Novel Residues Lining the CFTR Chloride Channel Pore Identified by Functional Modification of Introduced Cysteines
Novel Residues Lining the CFTR Chloride Channel Pore Identified by Functional Modification of Introduced Cysteines
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DOI：
10.1007/s00232-009-9167-3
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发表时间：
2009-04-01
影响因子：
2.4
通讯作者：
Linsdell, Paul
中科院分区：
文献类型：
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作者：
Fatehi, Mohammad;Linsdell, Paul
取代半胱氨酸可及性诱变（SCAM）已被广泛用于鉴定离子通道蛋白中的孔衬氨基酸侧链。然而，渗透和门控功能的影响可能难以分开，导致不确定性的反应性半胱氨酸侧链的位置。我们结合SCAM与调查的电荷依赖性影响的甲硫基磺酸盐（MTS）试剂的功能性渗透性能的囊性纤维化跨膜电导调节（CFTR）的Cl-通道。我们发现，在第十一和第十二跨膜（TM）区域中的21个连续氨基酸中的7个被取代的半胱氨酸可以通过外部施加带正电荷的[2-（三甲基铵）乙基] MTS溴化物（MTSET）和带负电荷的钠[2-磺酰乙基] MTS（MTSES）来修饰。这些半胱氨酸的修饰导致在宏观和单通道电流水平下的开放通道电流-电压关系的变化，其反映了对Cl-从外部溶液通过通道渗透的速率的特定的、电荷依赖性的影响。因此，该方法鉴定了位于渗透途径内的氨基酸侧链。孔衬残基的半胱氨酸诱变也影响孔内阴离子结合和阴离子选择性，提供更多关于这些残基的作用的信息。我们的研究结果表明，一个简单的方法筛选孔衬氨基酸的离子通道。我们认为，TM 11有助于CFTR孔和TM 11和12之间的细胞外环位于靠近孔的外口。
Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current-voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl- permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.