when the relative position of proline within the heptapeptide is varied.
Assuntos
Canais de Cloreto/química , Cloretos/química , Oligopeptídeos/química , Cátions , Clorofórmio , Dicroísmo Circular , Cinética , Modelos Moleculares , Ressonância Magnética Nuclear BiomolecularRESUMO
The synthetic peptide, R(2)N-COCH(2)OCH(2)CO-Gly-Gly-Gly-Pro-Gly-Gly-Gly-OR', was shown to be selective for Cl(-) over K(+) when R is n-octadecyl and R' is benzyl. Nineteen heptapeptides have now been prepared in which the N-terminal and C-terminal residues have been varied. All of the N-terminal residues are dialkyl but the C-terminal chains are esters, 2 degrees amides, or 3 degrees amides. The compounds having varied N-terminal anchors and C-terminal benzyl groups are as follows: 1, R = n-propyl; 2, R = n-hexyl; 3, R = n-octyl; 4, R = n-decyl; 5, R = n-dodecyl; 6, R = n-tetradecyl; 7, R = n-hexadecyl; 8, R = n-octadecyl. Compounds 9-19 have R = n-octadecyl and C-terminal residues as follows: 9, OR' = OCH(2)CH(3); 10, OR' = OCH(CH(3))(2); 11, OR' = O(CH(2))(6)CH(3); 12, OR' = OCH(2)-c-C(6)H(11); 13, OR' = O(CH(2))(9)CH(3); 14, OR' = O (CH(2))(17)CH(3); 15, NR'(2) = N[(CH(2))(6)CH(3)](2); 16, NHR' = NH(CH(2))(9)CH(3); 17, NR'(2) = N[(CH(2))(9)CH(3)](2); 18, NHR' = NH(CH(2))(17)CH(3); 19, NR'(2) = N[(CH(2))(17)CH(3)](2). The highest anion transport activities were observed as follows. For the benzyl esters whose N-terminal residues were varied, i.e.1-8, compound 3 was most active. For the C(18) anchored esters 10-14, n-heptyl ester 11 was most active. For the C(18) anchored, C-terminal amides 15-19, di-n-decylamide 17 was most active. It was concluded that both the C- and N-terminal anchors were important for channel function in the bilayer but that activity was lost unless only one of the two anchoring groups was dominant.
RESUMO
A small library of hydraphiles has been prepared that incorporates either 1,4-phenylenedioxy or 2,6-naphthalenedioxy within the spacer chains. The side chains attached to the distal macrocycles in these tris(macrocyclic) compounds are either n-dodecyl or benzyl. The presence of the arenes subunits significantly affect sodium cation release from vesicles. The efficacy of ion transport is paralleled by the toxicity of these compounds to Bacillus subtilis.
Assuntos
Bacillus subtilis/efeitos dos fármacos , Hidrocarbonetos Aromáticos/química , Bacillus subtilis/crescimento & desenvolvimento , Cátions/química , Cátions/metabolismo , Divisão Celular/efeitos dos fármacos , Hidrocarbonetos Aromáticos/toxicidade , Eletrodos Seletivos de Íons , Estrutura Molecular , Fosfolipídeos/química , Sódio/química , Sódio/metabolismo , Fatores de TempoRESUMO
Hydraphiles are synthetic ion channels that use crown ethers as entry portals and that span phospholipid bilayer membranes. Proton and sodium cation transport by these compounds has been demonstrated in liposomes and planar bilayers. In the present work, whole cell patch clamp experiments show that hydraphiles integrate into the membranes of human embryonic kidney (HEK 293) cells and significantly increase membrane conductance. The altered membrane permeability is reversible, and the cells under study remain vital during the experiment. Control compounds that are too short (C(8)-benzyl channel) to span the bilayer or are inactive owing to a deficiency in the central relay do not induce similar conductance increases. Control experiments confirm that the inactive channel analogues do not show nonspecific effects such as activation of native channels. These studies show that the combination of structural features that have been designed into the hydraphiles afford true, albeit simple, channel function in live cells.
Assuntos
Éteres de Coroa/química , Canais Iônicos/fisiologia , Bicamadas Lipídicas/metabolismo , Animais , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Permeabilidade da Membrana Celular , Éteres de Coroa/metabolismo , Hipocampo/citologia , Humanos , Canais Iônicos/química , Canais Iônicos/metabolismo , Ácido Caínico/farmacologia , Rim/citologia , Bicamadas Lipídicas/química , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp/métodos , Ratos , Receptores de Glutamato/química , Receptores de Glutamato/metabolismoRESUMO
The design, synthesis, and characterization of a synthetic chloride membrane transporter, SCMTR (synthetic chloride membrane transporter, "scimitar"), are presented. The compound [CH3(CH2)17]2NCOCH2OCH2CO-GGGPGGG-OBzl inserts rapidly into liposomes and planar lipid bilayers. SCMTR has a 1.3 +/- 0.01 nS chloride diffusion pathway (>10:1 Cl/K selectivity). Evidence was also obtained for voltage gating behavior.