Cysteine-independent inhibition of the CFTR chloride channel by the cysteine-reactive reagent sodium (2-sulphonatoethyl) methanethiosulphonate
Cysteine-independent inhibition of the CFTR chloride channel by the cysteine-reactive reagent sodium (2-sulphonatoethyl) methanethiosulphonate
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DOI：
10.1111/j.1476-5381.2009.00258.x
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发表时间：
2009-07-01
影响因子：
7.3
通讯作者：
Linsdell, P.
中科院分区：
文献类型：
--
作者：
Li, M-S;Demsey, A. F. A.;Linsdell, P.
背景和目的：甲硫基磺酸盐（MTS）试剂广泛用于离子通道结构-功能研究中半胱氨酸侧链的共价修饰。我们已经调查了广泛使用的带负电荷的MTS试剂，（2-sulphonatoethyl）methanethiosulfonate（MTSES），和囊性纤维化跨膜传导调节因子（CFTR）Cl-channel.Experimental方法之间的相互作用：膜片钳记录被用来研究一个'cys-少'的人CFTR的变体，其中所有18个内源性半胱氨酸残基已被删除突变，在哺乳动物细胞系中表达。使用切除的内翻外膜补丁允许MTS试剂被应用到细胞质面的active channels.Key结果：细胞内应用MTSES，但不带正电荷的MTSET，抑制cys-少CFTR的功能。抑制作用呈电压依赖性，Kd值从-80 mV时的1.97 mmol·L-1增加到+80 mV时的36 mmol·L-1。抑制作用在MTSES洗脱时完全逆转，与通道蛋白的共价修饰不一致。在单通道水平上，MTSES引起单位电流振幅的浓度依赖性降低。这种抑制被加强时，细胞外Cl-浓度decreased.Conclusions和影响：我们的研究结果表明，MTSES抑制CFTR的功能的方式是独立的，它的能力，共价修饰半胱氨酸残基。相反，我们认为MTSES作为一种开放通道阻滞剂，从其细胞质末端进入CFTR通道孔，以物理方式阻塞Cl-渗透。鉴于MTS试剂在功能研究中的广泛使用，我们的研究结果为解释此类研究结果提供了广泛适用的警告。英国药理学杂志（2009）157，1065 - 1071; doi：10.1111/j.1476 - 5381.2009.00258.x; 2009年5月19日在线发表
Background and purpose: Methanethiosulphonate (MTS) reagents are used extensively to modify covalently cysteine side chains in ion channel structure-function studies. We have investigated the interaction between a widely used negatively charged MTS reagent, (2-sulphonatoethyl) methanethiosulphonate (MTSES), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel.Experimental approach: Patch clamp recordings were used to study a 'cys-less' variant of human CFTR, in which all 18 endogenous cysteine residues have been removed by mutagenesis, expressed in mammalian cell lines. Use of excised inside-out membrane patches allowed MTS reagents to be applied to the cytoplasmic face of active channels.Key results: Intracellular application of MTSES, but not the positively charged MTSET, inhibited the function of cys-less CFTR. Inhibition was voltage dependent, with a K-d of 1.97 mmol.L-1 at -80 mV increasing to 36 mmol.L-1 at +80 mV. Inhibition was completely reversed on washout of MTSES, inconsistent with covalent modification of the channel protein. At the single channel level, MTSES caused a concentration-dependent reduction in unitary current amplitude. This inhibition was strengthened when extracellular Cl- concentration was decreased.Conclusions and implications: Our results indicate that MTSES inhibits the function of CFTR in a manner that is independent of its ability to modify cysteine residues covalently. Instead, we suggest that MTSES functions as an open channel blocker that enters the CFTR channel pore from its cytoplasmic end to physically occlude Cl- permeation. Given the very widespread use of MTS reagents in functional studies, our findings offer a broadly applicable caveat to the interpretation of results obtained from such studies. British Journal of Pharmacology (2009) 157, 1065-1071; doi:10.1111/j.1476-5381.2009.00258.x; published online 19 May 2009