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1.
Biophys J ; 95(7): 3278-86, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18599631

RESUMO

Using synchrotron grazing-incidence x-ray diffraction (GIXD) and reflectivity, the in-plane and out-of-plane structures of mixed-ganglioside GT(1b)-phospholipid monolayers were investigated at the air-liquid interface and compared with monolayers of the pure components. The receptor GT(1b) is involved in the binding of lectins and toxins, including botulinum neurotoxin, to cell membranes. Monolayers composed of 20 mol % ganglioside GT(1b), the phospholipid dipalmitoyl phosphatidylethanolamine (DPPE), and the phospholipid dipalmitoyl phosphatidylcholine (DPPC) were studied in the gel phase at 23 degrees C and at surface pressures of 20 and 40 mN/m, and at pH 7.4 and 5. Under these conditions, the two components did not phase-separate, and no evidence of domain formation was observed. The x-ray scattering measurements revealed that GT(1b) was intercalated within the host DPPE/DPPC monolayers, and slightly expanded DPPE but condensed the DPPC matrix. The oligosaccharide headgroups extended normally from the monolayer surfaces into the subphase. This study demonstrated that these monolayers can serve as platforms for investigating toxin membrane binding and penetration.


Assuntos
1,2-Dipalmitoilfosfatidilcolina/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Gangliosídeos/metabolismo , Fosfatidiletanolaminas/metabolismo , Difração de Raios X , Configuração de Carboidratos , Gangliosídeos/química , Bicamadas Lipídicas/metabolismo , Pressão , Propriedades de Superfície
2.
Biophys J ; 81(6): 3077-89, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11720976

RESUMO

Using periodic boundary conditions and a constant applied field, we have simulated current flow through an 8.125-A internal diameter, rigid, atomistic channel with polar walls in a rigid membrane using explicit ions and extended simple point charge water. Channel and bath currents were computed from 10 10-ns trajectories for each of 10 different conditions of concentration and applied voltage. An electric field was applied uniformly throughout the system to all mobile atoms. On average, the resultant net electric field falls primarily across the membrane channel, as expected for two conductive baths separated by a membrane capacitance. The channel is rarely occupied by more than one ion. Current-voltage relations are concentration dependent and superlinear at high concentrations.


Assuntos
Íons , Cloreto de Sódio/química , Cloreto de Sódio/metabolismo , Água/química , Simulação por Computador , Eletrólitos/química , Eletrofisiologia , Modelos Moleculares , Eletricidade Estática
3.
Biophys J ; 81(3): 1245-54, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11509341

RESUMO

Nonlinear least squares fitting was used to assign rate constants for the three-barrier, two-site, double-occupancy, single-filing kinetic model for previously reported current-voltage relations of (5F-Indole)Trp(13) gramicidin A and gramicidin A channels (, 75:2830-2844). By judicious coupling of parameters, it was possible to reduce the parameter space from 64 parameters to 24, and a reasonable fit consistent with other experimental data was obtained. The main features of the fit were that fluorination increased the rate constant for translocation by a factor of 2.33, consistent with a free energy change in the translocation barrier of -0.50 kcal/mol, and increased first-ion binding affinity by a factor of 1.13, primarily by decreasing the first-ion exit rate constant. The translocation rate constant was 5.62 times slower in diphytanoyl phosphatidylcholine (DPhPC) bilayers than in monoolein (GMO) bilayers (coupled for the four combinations of peptide and salt), suggesting a 44.2-mV difference in the projection of the interfacial dipole into the channel. Thus fluorination caused increased currents in DPhPC bilayers, where a high interfacial dipole potential makes translocation more rate limiting because the translocation barrier was reduced, and decreased currents in GMO bilayers, where ion exit or entry is rate limiting because these barriers were increased.


