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Mechanism of Block of the hERG K⁺ Channel by the Scorpion Toxin CnErg1

Adam P. Hill ^* , M. Sunde , T. J. Campbell ^*  and J. I. Vandenberg ^*  

^* Mark Cowley Lidwill Research Program in Electrophysiology and Biophysics, Victor Chang Cardiac Research Institute, New South Wales, Australia; St. Vincent's Clinical School, University of New South Wales, Australia; and School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Australia

Correspondence: Address reprint requests to J. Vandenberg, Tel.: 61-2-9295-8371; E-mail: j.vandenberg{at}victorchang.edu.au.

The scorpion toxin CnErg1 binds to human ether-a-go-go related gene (hERG) K⁺ channels with a 1:1 stoichiometry and high affinity. However, in contrast to other scorpion toxin-ion channel interactions, the inhibition of macroscopic hERG currents by high concentrations of CnErg1 is incomplete. In this study, we have probed the molecular basis for this incomplete inhibition. High concentrations of CnErg1 had only modest effects on hERG gating that could not account for the incomplete block. Furthermore, the residual current in the presence of 1 µM CnErg1 had normal single channel conductance. Analysis of the kinetics of CnErg1 interaction with hERG indicated that CnErg1 binding is not diffusion-limited. A bimolecular binding scheme that incorporates an initial encounter complex and permits normal ion conduction was able to completely reproduce both the kinetics and steady-state level of CnErg1-hERG binding. This scheme provides a simple kinetic explanation for incomplete block; that is, relatively fast backward compared to forward rate constants for the interconversion of the toxin-channel encounter complex and the blocked toxin-channel complex. We have also examined the temperature-dependence of CnErg1 binding to hERG. The dissociation constant, K_d, for CnErg1 increases from 7.3 nM at 22°C to 64 nM at 37°C (i.e., the affinity decreases as temperature increases) and the proportion of binding events that lead to channel blockade decreases from 70% to 40% over the same temperature range. These temperature-dependent effects on CnErg1 binding correlate with a temperature-dependent decrease in the stability of the putative CnErg1 binding site, the amphipathic -helix in the outer pore domain of hERG, assayed using circular dichroism spectropolarimetry. Collectively, our data provides a plausible kinetic explanation for incomplete blockade of hERG by CnErg1 that is consistent with the proposed highly dynamic conformation of the outer pore domain of hERG.

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