Functional evolution of scorpion venom peptides with an inhibitor cystine knot fold

Bin Gao,Peta J Harvey,David J Craik,Michel De Waard,Shunyi Zhu,Michel Ronjat

doi:10.1042/bsr20130052

Abstract

The ICK (inhibitor cystine knot) defines a large superfamily of polypeptides with high structural stability and functional diversity. Here, we describe a new scorpion venom-derived K+ channel toxin (named λ-MeuKTx-1) with an ICK fold through gene cloning, chemical synthesis, nuclear magnetic resonance spectroscopy, Ca2+ release measurements and electrophysiological recordings. λ-MeuKTx-1 was found to adopt an ICK fold that contains a three-strand anti-parallel β-sheet and a 310-helix. Functionally, this peptide selectively inhibits the Drosophila Shaker K+ channel but is not capable of activating skeletal-type Ca2+ release channels/ryanodine receptors, which is remarkably different from the previously known scorpion venom ICK peptides. The removal of two C-terminal residues of λ-MeuKTx-1 led to the loss of the inhibitory activity on the channel, whereas the C-terminal amidation resulted in the emergence of activity on four mammalian K+ channels accompanied by the loss of activity on the Shaker channel. A combination of structural and pharmacological data allows the recognition of three putative functional sites involved in channel blockade of λ-MeuKTx-1. The presence of a functional dyad in λ-MeuKTx-1 supports functional convergence among scorpion venom peptides with different folds. Furthermore, similarities in precursor organization, exon–intron structure, 3D-fold and function suggest that scorpion venom ICK-type K+ channel inhibitors and Ca2+ release channel activators share a common ancestor and their divergence occurs after speciation between buthidae and non-buthids. The structural and functional characterizations of the first scorpion venom ICK toxin with K+ channel-blocking activity sheds light on functionally divergent and convergent evolution of this conserved scaffold of ancient origin.

Highlights

Potassium channels are a large and diverse family of ion channels that selectively transport K + ions across membranes of excitable cells and non-excitable cells [1]
We describe a new scorpion inhibitor cystine knot (ICK) peptide, named λ-MeuKTx-1 that is designated as the first member of the λ-KTx family coming from Mesobuthus eupeus in terms of its genomic organization, solution structure, biological activity and structure–function relationship
Reverse-transcription of total RNA into the first-strand cDNA was performed by an RT-PreMix kit (SBS Genetech) and a universal dT3AP [oligo(dT)-containing adaptor primer], which were directly used as templates for RACE (3 rapid amplification of cDNA ends) with primers MeuICK-F1 and 3AP (Figure 1A)

Summary

Introduction

Potassium channels are a large and diverse family of ion channels that selectively transport K + ions across membranes of excitable cells (neuronal and muscle tissues) and non-excitable cells [1]. They control the flow of K + ions into and out of cells and thereby play key roles in generating action potentials and maintaining the resting membrane potential. Potassium channels regulate many key physiological processes, including neurotransmitter release, immune response, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction and cell proliferation [2,3,4].

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