Mapping of Maurotoxin Binding Sites on hKv1.2, hKv1.3, and hIKCa1 Channels

Abstract

Maurotoxin (MTX) is a potent blocker of human voltage-activated Kv1.2 and intermediate-conductance calcium-activated potassium channels, hIKCa1. Because its blocking affinity on both channels is similar, although the pore region of these channels show only few conserved amino acids, we aimed to characterize the binding sites of MTX in these channels. Investigating the pH o dependence of MTX block on current through hKv1.2 channels, we concluded that the block is less pH osensitive than for hIKCa1 channels. Using mutant cycle analysis and computer docking, we tried to identify the amino acids through which MTX binds to hKv1.2 and hIKCa1 channels. We report that MTX interacts with hKv1. , and the GYGD motif are conserved in hIKCa1 channels, and the replacement of His 399 from hKv1.3 channels with a threonine makes this channel MTXsensitive, we concluded that MTX binds to all three channels through the same amino acids. Glu 355, although important, is not essential in MTX recognition. A negatively charged amino acid in this position could better stabilize the toxin-channel interaction and could explain the pH o sensitivity of MTX block on current through hIKCa1 versus hKv1.2 channels.It is well recognized that scorpion venoms represent important sources of potassium channel peptide blockers. Interactions between scorpion toxins and potassium channels were investigated in numerous studies and provided insights into the structure and topology of the external pore vestibule of the channels (Hidalgo and MacKinnon, 1995;Aiyar et al., 1996;Rauer et al., 2000;Wrisch and Grissmer, 2000).Maurotoxin (MTX) is a scorpion toxin isolated recently from the venom of the Tunisian chactoid scorpion Scorpio maurus palmatus (Kharrat et al., 1996(Kharrat et al., , 1997Castle et al., 2003). It belongs to the ␣-KTX toxins family (Tytgat et al., 1999) (Fig. 1B). It is composed of a 34-amino-acid peptide and has four disulfide bridges with an atypical pattern organization (C1-C5, C2-C6, C3-C4, and C7-C8) compared with other toxins belonging to the same family (C1-C4, C2-C5, C3-C6 for three-disulfide-bridged toxins and C1-C5, C2-C6, C3-C7, and C4-C8 for four-disulfide-bridged toxins) (Kharrat et al., 1996). Despite its different disulfide bridge organization, MTX has the same three-dimensional structure of potassium channels toxin blockers formed by one ␣-helix and two ␤-sheets. Moreover, MTX has been reported to block, in Xenopus laevis oocytes, IKCa1, Kv1.2, and ShakerB channels with an affinity lower than 10 nM, whereas its affinity for other potassium channels, such as Kv1.1 and Kv1.3, was higher than 50 nM (Kharrat et al., 1996;Avdonin et al., 2000;Lecomte et al., 2000;Castle et al., 2003).hKv1.2 and hIKCa1 channels are distributed differently in different tissues. hKv1.2 channels are found predominantly in the brain, where they are most likely to be associated with Kv1.1 and Kv1.6 channel subunits accomplishing crucial roles in neuronal signal transmission (Coleman et al., 1999;Monaghan et al., 2001;Dodson et al., 2003). In co...

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