Charybdotoxin and Margatoxin Acting on the Human Voltage-Gated Potassium Channel hK v 1.3 and Its H399N Mutant: An Experimental and Computational Comparison Azadeh Nikouee, †,‡ Morteza Khabiri, †,§ Stephan Grissmer,* ,‡ and Rü diger Ettrich* ,§ ‡ Institute of Applied Physiology, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany § Institute of Nanobiology and Structural Biology of GCRC, Academy of Sciences of the Czech Republic, and Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic ABSTRACT: The effect of the pore-blocking peptides charybdotoxin and margatoxin, both scorpion toxins, on currents through human voltage-gated hK v 1.3 wild-type and hK v 1.3_H399N mutant potassium channels was characterized by the whole-cell patch clamp technique. In the mutant channels, both toxins hardly blocked current through the channels, although they did prevent C-type inactivation by slowing down the current decay during depolarization. Molecular dynamics simulations suggested that the fast current decay in the mutant channel was a consequence of amino acid reorientations behind the selectivity filter and indicated that the rigidity-flexibility in that region played a key role in its interactions with scorpion toxins. A channel with a slightly more flexible selectivity filter region exhibits distinct interactions with scorpion toxins. Our studies suggest that the toxin-channel interactions might partially restore rigidity in the selectivity filter and thereby prevent the structural rearrangements associated with C-type inactivation. ■ INTRODUCTION The voltage-gated potassium channel hK v 1.3, belongs to the mammalian Shaker-related family of potassium channels and has a critical role in the function of a number of organs, including the brain, lung, kidney, and olfactory bulbs. 1-10 Ion selectivity and voltage sensing are two important characteristics of this channel. The three-dimensional structure of a single hK v 1.3 channel consists of four identical subunits that surround a central channel pore. Each subunit contains six trans- membrane (TM) helices (S1-S6). The ion conduction pathway is located between the helices S5 and S6, an area termed the pore or P-region. The amino acid sequence of the P-region is conserved and includes a selectivity filter motif TVGYG. This selectivity filter motif coordinates dehydrated potassium ions passing through the channel. 11-16 As revealed by electrophysiological experiments, 17 the hK v 1.3 channels can, depending on the electrical potential of the cell membrane, adopt one of three functional states. At a hyperpolarized membrane potential, most of the hK v 1.3 channel molecules are present in a nonconducting closed state. Upon depolarization, the channel initially undergoes a conformational change of the intracellular gate region, leading to the channel adopting a conducting open state. Prolonged depolarization causes the hK v 1.3 channel to enter a long-lived nonconducting state called a C-type inactivated state. The monoexponential decay of K + currents during a sustained depolarization is called C-type inactivation. 18 This slow C-type inactivation limits or eliminates the channel conductivity to K + ions. All basic structural information about this process involves a conjunct change in the outer vestibule and/or a collapsed and constricted region in the selectivity filter of the channel, 19,20 as well as the pore helix and the first part of the transmembrane helix S6, which flanks the selectivity filter. Apart from the C-type inactivation, another characteristic feature of the hK v 1.3 channel distinguishes it from other voltage-gated potassium channels: high sensitivity to scorpion peptide toxins, 18 such as margatoxin (MgTX) and charybdo- toxin (CTX). Similar to other members of the scorpion toxin family, these two toxins act on potassium channels with a 1:1 stoichiometry 21,22 and high affinity. 21 The MgTX and CTX are similar in size (37 and 39 amino acids, respectively), and both adopt a fold structure named cysteine-stabilized alpha/beta motif (CSαβ). The secondary structure of these peptides consists of two or three β-strands and one α-helix and is stabilized by three disulfide bonds. 23 The key residue for blocking the ion flux through the channel (K27 in CTX and K28 in MgTX) lies within one of the β-sheets. 24,25 Other important functional residues of both CTX and MgTX are located on the flat surface of the β-sheet. In addition, S10, K11 in CTX and K11, K18 in MgTX, which have strong electrostatic interactions with K + channels, are placed in the α-helix. 25-29 It has been reported that most scorpion toxins share a functional dyad. This functional dyad is composed of a basic Received: October 25, 2011 Revised: March 15, 2012 Published: April 10, 2012 Article pubs.acs.org/JPCB © 2012 American Chemical Society 5132 dx.doi.org/10.1021/jp2102463 | J. Phys. Chem. B 2012, 116, 5132-5140