Dataset Information

Paradoxical one-ion pore behavior of the long ?-helical peptide of marine cytotoxic polytheonamide B.

ABSTRACT: The cytotoxic 48-mer peptide, polytheonamide B (pTB), from a marine sponge forms a ?(6.3)-helix with an inner diameter of 4 Å and a length of 45 Å, features that allow the selective permeation of monovalent cations across targeted cell membranes. To characterize this long, narrow pore, electrophysiological examination using a planar lipid bilayer method was performed. The single-channel current amplitude exhibited saturation for concentrated Cs(+) or K(+) solution, and the reversal potential in mixed solutions did not exhibit any anomalous mole-fraction behavior. These results suggest the one-ion permeation mechanism. This is in contrast to the short (26 Å) ?(6.3)-helical gramicidin channel, which holds two ions simultaneously. The paradoxical one-ion permeation through the long pTB channel was modeled with a discrete-state Markov model. Ions permeate through the channel by stepping between two binding sites in the pore, but never occupy these sites simultaneously in either pure or mixed ion solution.

SUBMITTER: Iwamoto M

PROVIDER: S-EPMC3887386 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Paradoxical one-ion pore behavior of the long β-helical peptide of marine cytotoxic polytheonamide B.

Iwamoto Masayuki M Matsunaga Shigeki S Oiki Shigetoshi S

Scientific reports 20140110

The cytotoxic 48-mer peptide, polytheonamide B (pTB), from a marine sponge forms a β(6.3)-helix with an inner diameter of 4 Å and a length of 45 Å, features that allow the selective permeation of monovalent cations across targeted cell membranes. To characterize this long, narrow pore, electrophysiological examination using a planar lipid bilayer method was performed. The single-channel current amplitude exhibited saturation for concentrated Cs(+) or K(+) solution, and the reversal potential in ...[more]

PMID: 24407373

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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

Paradoxical one-ion pore behavior of the long ?-helical peptide of marine cytotoxic polytheonamide B.

Publications

Paradoxical one-ion pore behavior of the long β-helical peptide of marine cytotoxic polytheonamide B.

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