Interactions of amino terminal domains of Shaker K channels with a pore blocking site studied with synthetic peptides.
Interactions of amino terminal domains of Shaker K channels with a pore blocking site studied with synthetic peptides.
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DOI：
10.1085/jgp.102.6.949
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发表时间：
1993-12
期刊：
影响因子：
--
通讯作者：
Aldrich RW
中科院分区：
文献类型：
--
作者：
Murrell-Lagnado RD;Aldrich RW
当将Shaker的5个备选nh2末端序列的合成肽应用于具有nh2末端缺失（ShB δ 6-46）的ShB通道的细胞质侧时，其阻断通道的效力与相应变体的失活率相关。这些肽没有序列相似性，但5个肽中有3个明显的解离常数在2到15微米之间，这表明结合的特异性要求很低。为了确定有效阻断ShB δ 6-46所需的主要结构决定因素，我们研究了对20个残基ShB肽进行取代对结合力和解离率的影响。肽内的非极性残基似乎在通过疏水相互作用稳定结合方面很重要。对亮氨酸-7的取代表明疏水性与解离速率常数（koff）有明显的相关性，而对缔合速率常数（kon）影响不大。用带电荷的残基取代4-8区疏水残基破坏了结合。在肽的cooh末端，增加净正电荷的取代增加了kon，而koff的变化相对较小，这表明远程静电相互作用参与了肽有效浓度的增加。中和带电残留物会产生koff的微小变化。12-20区域内的收费也是如此；使净电荷守恒的变化对kon和koff的影响都不大。结果与肽的该区域具有延伸构象一致，并表明当结合时，该区域与通道蛋白很少接触，并且保持相对不受约束。在完整ShB通道的nh2末端区域内，类似的突变对阻断和解阻断率产生了性质上相似的影响。
Synthetic peptides of the five alternative NH2-terminal sequences of Shaker when applied to the cytoplasmic side of ShB channels that have an NH2-terminal deletion (ShB delta 6-46) block the channel with potencies correlated with the rate of inactivation in the corresponding variant. These peptides share no sequence similarity and yet three out of the five have apparent dissociation constants between 2 and 15 microM, suggesting that the specificity requirements for binding are low. To identify the primary structural determinants required for effective block of ShB delta 6-46, we examined the effects of substitutions made to the 20 residue ShB peptide on association and dissociation rates. Nonpolar residues within the peptide appear to be important in stabilizing the binding through hydrophobic interactions. Substitutions to leucine-7 showed there was a clear correlation between hydrophobicity and the dissociation rate constant (koff) with little effect on the association rate constant (kon). Substituting charged residues for hydrophobic residues within the region 4-8 disrupted binding. Within the COOH-terminal half of the peptide, substitutions that increased the net positive charge increased kon with relatively small changes in koff, suggesting the involvement of long-range electrostatic interactions in increasing the effective concentration of the peptide. Neutralizing charged residues produced small changes in koff. Charges within the region 12-20 act equivalently; alterations which conserved net charge produced little effect on either kon or koff. The results are consistent with this region of the peptide having an extended conformation and suggest that when bound this region makes few contacts with the channel protein and remains relatively unconstrained. Analogous mutations within the NH2-terminal domain of the intact ShB channel produced qualitatively similar effects on blocking and unblocking rates.