Assuntos
Gramicidina/química , Gramicidina/metabolismo , Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/metabolismo , Triptofano/metabolismo , Algoritmos , Permeabilidade da Membrana Celular , Simulação por Computador , Condutividade Elétrica , Transporte de Íons , Cinética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Eletricidade Estática , Termodinâmica
4.
Biophys J ; 81(3): 1255-64, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11509342

RESUMO

Experimental and theoretical calculations indicate that the dipole moment of the four Trp side chains in gramicidin A (gA) channels modify channel conductance through long-range electrostatic interactions. Electrostatic ion/side-chain interaction energies along the channel were computed with CHARMM using ab initio atom charges for native and 4-, 5-, or 6-fluorinated Trp side chains. The bulk water reaction to the polar side chains was included using the method of images as implemented by, and channel waters in idealized structures were included. Ion/Trp interaction energies were approximately -0.6 kcal/mol throughout the channel for all four of the native Trp pairs. Channel waters produced a modest reduction in the magnitude of interactions, essentially offsetting images representing the bulk water outside the channel. The effects of side-chain fluorination depended on ring position and, to a lesser extent, residue number. Compared with native Trp, 5-fluorination reduces the translocation barrier with minor effects on the exit barrier. In contrast, 6-fluorination primarily reduces exit barrier. 4-Fluorination produces a more complex double-well energy profile. Effects of measured side-chain movements resulting from fluorination or change in lipid bilayer were negligible whereas thermal side chain librations cause large effects, especially in the region of the ion-binding sites.


Assuntos
Flúor/metabolismo , Gramicidina/química , Gramicidina/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Sítios de Ligação , Permeabilidade da Membrana Celular , Ligação de Hidrogênio , Potenciais da Membrana , Conformação Proteica , Rotação , Eletricidade Estática , Termodinâmica , Triptofano/metabolismo , Água/química , Água/metabolismo
5.
Biochim Biophys Acta ; 1513(2): 185-92, 2001 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-11470090

RESUMO

To explore the possible role of Trp side chains in gramicidin channel conductance dispersity, we studied the dispersity of gramicidin M (gM), a gramicidin variant in which all four tryptophan residues are replaced with phenylalanine residues, and its enantiomer, gramicidin M(-) (gM(-)), and compared them to that of gramicidin A (gA). The conductances of highly purified gM and gM(-) were studied in alkali metal solutions at a variety of concentrations and voltages, in seven different types of lipid, and in the presence of detergent. Like gA channels, the most common gM channel conductance forms a narrow band. However, unlike gA channels, where the remaining 5-30% of channel conductances are broadly distributed below (and slightly above) the main band, in gM there is a narrow secondary band with <50% of the main peak conductance. This secondary peak was prominent in NaCl and KCl, but significantly diminished in CsCl and RbCl. Under some conditions, minor components can be observed with conductances yet lower than the secondary peak. Interconversions between the primary conductance state and these yet lower conductance states were observed. The current-voltage relations for both primary and secondary gM channel types have about the same curvature. The mean lifetime of the secondary channel type is below one third that of the primary type. The variants represent state deviations in the peptide or adjacent lipid structure.


Assuntos
Gramicidina/química , Canais Iônicos/química , Condutividade Elétrica , Glicerídeos , Gramicidina/análogos & derivados , Bicamadas Lipídicas/química , Octoxinol , Cloreto de Potássio
6.
Phys Rev Lett ; 86(11): 2467-70, 2001 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-11289956

RESUMO

The conductance of sodium ions through a simplified channel-membrane system immersed in a reservoir of 1M NaCl in SPC/E water is examined by molecular dynamics simulation. An applied external potential of 1.1 V drives the ions and water through a channel of length 25 A producing a current of 19.6 pA, in reasonable agreement with experimental findings. The stream of ions and water molecules flows continuously because of the constant applied field and periodic boundary conditions. We also examine the potential profile across the simulation cell, the average density distributions of the various species in the reservoir and radially in the channel, and the ion velocity in the channel.


Assuntos
Canais Iônicos/metabolismo , Modelos Biológicos , Fenômenos Biofísicos , Biofísica , Cloretos/metabolismo , Canais Iônicos/química , Modelos Moleculares , Sódio/metabolismo , Termodinâmica
7.
Novartis Found Symp ; 225: 74-87; discussion 87-92, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10472049

RESUMO

Seoh & Busath (1995) showed that in the presence of formamidinium, single gramicidin A channels were lengthened, had uniformly noisy currents at low voltages and had superlinear current-voltage relationships, all three properties being absent in gramicidin M- channels in which the interfacial tryptophan residues in gramicidin A are all replaced by phenylalanine. We measured the single channel noise power spectra (PSDs) in small monoolein (GMO) bilayers with formamidinium chloride solutions to help identify the mechanism of noise process. PSDs were Lorentzian with characteristic frequencies of 0.1-1.0 kHz in 0.1 and 0.3 M formamidinium chloride solutions, and from. 1-6 kHz in 1 M solution. Si(0), where measurable, ranged from approximately 50-200 fA2/Hz. The time course of the noise process could not be detected in these experiments. The low fc suggests slow motions or rare states of the blocking 'gates' which, judging from the result with gramicidin M-, must be equal to or related to the Trp residues.


