Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel.
Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel.
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DOI：
10.1085/jgp.200609516
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发表时间：
2006-11
期刊：
影响因子：
--
通讯作者：
Linsdell P
中科院分区：
文献类型：
--
作者：
Aubin CN;Linsdell P
许多不同的离子通道孔被认为具有聚集在其入口周围的带电氨基酸残基。这些残留物贡献的所谓表面电荷可以在从膜一侧的本体溶液中吸引带相反电荷的离子、增加有效的局部抗衡离子浓度并有利于离子通过通道快速移动方面发挥重要作用。在这里，我们使用定点诱变来识别在囊性纤维化跨膜电导调节器（CFTR）Cl−通道孔的细胞内口中贡献重要表面电荷的精氨酸残基。虽然野生型 CFTR 与对称溶液的线性电流-电压关系相关，但在位于第五和第六跨膜区细胞内末端附近的两个精氨酸残基（R303 和 R352）诱变后观察到强烈的向外整流。电流整流取决于这些位置处存在的电荷，与静电效应一致。此外，诱变诱导的整流在较低的 Cl− 浓度下更为明显，表明这些突变体在内孔口附近集中 Cl− 离子的能力降低。 R303和R352突变体表现出单通道电导降低，特别是在负膜电位下，这取决于这些位置上存在的氨基酸残基的电荷。然而，R303E 和 R352E-CFTR 的电导率非常低，可以通过提高细胞内 Cl− 浓度来大大增加。通过带电荷的甲硫代磺酸盐试剂对303位处引入的半胱氨酸残基的修饰再现了对电流整流的电荷依赖性效应。第二和第十跨膜区中精氨酸残基的诱变也改变了通道渗透特性，但是这些效应与通道表面电荷的变化不一致。这些结果表明，带正电的精氨酸残基起到将 Cl- 离子集中在 CFTR 孔内口的作用，这有助于最大限度地提高 Cl- 离子通过孔的渗透率。
Many different ion channel pores are thought to have charged amino acid residues clustered around their entrances. The so-called surface charges contributed by these residues can play important roles in attracting oppositely charged ions from the bulk solution on one side of the membrane, increasing effective local counterion concentration and favoring rapid ion movement through the channel. Here we use site-directed mutagenesis to identify arginine residues contributing important surface charges in the intracellular mouth of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel pore. While wild-type CFTR was associated with a linear current–voltage relationship with symmetrical solutions, strong outward rectification was observed after mutagenesis of two arginine residues (R303 and R352) located near the intracellular ends of the fifth and sixth transmembrane regions. Current rectification was dependent on the charge present at these positions, consistent with an electrostatic effect. Furthermore, mutagenesis-induced rectification was more pronounced at lower Cl− concentrations, suggesting that these mutants had a reduced ability to concentrate Cl− ions near the inner pore mouth. R303 and R352 mutants exhibited reduced single channel conductance, especially at negative membrane potentials, that was dependent on the charge of the amino acid residue present at these positions. However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl− concentration. Modification of an introduced cysteine residue at position 303 by charged methanethiosulfonate reagents reproduced charge-dependent effects on current rectification. Mutagenesis of arginine residues in the second and tenth transmembrane regions also altered channel permeation properties, however these effects were not consistent with changes in channel surface charges. These results suggest that positively charged arginine residues act to concentrate Cl− ions at the inner mouth of the CFTR pore, and that this contributes to maximization of the rate of Cl− ion permeation through the pore.
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影响因子：
64.8
作者：
IMOTO, K;BUSCH, C;NUMA, S
通讯作者：
NUMA, S
影响因子：
2.9
作者：
MACKINNON, R;LATORRE, R;MILLER, C
通讯作者：
MILLER, C
影响因子：
2.9
作者：
Nimigean, CM;Chappie, JS;Miller, C
通讯作者：
Miller, C
影响因子：
3.8
作者：
Moorhouse, AJ;Keramidas, A;Barry, PH
通讯作者：
Barry, PH
影响因子：
4.8
作者：
Seibert, FS;Linsdell, P;Clarke, DM
通讯作者：
Clarke, DM