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Electroosmotic enhancement of the binding of a neutral molecule to a transmembrane pore.

ABSTRACT: The flux of solvent water coupled to the transit of ions through protein pores is considerable. The effect of this electroosmotic solvent flow on the binding of a neutral molecule [beta-cyclodextrin (betaCD)] to sites within the staphylococcal alpha-hemolysin pore was investigated. Mutant alpha-hemolysin pores were used to which betaCD can bind from either entrance and through which the direction of water flow can be controlled by choosing the charge selectivity of the pore and the polarity of the applied potential. The Kd values for betaCD for individual mutant pores varied by >100-fold with the applied potential over a range of -120 to +120 mV. In all cases, the signs of the changes in binding free energy and the influence of potential on the association and dissociation rate constants for betaCD were consistent with an electroosmotic effect.

SUBMITTER: Gu LQ

PROVIDER: S-EPMC307596 | biostudies-literature | 2003 Dec

REPOSITORIES: biostudies-literature

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Electroosmotic enhancement of the binding of a neutral molecule to a transmembrane pore.

Gu Li-Qun LQ Cheley Stephen S Bayley Hagan H

Proceedings of the National Academy of Sciences of the United States of America 20031215 26

The flux of solvent water coupled to the transit of ions through protein pores is considerable. The effect of this electroosmotic solvent flow on the binding of a neutral molecule [beta-cyclodextrin (betaCD)] to sites within the staphylococcal alpha-hemolysin pore was investigated. Mutant alpha-hemolysin pores were used to which betaCD can bind from either entrance and through which the direction of water flow can be controlled by choosing the charge selectivity of the pore and the polarity of t ...[more]

PMID: 14676320

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Project description:Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene affect CFTR protein biogenesis or its function as a chloride channel, resulting in dysregulation of epithelial fluid transport in the lung, pancreas and other organs in cystic fibrosis (CF). Development of pharmaceutical strategies to treat CF requires understanding of the mechanisms underlying channel function. However, incomplete 3D structural information on the unique ABC ion channel, CFTR, hinders elucidation of its functional mechanism and correction of cystic fibrosis causing mutants. Several CFTR homology models have been developed using bacterial ABC transporters as templates but these have low sequence similarity to CFTR and are not ion channels. Here, we refine an earlier model in an outward (OWF) and develop an inward (IWF) facing model employing an integrated experimental-molecular dynamics simulation (200 ns) approach. Our IWF structure agrees well with a recently solved cryo-EM structure of a CFTR IWF state. We utilize cysteine cross-linking to verify positions and orientations of residues within trans-membrane helices (TMHs) of the OWF conformation and to reconstruct a physiologically relevant pore structure. Comparison of pore profiles of the two conformations reveal a radius sufficient to permit passage of hydrated Cl- ions in the OWF but not the IWF model. To identify structural determinants that distinguish the two conformations and possible rearrangements of TMHs within them responsible for channel gating, we perform cross-linking by bifunctional reagents of multiple predicted pairs of cysteines in TMH 6 and 12 and 6 and 9. To determine whether the effects of cross-linking on gating observed are the result of switching of the channel from open to close state, we also treat the same residue pairs with monofunctional reagents in separate experiments. Both types of reagents prevent ion currents indicating that pore blockage is primarily responsible.

Dataset Information

Electroosmotic enhancement of the binding of a neutral molecule to a transmembrane pore.

Publications

Electroosmotic enhancement of the binding of a neutral molecule to a transmembrane pore.

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