RESUMO
SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is presently undergoing investigation as an antitumor agent because of its high selective toxicity for hypoxic cells in vitro and in vivo. It has been found to be 15 to 200 times more toxic to hypoxic rodent and human cell lines than their normoxic counterparts. We investigated the toxicity of SR 4233 in primary cultures of hepatocytes under various oxygen tensions, ranging from 1% to 20% oxygen. The 50% lethal dose of SR 4233 was found to be 50 times lower in hepatocyte monolayers at 1% O2 versus 20% O2. Even at 4% O2, a concentration that prevails in the pericentral area of the liver under conditions of normal blood flow, SR 4233 was an order of magnitude more toxic than at 20% O2. All samples were analyzed for metabolites, and metabolism was found to be dependent on both the SR 4233 concentration and the oxygen tension. Formation of the major metabolite SR 4317 occurred to the greatest extent at the lowest oxygen concentration and the highest SR 4233 concentration. Very little metabolism occurred at 10 to 20% O2, which is in agreement with data in Chinese hamster ovary cells under aerobic conditions.
Assuntos
Antineoplásicos/toxicidade , Fígado/patologia , Triazinas/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Hipóxia/fisiopatologia , Cinética , Fígado/efeitos dos fármacos , Masculino , Oxigênio/análise , Ratos , Ratos Endogâmicos F344 , TirapazaminaRESUMO
The GABA(A) receptor is an important target for a variety of general anesthetics (Franks and Lieb, 1994) and for benzodiazepines such as diazepam. Specific point mutations in the GABA(A) receptor selectively abolish regulation by benzodiazepines (Rudolph et al., 1999; McKernan et al., 2000) and by anesthetic ethers (Mihic et al., 1997; Krasowski et al., 1998; Koltchine et al., 1999), suggesting the existence of discrete binding sites on the GABA(A) receptor for these drugs. Using anesthetics of different molecular size (isoflurane > halothane > chloroform) together with complementary mutagenesis of specific amino acid side chains, we estimate the volume of a proposed anesthetic binding site as between 250 and 370 A(3). The results of the "cutoff" analysis suggest a common site of action for the anesthetics isoflurane, halothane, and chloroform on the GABA(A) receptor. Moreover, the data support a crucial role for Leu232, Ser270, and Ala291 in the alpha subunit in defining the boundaries of an amphipathic cavity, which can accommodate a variety of small general anesthetic molecules.
Assuntos
Anestésicos/metabolismo , Rim/metabolismo , Receptores de GABA-A/metabolismo , Anestésicos/química , Sítios de Ligação/genética , Linhagem Celular , Clorofórmio/química , Clorofórmio/metabolismo , Relação Dose-Resposta a Droga , Halotano/química , Halotano/metabolismo , Humanos , Isoflurano/química , Isoflurano/metabolismo , Rim/citologia , Rim/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Receptores de GABA-A/química , Receptores de GABA-A/genética , Relação Estrutura-Atividade , TransfecçãoRESUMO
Seeman and coworkers (Seeman, P. (1972) Pharmacol. Rev. 24, 583--655) calculated that anesthetic agents expand membrane volume ten times more than the van der Waals volume of the agent alone. Their calculation was based on the assumption that the thickness of the erythrocyte membrane expands at the same rate as the surface area. However, recent data on bilayer membranes demonstrate that an expansion of membrane surface area is accompanied by a decrease in membrane thickness. A reinterpretation of their erythrocyte area expansion data using an appropriate contraction of membrane thickness suggests the volume in a membrane occupied by anesthetic molecules is approximately equal to their van der Waals volume.
Assuntos
Anestésicos/farmacologia , Membrana Eritrocítica/ultraestrutura , Eritrócitos/ultraestrutura , Membrana Eritrocítica/efeitos dos fármacos , Humanos , Proteínas de Membrana/fisiologiaRESUMO
The effect of 100 atm pressure on the organization of the lipid-peptide complex formed between polymyxin and dipalmitoyl phosphatidic acid has been investigated. Phase transition curves were obtained by electron paramagnetic resonance by measuring the partition coefficient of the spin label, 2, 2, 5, 5-tetramethylpiperidine-N-oxyl. The three-step phase transition curve previously obtained with fluorescence polarization measurements was confirmed, demonstrating three distinct phosphatidic acid domains in the bilayer. Pressure increases binding of polymyxin to phosphatidic acid bilayers and alters the proportions of the two domains that differ in the mode of binding between phosphatidic acid and polymyxin. The binding curves of polymyxin to phosphatidic acid bilayers wre determined and it was shown that application of pressure reduces the cooperativity of the binding curve.
