N-Terminally extended analogues of the K⁺ channel toxin from Stichodactyla helianthus as potent and selective blockers of the voltage-gated potassium channel Kv1.3

Abstract

The voltage-gated potassium channel Kv1.3 is an important target for the treatment of autoimmune diseases and asthma. Blockade of Kv1.3 by the sea anemone peptide K⁺-channel toxin from Stichodactyla helianthus (ShK) inhibits the proliferation of effector memory T lymphocytes and ameliorates autoimmune diseases in animal models. However, the lack of selectivity of ShK for Kv1.3 over the Kv1.1 subtype has driven a search for Kv1.3-selective analogues. In the present study, we describe N-terminally extended analogues of ShK that contain a negatively-charged Glu, designed to mimic the phosphonate adduct in earlier Kv1.3-selective analogues, and consist entirely of common protein amino acids. Molecular dynamics simulations indicated that a Trp residue at position [-3] of the tetrapeptide extension could form stable interactions with Pro377 of Kv1.3 and best discriminates between Kv1.3 and Kv1.1. This led to the development of ShK with an N-terminal Glu-Trp-Ser-Ser extension ([EWSS]ShK), which inhibits Kv1.3 with an IC₅₀ of 34 pm and is 158-fold selective for Kv1.3 over Kv1.1. In addition, [EWSS]ShK is more than 2900-fold more selective for Kv1.3 over Kv1.2 and KCa3.1 channels. As a highly Kv1.3-selective analogue of ShK based entirely on protein amino acids, which can be produced by recombinant expression, this peptide is a valuable addition to the complement of therapeutic candidates for the treatment of autoimmune diseases.

Keywords: N-terminal extension; ShK; electrophysiology; molecular dynamics; potassium channels.

Figure 1. Homology modelling of ShK analogues in complex with Kv1.3

(A) ShK-192 in complex with Kv1.3; view perpendicular to the membrane plane with the channel represented as a white and tan ribbon, and the ShK analogue in blue with a transparent surface. The side chain atoms of residues on the surface of the channel that differ between Kv1.1 and Kv1.3 are illustrated as spheres (atom coloring with carbon tan). (B) The phosphono group of the N-terminal Ppa extension in ShK-192 lies ∼8 Å from the N-terminus of the native ShK and forms a salt bridge with the ammonium of Lys 411. (C) Comparison of MODELLER energies and separation between the N-terminal Glu of the ShK analogue and Lys411 of the channel. Extensions to ShK of 1 (green triangle, E), 2 (red square, ES), 3 (blue diamond, ESS) and 4 (purple triangle, ESSS) residues. (D) Homology model of [ESSS]ShK in complex with Kv1.3. The side-chain atoms of Pro377 and Lys411 of the channel are represented as spheres (atom coloring with carbon tan). The side-chain atoms of the 4-residue extension, ESSS, are represented as spheres (atom coloring with carbon cyan). (E) Homology model of [EWSS]ShK in complex with Kv1.3. The side-chain atoms of Pro377 and Lys411 of the channel are highlighted. The side-chain atoms of the first two residues (EW) of the 4-residue extension are represented as spheres (atom coloring with carbon cyan).

Figure 2. Sequence alignment of the transmembrane region of Kv1.1 and Kv1.3

Annotation highlights sequence conservation between Kv1.1 (residues 325 to 415) and Kv1.3 (residues 350 to 440) sequences (asterisks=conserved); surface-exposed residues that differ between the two sequences are highlighted in yellow, residues in the selectivity filter are highlighted in green, and shaded areas indicate helices.

Figure 3. N-terminal extensions of ShK analogues

Schematic illustrating the structural similarity and physico-chemical properties of Ppa moiety of ShK-192 and various amino acid extensions.

Figure 4. Purification and characterization of [EWSS]ShK

(A) Analytical RP-HPLC chromatograph for the final purified [EWSS]ShK with absorbance at 214 nm with purity ≥ 95 %. (B) ESI-MS analysis of [EWSS]ShK. The multi-charged ions are deconvoluted to a mass of 4544 Da, which is consistent with the theoretical mass.

Figure 5. Selectivity of N-terminally extended ShK analogues

(A) Effect of [ESSS]ShK (top), [EESS]ShK (middle) and [EWSS]ShK (bottom) on Kv1.3 and Kv1.1 currents. (B) Effects of [ESSS]ShK (○ dotted line), [EESS]ShK (■ solid line), [EWSS]ShK (● solid line), and ShK (□ dotted line) on Kv1.3 or Kv1.1 currents measured by whole-cell patch-clamp on L929 fibroblasts stably transfected with mKv1.3 or mKv1.1, respectively, and fitted to a Hill equation (N = 3-5 cells per concentration). The panel on the left shows whole-cell Kv1.3 currents and the panel on the right shows whole-cell Kv1.1 currents. Data are presented as mean ± s.e.m.

Figure 6. Effects of [EWSS]ShK on Kv1.2 and KCa3.1

(A) The currents of Kv1.2 (Left) and (B) KCa3.1 (Right) were measured by whole-cell patch-clamp on B82 fibroblasts stably transfected with mKv1.2 or HEK293 cells stably transfected with hKCa3.1, respectively, and fitted to a Hill equation (N = 3-4 cells per concentration). All current traces were recorded at equilibrium block.