A Variable Residue in the Pore of Kv1 Channels Is Critical for the High Affinity of Blockers from Sea Anemones and Scorpions

Abstract

Animal toxins are associated with well defined selectivity profiles; however the molecular basis for this property is not understood. To address this issue we refined our previous three-dimensional models of the complex between the sea anemone toxin BgK and the S5-S6 region of Kv1.1 (Gilquin, B., Racape, J., Wrisch, A., Visan, V., Lecoq, A., Grissmer, S., Mé nez, A., and Gasparini, S. (2002) J. Biol. Chem. 277, 37406 -37413) using a docking procedure that scores and ranks the structures by comparing experimental and back-calculated values of coupling free energies ⌬⌬G int obtained from doublemutant cycles. These models further highlight the interaction between residue 379 of Kv1.1 and the conserved dyad tyrosine residue of BgK. Because the nature of the residue at position 379 varies from one channel subtype to another, we explored how these natural mutations influence the sensitivity of Kv1 channel subtypes to BgK using binding and electrophysiology experiments. We demonstrated that mutations at this single position indeed suffice to abolish or enhance the sensitivity of Kv1 channels for BgK and other sea anemone and scorpion toxins. Altogether, our data suggest that the residue at position 379 of Kv1 channels controls the affinity of a number of blocking toxins.Molecular recognition and specific association of protein ligands and protein targets are central to most biological processes. Understanding the molecular basis of these interactions is critical for engineering novel protein-protein interactions. In particular, understanding how protein ligands bind with high affinity to only a subset of closely related receptors may help to design ligands with novel selectivities.A number of studies have been carried out to identify the sites used by protein ligands to bind to several related receptors. These sites were shown to be composed of a core formed by conserved hot spot residues together with target-specific residues (1-5). However, how these sites accommodate the different receptors subtypes is still poorly understood. In many cases the molecular determinants responsible for protein ligand discrimination remain to be identified.Toxins from sea anemones and scorpions that block currents through Kv1 voltage-gated potassium channels are particularly appropriate to investigate the molecular basis of selectivity of protein-protein interactions since each toxin binds to only

Molecular Targets for Antiepileptic Drug Development

“…These toxins bind with high affinity near the pore region of the K v channel, blocking current flow. 133,134 The best known of these toxins are the dendrotoxins found in the Dendroaspis genus of the African mamba snake, which induce behavioral and electrographic seizure activity when injected intracerebrally and are also active in brain slice preparations. 135,136 The dendrotoxins block K v 1.1 and also K v 1.2 and K v 1.6.…”

A bifunctional sea anemone peptide with Kunitz type protease and potassium channel inhibiting properties

“…The Phe13 will most likely interact through hydrophobic forces with the aromatic cluster formed by Phe356, Trp364, Trp365, and Tyr375. Previous work has highlighted Tyr375 in K V 1.1 as a crucial residue for the selectivity of scorpion and sea anemone toxins [44]. The amino acid at this position is highly variable between K V 1 isoforms and the nature of the side chain of this residue contributes significantly to the subtype selectivity of toxins.…”

Tetraphenylporphyrin derivative specifically blocks members of the voltage-gated potassium channel subfamily Kv1

“…To investigate a gating modifying effect of por3, we mutated Kv1.3 Val 430 to Trp by analogy to the Shaker channel mutant V478W 20,24 expecting that the mutation diminishes ionic Kv1.3 currents and, thereby, facilitates measurement of gating currents. 21,22 Indeed, the mutant Kv1.3V430W channel no longer mediated measurable ionic current.…”

“…A positively charged amino acid chain near the extracellular pore entrance, like Kv1.5 Arg 487, hinders binding of pore blockers to Kv1 channels, e.g., that of TEA and scorpion toxins. 24 Hence, we replaced Arg 487 in Kv1.5 by tyrosine (Kv1.5R487Y), which occurs in an analogous location at the por3 sensitive Kv1.1 pore entrance. Por3 sensitivity of Kv1.5R487Y channels was investigated at +80 mV, where the channels are fully activated.…”