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

E. Dietlind Koch^*, Baldomero M. Olivera, Heinrich Terlau^* and Franco Conti

^* Max-Planck-Institute for Experimental Medicine, Molecular and Cellular Neuropharmacology Group, Göttingen, Germany; Department of Biology, University of Utah, Salt Lake City, Utah; and Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Genova, Italy

Correspondence: Address reprint requests to Heinrich Terlau, Max-Planck-Institute for Experimental Medicine, Molecular and Cellular Neuropharmacology Group, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany. Fax: ++49-551-389-9475; E-mail: hterlau{at}gwdg.de.

-Conotoxin PVIIA (-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 -PVIIA binding. Inactivation is slowed by the necessary preliminary unbinding of -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 -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.

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