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1.
Biochemistry ; 48(42): 10078-88, 2009 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-19754156

RESUMO

The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5-6 loop. Sites where cysteine reactivity is limited to channel-permeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Modelos Moleculares , Compostos de Sulfidrila/química , Animais , Ânions/química , Cisteína/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citoplasma/metabolismo , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Xenopus laevis
2.
Biophys J ; 91(5): 1737-48, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16766608

RESUMO

Previous attempts to identify residues that line the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have utilized cysteine-substituted channels in conjunction with impermeant, thiol-reactive reagents like MTSET+ and MTSES-. We report here that the permeant, pseudohalide anion [Au(CN)2]- can also react with a cysteine engineered into the pore of the CFTR channel. Exposure of Xenopus oocytes expressing the T338C CFTR channel to as little as 100 nM [Au(CN)2]- produced a profound reduction in conductance that was not reversed by washing but was reversed by exposing the oocytes to a competing thiol like DTT (dithiothreitol) and 2-ME (2-mercaptoethanol). In detached, inside out patches single-channel currents were abolished by [Au(CN)2]- and activity was not restored by washing [Au(CN)2]- from the bath. Both single-channel and macroscopic currents were restored, however, by exposing [Au(CN)2]- -blocked channels to excess [CN]-. The results are consistent with the hypothesis that [Au(CN)2]- can participate in a ligand exchange reaction with the cysteine thiolate at 338 such that the mixed-ligand complex, with a charge of -1, blocks the anion conduction pathway.


Assuntos
Permeabilidade da Membrana Celular/fisiologia , Cianetos/metabolismo , Cisteína/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Compostos de Ouro/metabolismo , Ativação do Canal Iônico , Oócitos/fisiologia , Substituição de Aminoácidos , Animais , Ânions , Células Cultivadas , Cianatos , Regulador de Condutância Transmembrana em Fibrose Cística/química , Condutividade Elétrica , Ouro , Ligantes , Mutagênese Sítio-Dirigida , Engenharia de Proteínas/métodos , Relação Estrutura-Atividade , Xenopus laevis
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