Elsevier

Toxicon

Volume 39, Issue 6, June 2001, Pages 739-748
Toxicon

Review
Potassium channels: from scorpion venoms to high-resolution structure

https://doi.org/10.1016/S0041-0101(00)00214-2Get rights and content

Introduction

In 1998, the field of ion channel research entered a new era when the first, high-resolution crystal structure of one of these proteins was solved (Doyle et al., 1998). For the first time, it was possible to understand, at a molecular level, the mechanisms that control ion selectivity and conduction in potassium channels. The protein whose structure had been determined, the KcsA K+ channel, is a two transmembrane spanning domain, potassium selective channel from Streptomyces lividans that gates in response to H+ when reconstituted in artificial lipid bilayers (Cuello et al., 1998, Heginbotham et al., 1999). The physiologic role of the KcsA channel in Streptomyces is not well understood. The amino acid sequence of the KcsA channel is closely related to that of some eukaryotic, six-transmembrane spanning domain, potassium channels (Doyle et al., 1998). In particular, the sequence in the pore region, containing the K+ channel signature sequence, is almost identical to that found in vertebrate voltage-gated K+ channels. An exciting aspect of the KcsA channel study was the finding that a number of structural features predicted from functional studies with eukaryotic six-transmembrane domain voltage-gated K+ channels were present in the determined structure of the prokaryotic channel. This provided compelling evidence that the overall architecture of K+ channels may be conserved through evolution, and that the structure of the KcsA channel can be used as a paradigm to probe the properties of other K+ channels.

The interpretation of structural features of the KcsA channel was greatly facilitated by a large amount of functional data accumulated in previous years with several eukaryotic K+ channels. Two major factors have facilitated the efforts of the many research laboratories that have focused on the study of K+ channels: (1) the extensive cloning and functional expression of these proteins; and (2) the existence of a large number of high affinity peptidyl inhibitors of these proteins, isolated from different scorpion and spider venoms (Tytgat et al., 1999). In fact, the use of peptidyl inhibitors derived from scorpion venoms provided the first indirect information concerning K+ channel structure. For instance, both identification of the pore region of the channel (MacKinnon and Miller, 1989b), and determination of the tetrameric composition of K+ channels (MacKinnon, 1991) were made possible with the use of scorpion toxins. In addition, these peptides have been invaluable tools for purifying channels from native tissues, as well as determining their subunit composition (Garcia-Calvo et al., 1994), and also for understanding the physiological role of specific channel proteins (Garcia et al., 1997). Clearly, scorpion toxins have played important, crucial roles in determining potassium channel structure and function. In this review, we will discuss how work with scorpion venom peptides have progressed from their initial discovery as K+ channel blockers, to their prominent role in interpreting the structure of the KcsA channel, and to the role that they may play in the future. Other aspects of work with these inhibitors, such as those concerning their role in developing the molecular pharmacology of potassium channels have previously been discussed extensively (Garcia et al., 1997, Garcia and Kaczorowski, 1998), and, therefore, will not be reviewed here.

Section snippets

Potassium channel blocking peptides

All potassium channel blocking peptides that have been purified from scorpion venoms contain 30–40 amino acids with three or four disulfide bridges. Based on primary sequence homology, 12 sub-families containing 49 different peptides, termed α-KTX1–12, have been described (Tytgat et al., 1999). Out of these 49 peptides, only a restricted number have been studied in detail with respect to their interaction with K+ channels. Noxiustoxin, α-KTX2.1, purified from venom of the scorpion Centruroides

Characterization of peptide-channel interaction residues

Site-directed mutagenesis studies were carried out with ChTX and AgTX2 to identify those residues which are important for interaction with potassium channels. These studies were enabled by the ability to biosynthetically produce large quantities of the relevant peptides, whereby recombinant peptides are made in E. coli as soluble fusion proteins (Park et al., 1991). Purification of the fusion protein, folding of peptide and its cleavage from the fusion protein can be easily accomplished to

High-resolution structure of potassium channels

The X-ray structure of the KcsA channel at 3.2 Å reveals that the channel is a tetramer with four-fold symmetry around a central pore (Doyle et al., 1998). Each subunit consists of two α-helical transmembrane domains connected by the pore region, which consists of the turret, pore helix, and selectivity filter. In the tetrameric structure, each subunit contributes one transmembrane helix to form the central pore, while the other transmembrane domain faces the lipid environment. The inner helices

Rationale drug design: peptidyl blockers of potassium channels

The finding that the overall architecture of various K+ channels is similar has obvious implications. With the use of molecular modeling and constraints derived from peptide–channel interaction studies, it should be feasible to derive a picture of the interaction surface of other channels, and to design peptides that target toxin-insensitive K+ channels. For instance, Kv2, Kv3 and Kv4 potassium channels are all insensitive to known peptide inhibitors derived from scorpion venoms (Garcia et al.,

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