External tetraethylammonium as a molecular caliper for sensing the shape of the outer vestibule of potassium channels.

External tetraethylammonium (TEA+) blocked currents through Kv1.1 channels in a voltage-independent manner between 0 and 100 mV. Lowering extracellular pH (pHo) increased the Kd for TEA+ block. A histidine at position 355 in the Kv1.1 channel protein (homologous to Shaker 425) was responsible for this pH-dependent reduction of TEA+ sensitivity, since the TEA+ effect became independent of pHo after chemical modification of the Kv1.1 channel at H355 and in the H355G and H355K mutant Kv1.1 channels. The Kd values for TEA+ block of the two mutant channels (0.34 +/- 0.06 mM, n = 7 and 0.84 +/- 0. 09 mM, n = 13, respectively) were as expected for a vestibule containing either no or a total of four positive charges at position 355. In addition, the pH-dependent TEA+ effect in the wt Kv1.1 channel was sensitive to the ionic strength of the solution. All our observations are consistent with the idea that lowering pHo increased protonation of H355. This increase in positive charge at H355 will repel TEA+ electrostatically, resulting in a reduction of the effective [TEA+]o at the receptor site. From this reduction we can estimate the distance between TEA+ and each of the four histidines at position 355 to be approximately 10 A, assuming fourfold symmetry of the channel and assuming that TEA+ binds in the central axis of the pore. This determination of the dimensions of the outer vestibule of Kv1.1 channels confirms and extends earlier reports on K+ channels using crystal structure data as well as peptide toxin/channel interactions and points out a striking similarity between vestibules of Kv1.1 and KcsA channels

Structural interactions between BgK and Kv1.1, a voltage gated potassium channel

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Identification of 1-lysophosphatidylethanolamine (C16:1) as an antimicrobial compound in the housefly, Musca domestica

We observed that a methanolic whole body extract of uninfected last instar larvae of the housefly, Musca domestica, displayed antifungal and antibacterial activity. We have further purified this extract to a single active fraction using reversed phase high performance liquid chromatography. The pure fraction inhibited growth of the Gram-positive bacteria Bacillus thuringiensis and the yeast Saccharomyces cerevisiae, but not the Gram-negative bacteria Escherichia coli. The active compound was determined to have a molecular mass of 451.2 Da. Further analysis by nuclear magnetic resonance identified the substance as mono-unsaturated 1-lysophosphatidylethanolamine (C16:1) (1-LPE). The structurally different and more common 2-LPE have been described as mediators of the antimicrobial activity of rimenophenazine antibiotic agents (Van Rensburg et al. 1992). Our results suggest that the isolated 1-LPE displays a higher activity in comparison, possibly based on structure-specific differences in activity. © 2003 Elsevier Ltd. All rights reserved.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Structure of the BgK-Kv1.1 complex based on distance restraints identified by double mutant cycles. Molecular basis for convergent evolution of Kv1 channel blockers.

A structural model of BgK, a sea anemone toxin, complexed with the S5-S6 region of Kv1.1, a voltage-gated potassium channel, was determined by flexible docking under distance restraints identified by a double mutant cycles approach. This structure provides the molecular basis for identifying the major determinants of the BgK-Kv1.1 channel interactions involving the BgK dyad residues Lys(25) and Tyr(26). These interactions are (i) electrostatic interactions between the extremity of Lys(25) side chain and carbonyl oxygen atoms of residues from the channel selectivity filter that may be strengthened by solvent exclusion provided by (ii) hydrophobic interactions involving BgK residues Tyr(26) and Phe(6) and Kv1.1 residue Tyr(379) whose side chain protrudes in the channel vestibule. In other Kv1 channel-BgK complexes, these interactions are likely to be conserved, implicating both conserved and variable residues from the channels. The data suggest that the conservation in sea anemone and scorpion potassium channel blockers of a functional dyad composed of a lysine, and a hydrophobic residue reflects their use of convergent binding solutions based on a crucial interplay between these important conserved interactions.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe