Lanigan M D, Pennington M W, Lefievre Y, Rauer H, Norton R S
Biomolecular Research Institute, 343 Royal Parade, Parkville 3052, Australia.
Biochemistry. 2001 Dec 25;40(51):15528-37. doi: 10.1021/bi011300b.
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.
ShK毒素是一种来自海葵日光海葵的钾通道阻滞剂，是一种由3个二硫键交联的35个残基的多肽。为了生成这种强效通道阻滞剂的截短肽类似物，我们评估了三种类似物，一种是将天然序列截短，然后通过引入额外的共价连接（一个非天然二硫键和两个内酰胺桥）使其稳定，另外两种是将由二硫键和/或内酰胺桥稳定的非天然结构支架进行修饰，以包含天然毒素的关键氨基酸残基。还研究了在ShK毒素的第一个螺旋中引入内酰胺桥（以创建环14/18[赖氨酸14，天冬氨酸18]ShK）的效果，以确认这种修饰与活性兼容。所有四种类似物都在体外测试了它们阻断非洲爪蟾卵母细胞中Kv1.3钾通道的能力，并使用1H NMR光谱确定了它们的溶液结构。全长ShK中的内酰胺桥耐受性良好，与Kv1.3的结合仅降低了5倍。截短并稳定的类似物无活性，显然是由于与天然结构略有偏差以及结合所需侧链改变的综合作用。其中一种肽支架也无活性，因为它未能采用所需的结构，但另一种的解离常数为92微摩尔。这种活性肽包含对ShK活性至关重要的赖氨酸22和酪氨酸23的模拟物，以及一个可以模拟天然毒素中精氨酸11或精氨酸24的精氨酸残基。对这种肽进行修饰应该会产生一种更有效的低分子量肽类似物，这不仅将有助于进一步进行体外和体内研究阻断Kv1.3的效果，还将有助于绘制与该钾通道的孔和前庭的相互作用图谱，而这些相互作用是强效阻断所必需的。
