Elsevier

Toxicon

Volume 60, Issue 5, October 2012, Pages 840-850
Toxicon

Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria

https://doi.org/10.1016/j.toxicon.2012.05.017Get rights and content

Abstract

The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which play a crucial role in the activation of human effector memory T-cells (TEM). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by TEM cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. We have established a recombinant peptide expression system in order to generate isotopically-labelled ShK and various ShK analogues for in-depth biophysical and pharmacological studies. ShK was expressed as a thioredoxin fusion protein in Escherichia coli BL21 (DE3) cells and purified initially by Ni2+ iminodiacetic acid affinity chromatography. The fusion protein was cleaved with enterokinase and purified to homogeneity by reverse-phase HPLC. NMR spectra of 15N-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv1.3 channels with a Kd of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for TEM cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of 15N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels.

Highlights

► ShK is a potent potassium channel blocker. ► A recombinant expression system for ShK has been established. ► Recombinant ShK is equipotent with synthetic ShK in a range of bioassays. ► Expression yield is maintained in minimal media suitable for isotopic labelling.

Introduction

Sea anemones produce many polypeptide toxins and proteins that are potent ion channel blockers and cytolysins. The first potassium channel blocker to be isolated and characterized from a sea anemone was ShK toxin, from Stichodactyla helianthus (Castaneda et al., 1995, Pennington et al., 1995), which is a 35-residue peptide containing six half-cystines that form three disulfide bonds (Pohl et al., 1995) (Fig. 1A). Its solution structure, determined by NMR spectroscopy (Tudor et al., 1996, 1998), consists of two short α-helices encompassing residues 14–19 and 21–24, and an N-terminus with an extended conformation up to residue 8, followed by a pair of interlocking turns that resembles a 310-helix (Fig. 1B).

The surface of ShK involved in binding to voltage-activated (Kv) potassium channels has been mapped using alanine scanning and selected synthetic analogues (Pennington et al., 1996a, Pennington et al., 1996b). Alanine scanning mutagenesis identified the conserved dyad Lys22 and Tyr23 as key functional residues (Fig. 1C). Other residues contributing to Kv1.3 binding include Arg11, His19, Ser20 and Arg24 (Pennington et al., 1996a, Rauer et al., 1999). These essential residues were found to be clustered on a surface of the peptide that binds to a shallow vestibule at the outer entrance to the ion conduction pathway and occludes the entrance to the pore (Pennington et al., 1996a, Rauer et al., 1999). To examine this interaction in more detail, the solution structure of ShK (Kalman et al., 1998, Tudor et al., 1996) was docked to a homology model of the Kv1.3 channel based on the crystal structure of the bacterial potassium channel KcsA (Doyle et al., 1998, Rauer et al., 2000), using restrained molecular dynamics simulations guided by data from complementary mutational analyses (Lanigan et al., 2002, Rauer et al., 2000). The model reveals that Lys22 of ShK projects into the ion conduction pathway while Arg11 is in close proximity to His404 in one of the Kv1.3 subunits.

All human T lymphocytes express two types of K+ channels, the voltage-gated Kv1.3 and the Ca2+-activated KCa3.1 channels, which play crucial roles in human T-cell activation (Leonard et al., 1992, Price et al., 1989). The expression levels of these two K+ channels are dependent upon the state of T-cell activation and differentiation (Wulff et al., 2003a). Naïve CD4+ or CD8+ T cells initially differentiate into long-lived central memory (TCM) T cells, which then differentiate into terminally-differentiated effector memory (TEM) cells upon repeated stimulation. Kv1.3 channels are significantly up-regulated in activated TEM cells, leading to a heightened sensitivity to Kv1.3 channel blockers (Beeton et al., 2006, Wulff et al., 2003b). Activation of naïve and central-memory (TCM) cells, by contrast, results in up-regulation of KCa3.1 channel expression and decreased sensitivity to Kv1.3 channel blockade (Wulff et al., 2003a). The differential expression of Kv1.3 and KCa3.1 K+ channels in activated TEM and TCM cells implies that it may be possible to selectively suppress TEM cells using a Kv1.3-specific inhibitor without causing generalized immunosuppression. Kv1.3 blockers therefore constitute valuable new therapeutic leads for the treatment of autoimmune diseases mediated by TEM cells, such as multiple sclerosis (MS) and rheumatoid arthritis (Beeton et al., 2011, Beeton et al., 2006, Chi et al., 2012, Wulff et al., 2003b).