Assuntos
Amidinas/química , Antibacterianos/química , Gramicidina/química , Canais Iônicos , Artefatos , Potenciais da Membrana/fisiologia
8.
Biochemistry ; 38(29): 9185-97, 1999 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-10413493

RESUMO

Dipoles of the tryptophan indole side chains have a direct impact on ion conductance in the gramicidin channel. Here, fluorination of the indoles (both 5- and 6-fluoro) is used to manipulate both the orientations and the magnitudes of the dipoles. The orientations and positions with respect to the channel axis were determined using (2)H solid state NMR of uniformly aligned lipid bilayer preparations. By exchange of the remaining four protons in the indole ring for deuterium, comparison could be made to d(5)-indole spectra that have previously been recorded for each of the four indoles of gramicidin A. After making the assignments which were aided by the observation of (19)F-(2)H dipolar interactions, we found that fluorination caused only minor changes in side chain conformation. With the high-resolution structural characterization of the fluorinated indoles in position 11, 13, and 15, the electrostatic interactions with a cation at the channel and bilayer center can be predicted and the influence of the modified dipoles on ion conductance estimated. The importance of the long-range electrostatic interaction was recently documented with the observation of alpha-helical dipoles oriented toward the bilayer center on the ion conductance pathway for the Streptomyces K(+) channel. We present direct measurements of the orientation of gramicidin channel F-Trp positions for use in analysis of dipole effects on channel permeation.


Assuntos
Gramicidina/química , Canais Iônicos/química , Bacillus/química , Proteínas de Bactérias/química , Deutério , Dimiristoilfosfatidilcolina/química , Gramicidina/análogos & derivados , Indóis/química , Bicamadas Lipídicas/química , Computação Matemática , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Prótons , Eletricidade Estática , Relação Estrutura-Atividade , Triptofano/análogos & derivados , Triptofano/química
10.
Biophys J ; 76(4): 1897-908, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10096887

RESUMO

The four Trp dipoles in the gramicidin A (gA) channel modulate channel conductance, and their side chain conformations should therefore be important, but the energies of different conformations are unknown. A conformational search for the right-handed helix based on molecular mechanics in vacuo yielded 46 conformations within 20 kcal/mol of the lowest energy conformation. The two lowest energy conformations correspond to the solid-state and solution-state NMR conformations, suggesting that interactions within the peptide determine the conformation. For representative conformations, the electrostatic potential of the Trp side chains on the channel axis was computed. A novel application of the image-series method of. Biophys. J. 9:1160-1170) was introduced to simulate the polarization of bulk water by the Trp side chains. For the experimentally observed structures, the CHARm toph19 potential energy (PE) of a cation in the channel center is -1.65 kcal/mol without images. With images, the PE is -1.9 kcal/mol, demonstrating that the images further enhance the direct dipole effect. Nonstandard conformations yielded less favorable PEs by 0.4-1.1 kcal/mol.


Assuntos
Gramicidina/química , Canais Iônicos/química , Sequência de Aminoácidos , Fenômenos Biofísicos , Biofísica , Condutividade Elétrica , Dados de Sequência Molecular , Conformação Proteica , Eletricidade Estática , Termodinâmica , Triptofano/química
11.
Biophys J ; 77(5): 2492-501, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20540928

RESUMO

Proton transport on water wires, of interest for many problems in membrane biology, is analyzed in side-chain analogs of gramicidin A channels. In symmetrical 0.1N HCl solutions, fluorination of channel Trp(11), Trp-(13), or Trp(15) side chains is found to inhibit proton transport, and replacement of one or more Trps with Phe enhances proton transport, the opposite of the effects on K(+) transport in lecithin bilayers. The current-voltage relations are superlinear, indicating that some membrane field-dependent process is rate limiting. The interfacial dipole effects are usually assumed to affect the rate of cation translocation across the channel. For proton conductance, however, water reorientation after proton translocation is anticipated to be rate limiting. We propose that the findings reported here are most readily interpreted as the result of dipole-dipole interactions between channel waters and polar side chains or lipid headgroups. In particular, if reorientation of the water column begins with the water nearest the channel exit, this hypothesis explains the negative impact of fluorination and the positive impact of headgroup dipole on proton conductance.


