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 BgKKv1.1 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.Although most biological processes are governed by proteinprotein recognition phenomena, the molecular determinants of the specificity of these interactions are yet poorly understood. In particular, one protein ligand can bind multiple receptors and a receptor can be bound by several protein ligands of similar or even different structures (l-3), and the molecular mechanisms underlying these multiple bindings are not well understood. Kv1 voltage-gated potassium channels and toxins from scorpions, snakes, sea anemones, and conus that block currents through these channels offer an appropriate mean to investigate the molecular basis of such pleiotropic aspects of protein-protein interactions. Indeed, a potassium channel can be blocked by different toxins of similar or different folds, and conversely a toxin can bind to several members of Kv1 channels (3). Two lines of evidence suggest that the four groups of toxins use a convergent solution to block Kv1 channels. First, despite their unrelated structures, they all exert their function by binding to the same P-region of the channels, comprised between transmembrane segments S5 and S6 (4 -12). Second, the binding sites of all these toxins contain a functional dyad composed of a lysine and a hydrophobic residue that might constitute a minimal functional core for the toxins to bind Kv1 channels, whereas additional residues might provide each toxin with a specific binding profile (3,(13)(14)(15)(16)(17).To get further insights into the toxin-channel interactions and in particular into the role of the dyad residues, we used double mutant cycle analysis to identify proximities between residues of BgK, a sea anemone toxin, and Kv1.1. Then, these proximities were used to determine the structure of BgK complexed with the S5-S6 region of Kv1.1. Analysis of this model provides ...

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.

Using a peptide toxin, kaliotoxin (KTX), we gained new insight into the topology of the pore region of a voltage-gated potassium channel, mKv1.

A series of psoralens and structurally related 5,7-disubstituted coumarins was synthesized and investigated for their K+ channel blocking activity as well as for their phototoxicity to Artemia salina and their ability to generate singlet oxygen and to photomodify DNA. After screening the compounds on Ranvier nodes of the toad Xenopus laevis, the affinities of the most promising compounds, which proved to be psoralens bearing alkoxy substituents in the 5-position or alkoxymethyl substituents in the neighboring 4- or 4'-position, to a number of homomeric K+ channels were characterized. All compounds exhibited the highest affinity to Kv1.2. 5,8-Diethoxypsoralen (10d) was found to be an equally potent inhibitor of Kv1.2 and Kv1.3, while lacking the phototoxicity normally inherent in psoralens. The reported compounds represent a novel series of nonpeptide blockers of Shaker-type K+ channels that could be further developed into selective inhibitors of Kv1.2 or Kv1. 3.