The role of cystic fibrosis transmembrane conductance regulator phenylalanine 508 side chain in ion channel gating
The role of cystic fibrosis transmembrane conductance regulator phenylalanine 508 side chain in ion channel gating
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DOI：
10.1113/jphysiol.2005.099457
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发表时间：
2006-04-15
影响因子：
5.5
通讯作者：
Aleksandrov, AA
中科院分区：
文献类型：
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作者：
Cui, L;Aleksandrov, L;Aleksandrov, AA
囊性纤维化跨膜传导调节因子（CFTR）是一种利用ABC转运蛋白结构基序的离子通道，第一核苷酸结合域（NBD1）中单个残基（Phe508）的缺失发生在大多数囊性纤维化患者中，损害了该蛋白的成熟和功能。然而，用小的不带电氨基酸（包括半胱氨酸）取代Phe508是允许成熟的。为了探索苯丙氨酸芳香侧链在通道门控中的可能作用，我们在无环CFTR的这个位置引入了半胱氨酸，使其能够通过巯基试剂进行选择性化学修饰。当被不同的核苷酸配体激活时，无cysless和野生型CFTR离子通道具有相同的平均打开时间。此外，通过引入ATPase抑制突变（E1371S），这两个通道都可以被锁定在开放状态。然而，单个半胱氨酸（F508C）的引入阻止了无槽E1371S通道保持永久打开状态，从而允许闭合发生。采用巯基试剂对无晶体E1371S/F508C进行化学改性，探讨了侧链在离子通道功能中的作用。具体来说，甲基乙硫磺酸对该变异的修饰使其恢复到含有508位野生型苯丙氨酸的无胱氨酸E1371S的门控行为。这提供了第一个直接证据，证明Phe508芳香侧链的特定相互作用在决定封闭状态的停留时间中起作用。因此，尽管芳香族侧链对CFTR折叠不是必需的，但它在离子通道功能中很重要。
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel employing the ABC transporter structural motif Deletion of a single residue (Phe508) in the first nucleotide-binding domain (NBD1), which occurs in most patients with cystic fibrosis, impairs both maturation and function of the protein. However, substitution of the Phe508 with small uncharged amino acids, including cysteine, is permissive for maturation. To explore the possible role of the phenylalanine aromatic side chain in channel gating we introduced a cysteine at this position in cysless CFTR, enabling its selective chemical modification by sulfhydryl reagents. Both cysless and wild-type CFTR ion channels have identical mean open times when activated by different nucleotide ligands. Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Specifically, benzyl-methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild-type phenylalanine at position 508. This provides the first direct evidence that a specific interaction of the Phe508 aromatic side chain plays a role in determining the residency time in the closed state. Thus, despite the fact that this aromatic side chain is not essential for CFTR folding, it is important in the ion channel function.