Changes in Accessibility of Cytoplasmic Substances to the Pore Associated with Activation of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel
Changes in Accessibility of Cytoplasmic Substances to the Pore Associated with Activation of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel
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DOI：
10.1074/jbc.m110.113332
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发表时间：
2010-10-15
影响因子：
4.8
通讯作者：
Linsdell, Paul
中科院分区：
文献类型：
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作者：
El Hiani, Yassine;Linsdell, Paul
囊性纤维化跨膜电导调节剂Cl-通道的打开既依赖于磷酸化，也依赖于ATP结合和水解。然而，这些细胞质调节因子打开Cl通道孔的机制尚不清楚。我们使用膜片钳记录来研究细胞质上应用的半胱氨酸反应试剂对沿着囊性纤维化跨膜电导调节剂的半胱氨酸无半胱氨酸变异的孔内衬第六跨膜区（TM6）长度引入的半胱氨酸的可及性。我们发现甲基乙硫代磺酸盐（MTS）试剂在开放通道细胞质侧作用时，可以不可逆地修饰取代TM6残基的半胱氨酸，如phe337、Thr-338、Ser-341、Ile-344、val345、Met-348、Ala-349、Arg-352和Gln-353。然而，内部[2-磺酰基]甲乙硫代磺酸盐（MTSES）（一种带负电荷的MTS试剂）的表观修饰速率取决于通道的激活状态。特别是，从内部沿TM6轴引入的半胱氨酸（T338C、S341C、I344C），即使在使用内部MTSES对非激活通道进行长时间预处理后，也没有明显的修饰证据。相比之下，靠近TM6内部的半胱氨酸（V345C, M348C）在激活和非激活通道中都很容易被修饰。平均阴离子Au(CN)(2)（-）对T338C的访问同样取决于通道激活状态。我们观察到的MTS修饰模式使我们能够将不同的孔衬氨基酸侧链指定为通道孔的不同功能区域。对这些发现的一种合理解释是，细胞质在孔的最窄区域接触残基的途径随着活化而改变。
Opening of the cystic fibrosis transmembrane conductance regulator Cl- channel is dependent both on phosphorylation and on ATP binding and hydrolysis. However, the mechanisms by which these cytoplasmic regulatory factors open the Cl- channel pore are not known. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining sixth transmembrane region (TM6) of a cysteine-less variant of cystic fibrosis transmembrane conductance regulator. We find that methanethiosulfonate (MTS) reagents modify irreversibly cysteines substituted for TM6 residues Phe-337, Thr-338, Ser-341, Ile-344, Val-345, Met-348, Ala-349, Arg-352, and Gln-353 when applied to the cytoplasmic side of open channels. However, the apparent rate of modification by internal [2-sulfonatoethyl] methanethiosulfonate (MTSES), a negatively charged MTS reagent, is dependent on the activation state of the channels. In particular, cysteines introduced far along the axis of TM6 from the inside (T338C, S341C, I344C) showed no evidence of significant modification even after prolonged pretreatment of non-activated channels with internal MTSES. In contrast, cysteines introduced closer to the inside of TM6 (V345C, M348C) were readily modified in both activated and non-activated channels. Access of a per-meant anion, Au(CN)(2)(-), to T338C was similarly dependent upon channel activation state. The pattern of MTS modification we observe allows us to designate different pore-lining amino acid side chains to distinct functional regions of the channel pore. One logical interpretation of these findings is that cytoplasmic access to residues at the narrowest region of the pore changes concomitant with activation.