Assuntos
Lipossomos , Ácidos Fosfatídicos , Polimixina B , Polimixinas , Sítios de Ligação , Lipídeos , Conformação Molecular , Peptídeos , Pressão , TermodinâmicaRESUMO
The rate of transfer of spin-labeled phospholipid from donor vesicles of sonicated 1-acyl-2-(10-doxylstearoyl)-sn-glycero-3-phosphocholine to other vesicle was determined as a function of content of cytochrome P-450 and the phosphatidylcholine/phosphatidylethanolamine ratio in the acceptor vesicles. The transfer rate was measured as an increase in intensity that resulted from a decrease in the line width in the EPR spectrum of the spin-labeled phospholipids as they was transferred to the nonspin-labeled acceptor vesicles. A lower transfer rate was observed for acceptor vesicles of pure egg phosphatidylcholine vesicles than for vesicles for a mixture of phosphatidylcholine and phosphatidylethanolamine. The presence of cytochrome P-450 in the acceptor vesicles further increased the transfer rate. Those alterations in the mole ratios of the protein and the two phospholipids that made the bilayer of the reconstituted vesicles more like the membrane of the endoplasmic reticulum resulted in an increase in phospholipid-transfer rate. The mole ratios of components that produce high phospholipid-transfer rates were similar to those that in an earlier study produced a 31P-NMR spectrum characteristic of a nonbilayer phase. These findings suggest that, in the membrane of the endoplasmic reticulum, phospholipid exchange may be an important element in function and interaction with other intracellular organelles.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Bicamadas Lipídicas , Fosfatidilcolinas/farmacologia , Fosfatidiletanolaminas/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Marcadores de SpinRESUMO
The phase transition temperature (Tt) of dipalmitoyl phosphatidic acid multilamellar liposomes is depressed 10 degrees C by the inhalation anesthetic methoxyflurane at a concentration of 100 mmol/mol lipid. Application of 100 atm of helium pressure to pure phosphatidic acid liposomes increased Tt only 1.5 degrees C. However, application of 100 atm helium pressure to dipalmitoyl phosphatidic acid lipsomes containing 100 mmol methoxyflurane/mol lipid almost completely antagonized the effect of the anesthetic. A non-linear pressure effect is observed. In a previous study, a concentration of 60 mmol methoxyflurane/mol dipalmitoyl phosphatidylcholine depressed Tt only 1.5 degrees C, exhibiting a linear pressure effect. The completely different behavior in the charged membrane is best explained by extrusion of the anesthetic from the lipid phase.
Assuntos
Pressão Atmosférica , Bicamadas Lipídicas/metabolismo , Fluidez de Membrana , Metoxiflurano/farmacologia , Fluidez de Membrana/efeitos dos fármacos , Ácidos FosfatídicosRESUMO
Leukotriene B4 was found to be metabolized by rat hepatocyte monolayers at a rate that was linear with increasing substrate concentration from 74 to 740 nM leukotriene B4. The rates of metabolism were dependent on the O2 concentration and were 315, 213, 80, and 36 pmol leukotriene B4 per min per nmol cytochrome P-450 at 20% (212 microM), 4% (42.5 microM), 2% (21.2 microM), and 1% (10.6 microM) O2, respectively. The metabolic rate was not linear with respect to O2 concentration; however, half maximal rate occurred at 4% O2, and O2 concentration found in the pericentral region of normally oxygenated liver. These results suggest that in vivo conditions of hypoxia or ischemia that lead to blood O2 concentrations less than 4% may drastically decrease hepatic clearance of leukotriene B4.
