Pore-modulating toxins exploit inherent slow inactivation to block K ⁺ channels

Abstract

Voltage-dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K+conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+channels as a novel pharmacological target and provides a rational framework for drug design.

A molecular lid mechanism of K+channel blocker action revealed by a cone peptide

“…Conkunitzin-C1 (Conk-C1) and Conkunitzin-C3 (Conk-C3). Both peptides exhibited high similarity to Conkunitzin-S1 (Conk-S1) from Conus Striatus, which was described previously 20,22 (85% and 86% similarity for Conk-C1 and Conk-C3, respectively, Fig. 1A).…”

“…Classical pore blockers acting via a "molecular plug" mechanism directly compete with ions bound at the S1/S0 coordination sites, thus readily dissociate from their binding site when the external [K + ] is raised or a strong depolarization is applied 27,28 . In contrast, Conk-S1, predicted to block ion conduction by triggering collapse of the pore thus avoiding any interaction with the permeant ions, was resilient to voltage-induced dissociation 20 . The "molecular lid" mechanism described herein for Conk-C3 lies between these two extremes: while bound toxin did not forbid ion occupation at the upper coordination sites of the SF, antagonistic interactions, which seem to originate from electrostatic repulsion between the toxin and these ions, were observed ( Fig.…”

“…Recently, we have described the mode of action of Conkunitzin-S1, a cone snail toxin that potently block the drosophila Shaker channel. We have found that instead of plugging the pore, the toxin trigger the collapse of the SF by targeting channel residues that gate the access of water molecules to the aqueous cavities around the pore, resembling the molecular events that take place during slow inactivation 20 .…”

“…Synthetic genes encoding for Conk-C1 and Conk-C3 were constructed, the peptides were expressed in E. coli and purified using an established method 20 . Both peptides were crystalized and high resolution structures were solved from x-ray diffraction, reveling a typical Kunitz fold composed of a double-stranded anti-parallel -sheet and N-and Cterminal -helices reticulated by two disulfide bridges ( Fig.…”

“…MD simulations performed with unmodified K + channels revealed remarkable stability of the selectivity filter region, which typically maintain its symmetrical conformation and bound ions up to several microseconds 20,23,24 . Recently we have demonstrated that Conk-S1, which highly resembles Conk-C3, could trigger an asymmetric collapse of the channel pore by introducing an ectopic traffic at the water-filled cavities that surround the channel pore 20 .…”