Designed Peptide Analogues of the Potassium Channel Blocker ShK Toxin

Abstract

ShK toxin, a potassium channel blocker from the sea anemone Stichodactyla helianthus, is a 35-residue polypeptide cross-linked by 3 disulfide bridges. In an effort to generate truncated peptidic analogues of this potent channel blocker, we have evaluated three analogues, one in which the native sequence was truncated and then stabilized by the introduction of additional covalent links (a non-native disulfide and two lactam bridges), and two in which non-native structural scaffolds stabilized by disulfide and/or lactam bridges were modified to include key amino acid residues from the native toxin. The effect of introducing a lactam bridge in the first helix of ShK toxin (to create cyclo14/18[Lys14,Asp18]ShK) was also examined to confirm that this modification was compatible with activity. All four analogues were tested in vitro for their ability to block Kv1.3 potassium channels in Xenopus oocytes, and their solution structures were determined using 1H NMR spectroscopy. The lactam bridge in full-length ShK is well tolerated, with only a 5-fold reduction in binding to Kv1.3. The truncated and stabilized analogue was inactive, apparently due to a combination of slight deviations from the native structure and alterations to side chains required for binding. One of the peptide scaffolds was also inactive because it failed to adopt the required structure, but the other had a K(d) of 92 microM. This active peptide incorporated mimics of Lys22 and Tyr23, which are essential for activity in ShK, and an Arg residue that could mimic Arg11 or Arg24 in the native toxin. Modification of this peptide should produce a more potent, low molecular weight peptidic analogue which will be useful not only for further in vitro and in vivo studies of the effect of blocking Kv1.3, but also for mapping the interactions with the pore and vestibule of this K(+) channel that are required for potent blockade.

Structurally Minimized μ‐Conotoxin Analogues as Sodium Channel Blockers: Implications for Designing Conopeptide‐Based Therapeutics

“…Two out of three disulfide‐deficient analogues of the ShK toxin were able to block K v 1.1 potassium channels with nanomolar affinity 42. Furthermore, a highly minimized analogue of ShK containing eight amino acid residues and cross‐linked by two disulfide bridges blocked K v 1.3 channels with a K d value of 92 μ M 41. The disulfide‐deficient analogue of the 34‐residue scorpion toxin, maurotoxin, also retained the overall α/β scaffold and the ability to block potassium channels,43 but no further efforts in minimizing the bioactive conformation of these neurotoxins have been reported yet.…”

K+ Channel Blockers: Novel Tools to Inhibit T Cell Activation Leading to Specific Immunosuppression

“…Small peptide analogs contain only a few amino acid residues but preserve strategically positioned residues essential for high affinity binding whose orientations and relative locations are fixed by disulfide and lactam bridges not present in the original peptide. Three such analogs of the high affinity blocker ShK toxin were produced of which only one was shown to block Kv1.3 channels with a K d of 92µM [90].…”

Endogenous animal toxin-like human β-defensin 2 inhibits own K+ channels through interaction with channel extracellular pore region

“…potency, such as scorpion toxins charybodotoxin (ChTX), OSK1, BmKTX and their analogs [3][4][5], and sea anemone toxin ShK and its derivatives [6]. Recently, scorpion Kunitz-type toxins were also found to block Kv1.3 channel [7].…”