Assuntos
Leucotrieno B4/metabolismo , Fígado/metabolismo , Oxigênio/farmacologia , Animais , Células Cultivadas , Sistema Enzimático do Citocromo P-450/análise , Hipóxia/metabolismo , Cinética , Masculino , Ratos , Ratos EndogâmicosRESUMO
Potentiation of chemical toxicity by hypoxia was studied in confluent hepatocyte monolayers. Addition of either hydroperoxyarachidonic acid (50 micrograms), leukotriene C4 (10 micrograms), or calcium ionophore A23187 (1.8 micrograms) to hepatocyte monolayers followed by incubation in 2% oxygen for 24 h killed 95% of the hypoxic cells, but was without effect on the normoxic cells. The greater than 10-fold increase in toxicity of A23187 suggests that hypoxic cells are less able to regulate intracellular calcium. The increased toxicity of hydroperoxyarachidonic acid and leukotriene C4 may be due to a related reduction in activity of protective enzymes.
Assuntos
Ácidos Araquidônicos/farmacologia , Calcimicina/farmacologia , Leucotrienos , Peróxidos Lipídicos/farmacologia , Fígado/citologia , Oxigênio/farmacologia , SRS-A/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Leucotrieno B4/farmacologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
We recently developed ultra-sensitive ethanol receptors (USERs) as a novel tool for investigation of single receptor subunit populations sensitized to extremely low ethanol concentrations that do not affect other receptors in the nervous system. To this end, we found that mutations within the extracellular Loop 2 region of glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs) can significantly increase receptor sensitivity to micro-molar concentrations of ethanol resulting in up to a 100-fold increase in ethanol sensitivity relative to wild-type (WT) receptors. The current study investigated: (1) Whether structural manipulations of Loop 2 in α1 GlyRs could similarly increase receptor sensitivity to other anesthetics; and (2) If mutations exclusive to the C-terminal end of Loop 2 are sufficient to impart these changes. We expressed α1 GlyR USERs in Xenopus oocytes and tested the effects of three classes of anesthetics, isoflurane (volatile), propofol (intravenous), and lidocaine (local), known to enhance glycine-induced chloride currents using two-electrode voltage clamp electrophysiology. Loop 2 mutations produced a significant 10-fold increase in isoflurane and lidocaine sensitivity, but no increase in propofol sensitivity compared to WT α1 GlyRs. Interestingly, we also found that structural manipulations in the C-terminal end of Loop 2 were sufficient and selective for α1 GlyR modulation by ethanol, isoflurane, and lidocaine. These studies are the first to report the extracellular region of α1 GlyRs as a site of lidocaine action. Overall, the findings suggest that Loop 2 of α1 GlyRs is a key region that mediates isoflurane and lidocaine modulation. Moreover, the results identify important amino acids in Loop 2 that regulate isoflurane, lidocaine, and ethanol action. Collectively, these data indicate the commonality of the sites for isoflurane, lidocaine, and ethanol action, and the structural requirements for allosteric modulation on α1 GlyRs within the extracellular Loop 2 region.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Hipnóticos e Sedativos/farmacologia , Receptores de Glicina/química , Receptores de Glicina/metabolismo , Animais , Biofísica , Relação Dose-Resposta a Droga , Estimulação Elétrica , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Isoflurano/farmacologia , Lidocaína/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Mutação/genética , Oócitos , Técnicas de Patch-Clamp , Propofol/farmacologia , Receptores de Glicina/genética , XenopusRESUMO
We have suggested the use of ethyl acetate for extraction of hydroxyl or superoxide radical adducts of the spin trap phenyl N-tert-butyl nitrone (PBM). The technique produced EPR spectra with narrow line widths, the radical adducts were more stable, and there were sufficiently large differences between the isotropic nitrogen hyperfine coupling constant (alpha N) and the beta hydrogen coupling constant (alpha H beta) for both the hydroxyl and superoxide radical adducts to allow their simultaneous quantitation in mixtures. However, Kalyanaraman, Mottley, and Mason have suggested that our assignments of alpha N and alpha H beta were incorrect and that extraction of spin-trapped adducts into ethyl acetate is not as useful as we had proposed. This paper demonstrates that their objections are unfounded and are based on a computational error that they made when they attempted to calculate the hyperfine splittings in their spectra.
Assuntos
Acetatos , Hidróxidos/isolamento & purificação , Óxidos de Nitrogênio , Marcadores de Spin , Superóxidos/isolamento & purificação , Óxidos N-Cíclicos , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Radical HidroxilaRESUMO
Rat hepatocyte homogenates convert 5-hydroperoxyeicosatetraenoic acid (5-HPETE) into biologically active leukotriene B4 (LTB4) as well as less active all-trans-LTB4 (i.e., 6-trans-LTB4 and 6-trans-12-epi-LTB4). Here, we present a hypothesis of the reaction mechanism and the minimal structural requirements of the active enzyme based on the following experimental evidence: The ED50 of the inhibitors 5,8,11,14-eicosatetraynoic acid (ETYA) and 5,6-dehydro-eicosatetraenoic acid was approximately 100-fold higher than for 5-lipoxygenase. Propanethiol and O2 were strong inhibitors of LTB4 formation, whereas butylated hydroxytoluene, nordihydroguaiaretic acid, metyrapone, Desferal and CO had no effect. Cytochrome c, catalase, hematin, and a Fe3+/Fe2+ couple, but not iron-free protoporphyrin IX, catalyzed the formation of only all-trans-LTB4. LTB4 formation in hepatocyte homogenates was heat- and trypsin-sensitive whereas all-trans-LTB4 formation was not. We propose that a ferric heme iron forms a ferryl-hydroxo complex upon homolytic scission of the oxygen-oxygen bond in 5-HPETE and the resulting 5,6-trans-epoxide radical is oxidized by the ferryl-hydroxo complex to yield LTA4. A mechanism for hydrolysis of LTA4 is described that results in formation of LTB4 (less than 1% yield) rather than all-trans-LTB4.
Assuntos
Leucotrieno B4/metabolismo , Leucotrienos/metabolismo , Fígado/enzimologia , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico , Ácidos Araquidônicos/antagonistas & inibidores , Ácidos Araquidônicos/metabolismo , Sítios de Ligação , Fenômenos Químicos , Química , Cromatografia Líquida de Alta Pressão , Retículo Endoplasmático/enzimologia , Radicais Livres , Leucotrieno A4 , Inibidores de Lipoxigenase , Fígado/ultraestrutura , Masculino , Modelos Biológicos , Estrutura Molecular , Oxigênio/farmacologia , Ratos , Ratos Endogâmicos F344RESUMO
This report describes the application of direct chemical ionization mass spectrometry (DCIMS) to the identification and quantification of 5- and 15-HPETEs. A unique feature of the method is use of a polyimide-coated fused silica fiber that allows vaporization of the hydroperoxides, with very low excess energy, into the plume of the chemical ionization reagent gas plasma. Mass spectra are obtained that allow identification of the nonreduced and nonderivatized free acid forms of 5- and 15-HPETE as well as their quantification from 1 microgram to 100 picograms.
Assuntos
Ácidos Araquidônicos/análise , Ácidos Hidroxieicosatetraenoicos/análise , Leucotrienos , Peróxidos Lipídicos/análise , Animais , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Espectrofotometria InfravermelhoRESUMO
1. Each residue in the second transmembrane segment (TM2) of the human GABA(A) receptor alpha(2) subunit was individually mutated to tryptophan. The wild-type or mutant alpha(2) subunits were expressed with the wild-type human GABA(A) receptor beta(2) subunit in Xenopus oocytes, and the effects of these mutations were investigated using two-electrode voltage-clamp recording. 2. Four mutations (V257W, T262W, T265W and S270W) produced receptors which were active in the absence of agonist, and this spontaneous open channel activity was blocked by both picrotoxin and bicuculline, except in the alpha(2)(V257W)beta(2) mutant receptor, which was not sensitive to picrotoxin. 3. Six mutations (V257W, V260W, T262W, T267W, S270W and A273W) enhanced the agonist sensitivity of the receptor, by 10 - 100 times compared with the wild-type alpha(2)beta(2) receptor. Other mutations (T261W, V263W, L269W, I271W and S272W) had little or no effect on the apparent affinity of the receptor to GABA. Eight of the tryptophan mutations (R255, T256, F258, G259, L264, T265, M266 or T268) resulted in undetectable GABA-induced currents. 4. The S270W mutation eliminated potentiation of GABA by ethanol, whereas T261W markedly increased the action of ethanol. The T262W mutation produced direct activation (10% of maximal GABA response) by ethanol in the absence of GABA, while other mutations did not alter the action of ethanol significantly. 5. These results are consistent with a unique role for S270 in the action of ethanol within the TM2 region, and with models of GABA(A) receptor channel function, in which specific residues within TM2 are critical for the regulation of channel gating (S270, L264), while other residues (L269, I271 and S272) have little effect on these functions and may be non-critical structural residues.
