title: The binding of kappa-Conotoxin PVIIA and fast C-type inactivation of Shaker K+ channels are mutually exclusive.
abstract: Kappa-conotoxin PVIIA (kappa-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 kappa-PVIIA binding. Inactivation is slowed by the necessary preliminary unbinding of kappa-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 kappa-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.
authors.affiliation: Max-Planck-Institute for Experimental Medicine, Molecular and Cellular Neuropharmacology Group, Göttingen, Germany.
authors.forename: E Dietlind
authors.initials: ED
authors.lastname: Koch
authors.forename: Baldomero M
authors.initials: BM
authors.lastname: Olivera
authors.forename: Heinrich
authors.initials: H
authors.lastname: Terlau
authors.forename: Franco
authors.initials: F
authors.lastname: Conti
mesh_terms: D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D020916:Conotoxins / Q000493:pharmacokinetics / Q000494:pharmacology*; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008954:Models, Biological; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D011485:Protein Binding; D051637:Shaker Superfamily of Potassium Channels; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D020916:Conotoxins; D015221:Potassium Channels; D051637:Shaker Superfamily of Potassium Channels; C108818:kappa-conotoxin PVIIA