Assuntos
Gramicidina/metabolismo , Prótons , Transporte Biológico , Gramicidina/química , Ácido Clorídrico/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Permeabilidade , Multimerização Proteica , Estrutura Quaternária de Proteína
12.
Biophys J ; 75(6): 2830-44, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9826605

RESUMO

Gramicidin A (gA), with four Trp residues per monomer, has an increased conductance compared to its Phe replacement analogs. When the dipole moment of the Trp13 side chain is increased by fluorination at indole position 5 (FgA), the conductance is expected to increase further. gA and FgA conductances to Na+, K+, and H+ were measured in planar diphytanoylphosphatidylcholine (DPhPC) or glycerylmonoolein (GMO) bilayers. In DPhPC bilayers, Na+ and K+ conductances increased upon fluorination, whereas in GMO they decreased. The low ratio in the monoglyceride bilayer was not reversed in GMO-ether bilayers, solvent-inflated or -deflated bilayers, or variable fatty acid chain monoglyceride bilayers. In both GMO and DPhPC bilayers, fluorination decreased conductance to H+ but increased conductance in the mixed solution, 1 M KCl at pH 2.0, where K+ dominates conduction. Eadie-Hofstee plot slopes suggest similar destabilization of K+ binding in both lipids. Channel lifetimes were not affected by fluorination in either lipid. These observations indicate that fluorination does not change the rotameric conformation of the side chain. The expected difference in the rate-limiting step for transport through channels in the two bilayers qualitatively explains all of the above trends.


Assuntos
Gramicidina/química , Canais Iônicos/química , Fenômenos Biofísicos , Biofísica , Condutividade Elétrica , Flúor/química , Glicerídeos/química , Técnicas In Vitro , Indóis/química , Cinética , Bicamadas Lipídicas/química , Potenciais da Membrana , Modelos Químicos , Conformação Molecular , Oniocompostos/química , Permeabilidade , Termodinâmica , Triptofano/química
13.
Biophys J ; 73(4): 1699-716, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9336167

RESUMO

The free energy profiles for four organic cations in right-handed single-helix gramicidin A dimers were computed by using umbrella sampling molecular dynamics with CHARMM. Ion-water column translocations were facilitated by using a novel "water-tunnel" approach. The overlapping pieces of free energy profile for adjacent windows were selected from three trajectories that differed in initial ion rotation and were aligned by the method of umbrella potential differences. Neglected long-range electrostatic energies from the bulk water and the bilayer were computed with DelPhi and added to the profile. The approach was corroborated for the formamidinium-guanidinium pair by using perturbation dynamics at axial positions 0, 6, 12, and 15 A from the channel center. The barrier to ethylammonium entry was prohibitive at 21 kcal/mol, whereas for methylammonium it was 5.5 kcal/mol, and the profile was quite flat through the channel, roughly consistent with conductance measurements. The profile for formamidinium was very similar to that of methylammonium. Guanidinium had a high entry barrier (deltaF = +8.6 kcal/mol) and a narrow deep central well (deltaF = -2.6 kcal/mol), qualitatively consistent with predictions from voltage-dependent potassium current block measurements. Its deep central well, contrasting with the flat profile for formamidinium, was verified with perturbation dynamics and was correlated with its high propensity to form hydrogen bonds with the channel at the dimer junction (not shared by the other three cations). Analysis of the ensemble average radial forces on the ions demonstrates that all four ions undergo compressive forces in the channel that are at maximum at the center of the monomer and relieved at the dimer junction, illustrating increased flexibility of the channel walls in the center of the channel.