Assuntos
Etanol/farmacologia , Receptores de GABA-A/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Depressores do Sistema Nervoso Central/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Dados de Sequência Molecular , Mutagênese , Oócitos , Receptores de GABA-A/química , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/genética , Homologia de Sequência de Aminoácidos , Transfecção , Triptofano/genética , Triptofano/metabolismo , Xenopus laevisRESUMO
Hepatocyte monolayer cultures were exposed to 6000 ppm styrene vapor at 20%, 2%, or 1% O2 and assayed for signs of cell damage immediately following the 2-hr exposure and then 24 hr later. Oxygen concentrations were used that were previously shown to maximize lipid peroxidation and to predispose hepatocyte monolayers to chemical injury. The use of two time points allowed assessment of acute injury as well as injury that requires several hours to manifest itself. The uptake of styrene into the buffer in the culture dishes was measured by gas chromatography and was found to be 0.49, 0.68, and 0.74 mM at 15, 60, and 120 min, respectively. However, as measured by release of aspartate aminotransferase and inclusion of trypan blue, no toxicity was evident at either time point, irrespective of the oxygen concentration. This study shows that despite the weakening of hepatocyte defense mechanisms by hypoxia, styrene is not acutely toxic to these cells. Furthermore, if any damage to DNA, RNA, or the capability for protein synthesis occurs during exposure to styrene, it is insufficient to cause lysis within 24 hr.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Oxigênio/fisiologia , Estirenos/toxicidade , Animais , Aspartato Aminotransferases/sangue , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Gasosa , Técnicas In Vitro , Masculino , Ratos , Ratos Endogâmicos F344 , Azul TripanoRESUMO
Volatile anesthetic concentrations have been difficult to measure, but are an important experimental parameter for in vitro studies of anesthetic actions. Calcium sensitive electrodes were investigated as a means of continuously monitoring anesthetic concentrations in artificial cerebrospinal fluids (ACSF). Anesthetic-induced Ca2+ electrode signals were compared at room (22 degrees C) and physiological (35 degrees C) temperatures. Electrophysiological measures of anesthetic effects on synaptic potentials provided a bioassay. Halothane and isoflurane produced negative changes in calcium electrode potentials which were linearly related to concentrations over a clinically useful range (0.5-1.5 MAC). Anesthetic-induced voltages persisted in nominally zero Ca2+ ACSF and even in deionized water. A good correlation (r>0.9) was found for calcium electrode measures of anesthetic concentration and synaptic response depression produced by halothane, at both 22 and 35 degrees C. These results support three conclusions: (1) calcium sensitive electrodes provide a useful measure of volatile anesthetic concentrations in aqueous solution. (2) Care must be taken when using these electrodes for Ca2+ concentration measurements, if a volatile anesthetic is also to be used, since the anesthetic could introduce an appreciable error (>50%). (3) A temperature change of 13 degrees C had surprisingly little effect on Ca2+ electrode responses or on synaptic depression produced by anesthetics.
Assuntos
Anestésicos Inalatórios/metabolismo , Cálcio/química , Eletrodos Seletivos de Íons , Animais , Encéfalo , Cultura em Câmaras de Difusão , Eletroquímica/instrumentação , Eletroquímica/métodos , Halotano/análise , Halotano/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Isoflurano/análise , Isoflurano/farmacologia , Perfusão , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Ratos , Soluções , Água/químicaRESUMO
Bcl-2 expression in neural cells has been shown to inhibit apoptotic death in association with a decrease in reactive oxygen species. We present the results of a study that used electron spin resonance (ESR) measurements to evaluate the level of hydroxyl radical production in bcl-2 expressing GT1-7 cells and control cells. Incubation of cell monolayers with the spin trap N-t-alpha-phenylnitrone (PBN), and measurements of the hydroxyl radical production at different timepoints, revealed a higher radical production in control cells than in bcl-2 expressing cells, even in the absence of insult. The ESR signal was suppressed by addition of ethanol, indicating that the trapped radical was indeed hydroxyl radical. The mechanism by which the expression of bcl-2 leads to a decrease in cellular production of hydroxyl radical is unknown.
Assuntos
Radical Hidroxila/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Animais , Linhagem Celular , Sobrevivência Celular , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/antagonistas & inibidores , Radicais Livres/metabolismo , Proteínas de Ligação ao GTP/biossíntese , Expressão Gênica , Neurônios , Proteínas Proto-Oncogênicas c-bcl-2 , Proto-Oncogenes , Marcadores de SpinRESUMO
Previous studies have emphasized the role of molecular polarizability and electric moments, especially dipole and quadrupole moments, in binding of drugs to sites of action. A recent publication of ED50s that prevent response to a noxious stimulus for eight fluorobenzenes has made it possible to compare anesthetic potency with ab initio Hartree-Fock calculations of molecular polarizability as well as dipole and quadrupole moments. Fluorobenzenes provide a stringent test of the role of electric moments in anesthetic potency because individual dipole moments range from 0 to 2.84 debye (D) while the quadrupole moment of benzene is large and negative (-30 x 10(-40) C m(2)), that of hexafluorobenzene is large and positive (30 x 10(-40) C m(2)), and that of 1,3,5-trifluorobenzene is nearly zero. We found that anesthetic potency of fluorobenzenes was not affected by the presence of either dipole or quadrupole moments. This result is surprising because fluoroalkanes and fluorocycloalkanes are most potent when half fluorinated and are usually not anesthetics when perfluorinated. The results suggest that electrostatic interactions are not important for binding of fluorobenzenes at sites of anesthetic action and that these sites are different from those that bind conventional anesthetics.
RESUMO
We built a model of a GABAA alpha1 receptor (GABAAR) that combines the ligand binding (LBD) and the transmembrane domains (TMD). We used six steps: (1) a four-alpha helical bundle in the crystal structure of bovine cytochrome c oxidase (2OCC) was identified as a template for the TMD of a single subunit. (2) The five pore-forming alpha helices of a bacterial mechanosensitive channel (1MSL) served as a template for the pentameric ion channel. (3) Five copies of the tetrameric template from 2OCC were superimposed on 1MSL to produce a homopentamer containing 20 alpha helices arranged around a funnel-shaped central pore. (4) Five copies of the GABAAR sequence were threaded onto the alpha-helical segments of this template and inter-helical loops were generated to produce the TMD model. (5) A model of the LBD was built by threading the aligned sequence of GABAAR onto the crystal structure of the acetylcholine binding protein (1I9B). (6) The models of the LBD and the TMD were aligned along a common five-fold axis, moved together along that axis until in vdW contact, merged, and then optimized with restrained molecular dynamics. Our model corresponds closely with recently published coordinates of the acetylcholine receptor (1OED) but also explains additional features. Our model reveals structures of loops that were not visible in the cryoelectron micrograph and satisfies most labeling and mutagenesis data. It also suggests mechanisms for ligand binding transduction, ion selectivity, and anesthetic binding.
Assuntos
Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de GABA-A/química , Animais , Sítios de Ligação , Bovinos , Canais Iônicos/química , Modelos Moleculares , Ligação Proteica , Isoformas de Proteínas/químicaRESUMO
(1) Successful application of molecular mechanics and molecular dynamics calculations to the binding of halogenated anesthetics requires forcefields with correct parameters for halocarbons. (2) Unfortunately, our survey of six popular forcefields revealed that some of them provide a very poor representation of electrostatic interactions for the halogens. (3) This problem is due to poor or missing assignments of partial atomic charges to the halogen atoms. (4) We describe the forcefields most appropriate for use with halogenated anesthetics and suggest a general method for editing the assignment of partial atomic charges by performing an initial quantum mechanics calculation.