Trans-toxin ion-sensitivity of charybdotoxin-blocked potassium-channels reveals unbinding transitional states

Abstract

In silico and in vitro studies have made progress in understanding protein–protein complex formation; however, the molecular mechanisms for their dissociation are unclear. Protein–protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.

Tethered peptide neurotoxins display two blocking mechanisms in the K + channel pore as do their untethered analogs

“…Here, as in a prior report (11), we demonstrate that SAK1 toxins can use a second binding orientation in some channels so that an Arg confers voltage dependence rather than the nearby canonical Lys. Recently, high-speed atomic force microscopy was used to show that the affinity of the peptide AgTx2 increases in the KcsA pore over time, consistent with an induced fit binding model (35), and another group demonstrated that CTX can wobble between several bound conformations in Shaker channels so that the pore occluding Lys detaches sufficiently to allow cis-K + ions to enter the pore and alter blockade (36). Here, we also extend the utility of T-toxins to screening and quantifying the parameters of SAK1 toxin-channel interactions to facilitate the study of variants, particularly those with low affinity, in a manner that is amenable to the study of other peptides and membrane receptors.…”

A Molecular Lid Mechanism of K+ Channel Blocker Action Revealed by a Cone Peptide

“…One such non-trivial prediction offered by MD simulations of K + channel blockers relies on their different levels of interaction with the permeant ions. 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 the collapse of the pore, thus avoiding any interaction with the permeant ions, was resilient to voltage-induced dissociation 20 .…”

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

“…One such non-trivial prediction offered by MD simulations of K + channel blockers rely on their different levels of interaction with the permeant ions. 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 .…”