Assuntos
Gramicidina/química , Canais Iônicos/química , Amidinas/química , Fenômenos Biofísicos , Biofísica , Cátions , Dimerização , Guanidina/química , Metilaminas/química , Modelos Moleculares , Conformação Proteica , Compostos de Amônio Quaternário/química , Eletricidade Estática , Termodinâmica , Água
14.
Biophys J ; 68(6): 2271-9, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7544164

RESUMO

Compared with alkali metal cations, formamidinium ions stabilize the gramicidin A channel molecule in monoolein bilayers (Seoh and Busath, 1993a). A similar effect is observed with N-acetyl gramicidin channel molecules in spite of the modified forces at the dimeric junction (Seoh and Busath, 1993b). Here we use electrophysiological measurements with tryptophan-to-phenylalanine-substituted gramicidin analogs to show that the formamidinium-induced channel molecule stabilization is eliminated when the four gramicidin tryptophans are replaced with phenylalanines in gramicidin M-. This suggests that the stabilization is mediated by the tryptophan side chains. Tryptophan residues 9, 13, and 15 must cooperate to produce the effect because replacement of any one of the three with phenylalanine significantly reduces stabilization; replacement of Trp-11 with phenylalanine causes negligible decrease in stabilization. In addition, formamidinium-related current-voltage supralinearity and open-channel noise are absent with gramicidin M-. When the lipid bilayer was formed with monoolein ether rather than monoolein ester, the channel lifetimes were reduced markedly and, at low voltage and relative to those in KCl solution, were decreased by a factor of 2, whereas the open-channel noise was unaffected and the current-voltage relation was only modestly affected. These results suggest that formamidinium modifies the state of the tryptophan side chains, which, in turn, affects channel lifetime, current-voltage supralinearity, and open-channel noise through interactions with water or lipid headgroup atoms including the lipid ester carbonyl.


Assuntos
Amidinas , Gramicidina , Canais Iônicos , Modelos Biológicos , Triptofano , Eletrofisiologia/métodos , Cinética , Bicamadas Lipídicas , Substâncias Macromoleculares , Potenciais da Membrana , Fenilalanina , Relação Estrutura-Atividade
15.
Biophys J ; 65(5): 1817-27, 1993 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7507714

RESUMO

Compared to the N-formyl gramicidin A (GA), the N-acetyl gramicidin A (NAG) channel has unchanged conductance in 1 M NH4+ (gamma NN/gamma GG = 1, conductance ratio) but reduced conductance in 1 M K+ (gamma NN/gamma GG = 0.6) methylammonium (gamma NN/gamma GG = 0.3), and formamidinium (gamma NN/gamma GG = 0.1) solutions. Except with formamidinium, "flicker blocks" are evident even at low cutoff frequencies. For all cations studied, channel lifetimes of N-acetyl homodimers (NN) are approximately 50-fold shorter than those of the GA homodimer (GG). The novel properties of GA channels in formamidinium solution (supralinear current-voltage relations and dimer stabilization (Seoh and Busath, 1993)) also appear in NN channels. The average single channel lifetime in 1 M formamidinium solution at 100 mV is 6-7-fold longer than in K+ and methylammonium solutions and, like in the GA channel, significantly decreases with increasing membrane potential. Experiments with mixtures of the two peptides, GA and NAG, showed three main conductance peaks. Oriented hybrids were formed utilizing the principle that monomers remain in one leaflet of the bilayer (O'Connell et al., 1990). With GA at the polarized side and NAG at the grounded side, at positive potentials (in which case hybrids were designated GN) and at negative potentials (in which case hybrids were designated NG), channels had the same conductances and channel properties at all potentials studied. Flicker blocks were not evident in the hybrid channels, which suggests that both N-acetyl methyl groups at the junction of the dimer are required to cause flickers. Channel lifetimes in hybrids are only approximately threefold shorter than those of the GG channels, and channel conductances are similar to those of GG rather than NN channels. We suggest that acetyl-acetyl crowding at the dimeric junction in NN channels cause dimer destabilization, flickers, and increased selectivity in N-acetyl gramicidin channels.


Assuntos
Gramicidina/química , Canais Iônicos/química , Amidinas/farmacologia , Fenômenos Biofísicos , Biofísica , Condutividade Elétrica , Ligação de Hidrogênio , Canais Iônicos/efeitos dos fármacos , Bicamadas Lipídicas/química , Potenciais da Membrana , Metilaminas/farmacologia , Modelos Moleculares , Potássio/farmacologia , Conformação Proteica/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia
16.
Biophys J ; 64(4): 1017-28, 1993 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7684267

RESUMO

The conductance properties of organic cations in single gramicidin A channels were studied using planar lipid bilayers. From measurements at 10 mM and at 27 mV the overall selectivity sequence was found to be NH4+ > K+ > hydrazinium > formamidinium > Na+ > methylammonium, which corresponds to Eisenman polyatomic cation sequence X'. Methylammonium and formamidinium exhibit self block, suggesting multiple occupancy and single filing. Formamidinium has an apparent dissociation constant (which is similar to those of alkali metal cations) for the first ion being 22 mM from the Eadie-Hofstee plot (G0 vs. G0/C), 12 mM from the rate constants of a three-step kinetic model. The rate-limiting step for formamidinium is translocation judging from supralinear I-V relations at low concentrations. 1 M formamidinium solutions yields exceptionally long single channel lifetimes, 20-fold longer than methylammonium, which yields lifetimes similar to those found with alkali metal cations. The average lifetime in formamidinium solution significantly decreases with increasing voltage up to 100 mV but is relatively voltage independent between 100 and 200 mV. At lower voltages (< or = 100 mV), the temperature and concentration dependences of the average lifetime of formamidinium were steep. At very low salt concentrations (0.01 M, 100 mV), there was no significant difference in average lifetime from that formed with 0.01 M methylammonium or hydrazinium. We conclude that formamidinium very effectively stabilizes the dimeric channel while inside the channel and speculate that it does so by affecting tryptophan-reorientation or tryptophan-lipid interactions at binding sites.


Assuntos
Gramicidina/metabolismo , Canais Iônicos/metabolismo , Amidinas/metabolismo , Fenômenos Biofísicos , Biofísica , Cátions , Condutividade Elétrica , Técnicas In Vitro , Transporte de Íons , Cinética , Bicamadas Lipídicas , Potenciais da Membrana , Metilaminas/metabolismo , Modelos Biológicos , Permeabilidade
17.
Annu Rev Physiol ; 55: 473-501, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-7682046

RESUMO

The various details provided by physical methods have been integrated into a coherent picture of the cation transport process. The free energy profile describes simultaneously the thermodynamic and kinetic factors that govern conductance. Localization of ion binding sites near each end of the channel by NMR and X-ray crystallographic techniques demonstrates that there is an energy barrier separating the two ends. There is no a priori reason to suspect any barrier to ion entry into the channel, and recent molecular modeling computations confirm this intuition. Therefore, the channel is best described as a two-site, one-barrier channel. However, the movement of ions across the central barrier over a distance of 1.9 nm is really a multistep process best described as Brownian motion (even the short steps from one pair of carbonyls to the next is more diffusive than activated for ions as large as Na+), the entry step is probably best considered as a compound step requiring correction for interfacial polarization and diffusion limitation, the binding sites in the channel ought not be considered to be in equilibrium with the bath, and quasi-knock off behavior (binding of a second ion at the entry facilitates release of a first ion from the exit) is probably the rule at physiological permeant ion concentrations and higher. Progress will probably focus on channel kinetics. The channel backbone probably undergoes conformational changes as the ions pass which, according to solid state NMR results, may last long enough to provide the channel with a sort of memory and which may give rise to excess single channel noise. These conformational changes are further reflected in shifts in side chain positions which, in turn, may be partly responsible for changes in the single-channel lifetime, which appears to be exquisitely sensitive to side chain-lipid interactions. Reasonable explanations for the impermeance of divalent cations, anions, and medium-sized organic cations such as guanidinium have proven elusive at first, but it now appears that divalent cations bind water too tightly, anions bind water in an orientation that produces unfavorable contacts between water oxygens and peptide carbonyl oxygens at the channel entry, and iminium ions bind strongly to the flexible peptide carbonyls at the channel entry.


