The Binding of κ-Conotoxin PVIIA and Fast C-Type Inactivation of Shaker K + Channels are Mutually Exclusive

• Published in: Biophysical journal 2004, Vol.86 (1), p.191-209
• Main Authors: Koch, E. Dietlind, Olivera, Baldomero M., Terlau, Heinrich, Conti, Franco
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Cell Membrane - drug effects; Cell Membrane - physiology; Cells, Cultured; Channels, Receptors, and Transporters; Conotoxins - pharmacokinetics; Conotoxins - pharmacology; Ion Channel Gating - drug effects; Ion Channel Gating - physiology; Kinetics; Models, Biological; Oocytes - drug effects; Oocytes - physiology; Potassium Channels - drug effects; Potassium Channels - physiology; Protein Binding; Shaker Superfamily of Potassium Channels; Structure-Activity Relationship; Xenopus laevis
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(04)74096-5

κ-Conotoxin PVIIA ( κ-PVIIA), a 27-amino acid peptide identified from the venom of Conus purpurascens, inhibits the Shaker K + channel by blocking its outer pore. The toxin appears as a gating modifier because its binding affinity decreases with relatively fast kinetics upon channel opening, but there is no indication that it interferes with the gating transitions of the wild-type channels (WT), including the structural changes of the outer pore that underlie its slow C-type inactivation. In this report we demonstrate that in two outer pore mutants of Shaker-IR (M448K and T449S), that have high toxin sensitivity and fast C-type inactivation, the latter process is instead antagonized by and incompatible with κ-PVIIA binding. Inactivation is slowed by the necessary preliminary unbinding of κ-PVIIA, whereas toxin rebinding must await recovery from inactivation causing a double-exponential relaxation of the second response to double-pulse stimulations. Compared with the lack of similar effects in WT, these results demonstrate the ability of peptide toxins like κ-PVIIA to reveal possibly subtle differences in structural changes of the outer pore of K + channels; however, they also warn against a naive use of fast inactivating mutants as models for C-type inactivation. Unfolded from the antagonistic effect of inactivation, toxin binding to mutant noninactivated channels shows state- and voltage-dependencies similar to WT: slow and high affinity for closed channels; relatively fast dissociation from open channels at rate increasing with voltage. This supports the idea that these properties depend mainly on interactions with pore-permeation processes that are not affected by the mutations. In mutant channels the state-dependence also greatly enhances the protection of toxin binding against steady-state inactivation at low depolarizations while still allowing large responses to depolarizing pulses that relieve toxin block. Although not obviously applicable to any known combination of natural channel and outer-pore blocker, our biophysical characterization of such highly efficient mechanism of protection from steady-state outer-pore inactivation may be of general interest.