Patch-clamp experiments on cloned potassium channels expressed in mammalian cells revealed that ShK blocked not only Kv1.3 (Kd 11 pM) but also Kv1.1 (Kd 16 pM), Kv1.7 (Kalman et al., 1998) and Kv3.2 channels (Beeton et al., 2005, Yan et al., 2005) in the nanomolar range. This lack of specificity constitutes a potential drawback for the use of ShK as a therapeutic agent. For example, lack of selectivity for Kv1.3 over the neuronal Kv1.1 channels could prove detrimental if ShK were to enter the brain through a compromised blood–brain barrier and induce neurotoxicity. It was therefore essential to develop ShK analogues that are selective for Kv1.3 over Kv1.1 and other K+ channels. Towards this goal, more selective analogues have been made by the incorporation of non-natural amino acids or adducts; these include ShK-Dap22, in which the critical Lys22 was replaced by the shorter, positively charged, non-natural residue 1,3-diaminopropionic acid (Dap) (Kalman et al., 1998); ShK-F6CA, a fluorescein-labelled analogue of ShK (Beeton et al., 2003); and analogues with either phospho-Tyr (ShK-186) or phosphono-Phe (ShK-192) attached via a hydrophilic linker (i.e. aminoethyloxyethyloxyacetyl) to Arg1 (Beeton et al., 2005, Pennington et al., 2009). However, these analogues have several potential limitations, for example, ShK-186 and ShK-192 contain non-protein adducts while the phosphorylated residue of ShK-186 is susceptible to hydrolysis. As a result, there is still scope for the development of new analogues with enhanced stability and increased specificity for Kv1.3.

In this study we report the cloning, expression and purification of ShK in Escherichia coli. The majority of the expressed protein formed insoluble inclusion bodies but the protein was successfully refolded and the final ShK yields obtained from rich and minimal media were 3 mg/L and 2.5 mg/L, respectively. NMR spectroscopic analyses of 15N-labelled ShK indicated that the protein was correctly folded while electrophysiological studies showed that recombinant ShK blocked Kv1.3 channels with a similar efficiency to that of the synthetic peptide.

Section snippets

Construction of pET32a/ShK expression plasmid

DNA encoding the amino acid sequence of ShK and an N-terminal enterokinase cleavage site was synthesized by PCR using two overlapping primers and was cloned into a pET-32a expression vector (Novagen, USA). For efficient protein expression in bacterial cells, the DNA template was designed based on optimized codons for expression in E. coli. The forward primer (5′-CCAAGAGAATTCGATGATGATGATAAACGCAGCTGCATTGATACCATTCCGAAAAGCCGCTGCACCGCGTTTCAGTGCAAACAT) contained an EcoRI restriction endonuclease

Expression of Trx-ShK

The Trx-ShK fusion protein (Fig. 2A) was highly expressed upon induction with 1 mM IPTG at 18 °C overnight. SDS-PAGE analysis showed the appearance of an intense band at about 25 kDa (Fig. 2B, lane 2). Protein was expressed as inclusion bodies (Fig. 2C, lane 2), with yields of 2 g per L from both LB and M9 media, while only a small portion of the Trx-ShK fusion protein was soluble (18 mg/L of LB and 10 mg/L of M9 media). The identity of the Trx-ShK fusion protein was confirmed by in-gel trypsin

Discussion

We have developed an efficient bacterial expression system for the sea anemone peptide ShK toxin. Following NTA chromatography, cleavage by enterokinase and RP-HPLC, we obtained pure peptide of the correct mass. Enterokinase has the advantage of leaving no extra residues at the N-terminus of the cleaved recombinant ShK peptide, thus allowing for direct comparison with the native ShK peptide. Our patch-clamp and T-lymphocyte proliferation data show that recombinant ShK has essentially identical

Acknowledgments

This work was supported in part by grants from the Australian Research Council (DP1093909 to RSN) and the National Institutes of Health (NS073712 to CB and RSN and AI084981 to CB). RSN acknowledges the award of a fellowship by the National Health and Medical Research Council of Australia.

References (44)

  • H. Rauer et al.

    Structure-guided transformation of charybdotoxin yields an analog that selectively targets Ca2+-activated over voltage-gated K+ channels

    J. Biol. Chem.

    (2000)
  • H. Rauer et al.

    Structural conservation of the pores of calcium-activated and voltage-gated potassium channels determined by a sea anemone toxin

    J. Biol. Chem.

    (1999)
  • M. Turkov et al.

    In vitro folding and functional analysis of an anti-insect selective scorpion depressant neurotoxin produced in Escherichia coli

    Protein Expr. Purif.

    (1997)
  • S. Yao et al.

    Exchange enhanced sensitivity gain for solvent-exchangeable protons in 2D 1H–15N heteronuclear correlation spectra acquired with band-selective pulses

    J. Magn. Reson.

    (2011)
  • C. Beeton et al.