Assuntos
Gramicidina/metabolismo , Canais Iônicos/metabolismo , Animais , Humanos , Cinética , Modelos Biológicos
18.
Biophys J ; 63(1): 152-61, 1992 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1384733

RESUMO

Empirical energy function calculations were used to evaluate the effects of minimization on the structure of a gramicidin A channel and to analyze the energies of interaction between three cations (guanidinium, acetamidinium, formamidinium) and the channel as a function of position along the channel axis. The energy minimized model of the gramicidin channel, which was based on the results of Venkatachalam and Urry (1983), has a constriction at the channel entrance. If the channel is not allowed to relax in the presence of the ions (rigid model), there is a large potential energy barrier for all three cations. The barrier varies with cation size and is due to high van der Waals and ion deformation energies. If the channel is minimized in the presence of the ions, the potential energy barrier to formamidinium entry is almost eliminated, but a residual barrier remains for guanidinium and acetamidinium. The residual barrier is primarily due, not to the expansion of the helix, but, to the disruption of hydrogen bonds between the terminal ethanoloamine and the next turn of the helix which occurs when the carbonyls of the outer turn of the helix librate inward toward the ion as it enters the channel. The residual potential energy barriers could be a possible explanation for the measured selectivity of gramicidin for formamidinium over guanidinium. The results of this full-atomic model address the applicability of the size-exclusion concept for the selectivity of the gramicidin channel.


Assuntos
Gramicidina/metabolismo , Canais Iônicos/metabolismo , Amidinas/metabolismo , Fenômenos Biofísicos , Biofísica , Gramicidina/química , Guanidina , Guanidinas/metabolismo , Modelos Biológicos , Modelos Moleculares , Sondas Moleculares , Permeabilidade , Termodinâmica
19.
Biochim Biophys Acta ; 1107(1): 31-8, 1992 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-1377493

RESUMO

Ondrias et al. ((1986) Stud. Biophys. 115, 17-22) found that dibucaine, butacaine, and tetracaine reduce the conductance of membranes containing multiple (greater than 10(6)) gramicidin channels. Similar experiments with local anesthetics (LA's) added to the bath while gently stirring showed that the inhibition developed slowly over a time course of 5-10 min. We developed a many (10-20) channel membrane technique which demonstrated that when LA's were added to the bath and the membrane was repeatedly broken and reformed, the channel occurrence frequency declined promptly. In standard single-channel membrane experiments at lower gramicidin densities, the mean single channel conductance and lifetime distributions with LA's present in the bath did not differ from the controls. The predominant channel conductance amplitude was lower by 9.1% than those of controls, but channel amplitude distributions were also modified so that the net reduction in overall population channel conductance was only about 2.0%. Channel currents showed no evidence of flicker blocks. The lifetime histograms of control and LA-exposed channel populations were both satisfactorily fit by a single-exponential function with the same mean. Thus, inhibition is due primarily to a reduction in the frequency of occurrence of conducting channels, implying a reduced concentration of active monomers in the membrane.


Assuntos
Anestésicos Locais/farmacologia , Gramicidina/metabolismo , Canais Iônicos/metabolismo , Ácido 4-Aminobenzoico/farmacologia , Dibucaína/farmacologia , Condutividade Elétrica , Canais Iônicos/efeitos dos fármacos , Cinética , Tetracaína/farmacologia
20.
Protein Eng ; 5(4): 285-93, 1992 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1409550

RESUMO

To examine the feasibility of a beta structure for the pore-lining region of the voltage-gated potassium channel, we have characterized a family of 12 antiparallel beta-barrels. Each is comprised of four identical pairs of beta-strands organized with approximate 4-fold symmetry about a channel axis. The C- and N-termini of the beta-strand pairs are assumed to be at the extracellular end of the channel, and each pair is connected by a hairpin turn at the intracellular end of the channel. The models differ in the residues located in the hairpin turn and in the orientation of the two strands of each pair in the barrel, i.e. whether the C-terminus of a pair is clockwise (CW) or counterclockwise (CCW) from the N-terminus when the channel is viewed from outside the cell. Following known structure precedents and potential energy predictions, the barrel is assumed to be right-twisting in all cases. All models have crowded layers of inward-projecting aromatic side-chains near the center of the channel which could regulate channel selectivity. The models with an odd number of amino acids in the hairpin turn have the advantage of predicting that F433 points into the barrel, but the disadvantage that V438 does not. Of these models, two of the models are most consistent with the external tetraethylammonium (TEA) block data, and of those, one (T439 CCW 3:5) is most consistent with the internal TEA block data.


Assuntos
Canais de Potássio/química , Sequência de Aminoácidos , Ligação de Hidrogênio , Ativação do Canal Iônico , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica
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