Learning how charybdotoxin dissociates from a voltage gated K-channel.

Abstract

Physical interactions between proteins are at the center of most living events. An extremely simplistic view of protein-protein interactions, the lock-and-key mechanism, consists of the association of the two complementary surfaces and their dissociation, determined by the superficial affinity. While experimental and computational chemists have given us some understanding of the protein-protein association, we lack molecular details of the events leading to the dissociation of the complex. As an excellent example of the lock-key binding mechanism, CTX is a scorpion toxin that binds to the external pore entrance of voltage-gated Shaker K-channels (KvC) with extreme simplicity. CTX binding prevents ion flow without significant conformational impact on either protein. Interestingly, the toxin's dissociation rate is sensitive to the ionic composition of the external solution, revealing the existence of dislocated binding states (wobbling). In tandem with MD simulations, we have investigated the effects of K+ ions, the electric field, and temperature on the CTX binding equilibrium with the aim of understanding the events leading to the CTX dissociation from Shaker-K427E. The K-channels were expressed heterologously in Xenopus oocytes and recorded with the two-electrode-voltage-clamp technique under continuous perfusion. The dissociation rate showed more temperature, voltage, and potassium sensitivity, suggesting that K+ ion access to the pore in the toxin-blocked channel is very sensitive to random thermal motions. Positive internal voltages promote CTX-wobbling and partial toxin dislodging and, possibly, equilibrating the K+ ions of the selectivity filter with the external solution. This observation is compatible with the external K+ sensitivity observed in oocytes. Thus, as proposed 35 years ago, the electric field can force potassium ions within the pore in the direction of the external entry, electrostatically destabilizing CTX. Funded by Fondecyt 1211366