    Selective blocking of voltage-gated K+ channels improves experimental autoimmune encephalomyelitis and inhibits T cell activation

    J. Immunol.

    (2001)
  • C. Beeton et al.

    Selective blockade of T lymphocyte K+ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis

    Proc. Natl. Acad. Sci. U.S.A.

    (2001)
  • C. Beeton et al.

    Induction and monitoring of adoptive delayed-type hypersensitivity in rats

    J. Vis. Exp.

    (2007)
  • C. Beeton et al.

    Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases

    Mol. Pharmacol.

    (2005)
  • C.W. Beeton et al.

    Analogs of the sea anemone potassium channel blocker ShK for the treatment of autoimmune diseases

    Inflamm. Allergy Drug Targets

    (2011)
  • C. Beeton et al.

    Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases

    Proc. Natl. Acad. Sci. U.S.A.

    (2006)
  • J.H. Chill et al.

    NMR study of the tetrameric KcsA potassium channel in detergent micelles

    Protein Sci.

    (2006)
  • D.A. Doyle et al.

    The structure of the potassium channel: molecular basis of K+ conduction and selectivity

    Science

    (1998)
  • Cited by (19)

    • Blocking Kv1.3 channels inhibits Th2 lymphocyte function and treats a rat model of asthma

      2014, Journal of Biological Chemistry
      Citation Excerpt :

      After 3 washes with FACS-PBS, cells were fixed for 20 min at room temperature with PBS + 1% paraformaldehyde, and data were acquired on a BD CantoII or a BD LSRII flow cytometer (BD Biosciences) using FACSDiva and analyzed using FlowJo (Treestar, Ashland, OR). Proliferation assays were performed as previously described (11–13, 23, 25, 26). Intracellular levels of IL-4, IL-5, and IL-13 were detected by flow cytometry after a 6-h allergen-induced activation in the presence of 100 nm ShK-186 or vehicle and of the protein transport inhibitor brefeldin A.

    • Production of recombinant venom peptides as tools for ion channel research

      2021, Methods in Enzymology
      Citation Excerpt :

      This is achieved by fusing a periplasmic export signal upstream of the gene of interest, in most cases in conjunction with additional tags to increase solubility and facilitate purification. This expression method gained much traction in recent years, owing to its amenability to venomics methods such as high-throughput expression (Turchetto et al., 2017) and in-vivo isotopic labeling (Chang et al., 2012). The reported success rates for periplasmic expression of venom peptides are relatively high, and it became the method of choice of several research groups (Geron, 2020; Klint et al., 2013; Vetter et al., 2011).

    • Prolonged immunomodulation in inflammatory arthritis using the selective Kv1.3 channel blocker HsTX1[R14A] and its PEGylated analog

      2017, Clinical Immunology
      Citation Excerpt :

      For example, fusing OsK1, OdK2, or ShK with Fc fragments of immunoglobulins, with PEG moieties, or with albumin reduced their affinity for Kv1.3 channels to varying degrees [40; 63]. A thioredoxin fusion of ShK or a biotin-tagged ShK bound to streptavidin had no effect on Kv1.3 currents at 100 nM, whereas ShK itself blocks the channel with low picomolar affinity [44,64]. Importantly, the PEGylation of HsTX1[R14A] retained a 250-fold or higher selectivity for Kv1.3 over closely-related Kv1.1, Kv1.2, Kv1.4, and Kv1.5 channels.

    • A C-terminally amidated analogue of ShK is a potent and selective blocker of the voltage-gated potassium channel Kv1.3

      2012, FEBS Letters
      Citation Excerpt :

      ShK-K-amide blocks Kv1.3 with an IC50 of 26 ± 3 pM, similar to the affinities for this channel of ShK (9 ± 2 pM) [12,28] and ShK-amide (35 ± 5 pM). However, with an IC50 of 942 ± 120 pM for Kv1.1, ShK-K-amide is far more selective for Kv1.3 over Kv1.1 than ShK, which blocks Kv1.1 with an IC50 of 23 ± 3 pM [12,28], or ShK-amide (37 ± 4 pM). This is the first report of an ShK analogue with enhanced Kv1.3 selectivity following modification of the C-terminus; all prior work that resulted in an increase in selectivity had focused on either the pore-blocking Lys22 or the N-terminus of ShK [3,4].

    • A bioengineered probiotic for the oral delivery of a peptide Kv1.3 channel blocker to treat rheumatoid arthritis

      2023, Proceedings of the National Academy of Sciences of the United States of America
    View all citing articles on Scopus
    View full text