Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore
Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore
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CFTR 氯离子通道孔外口正电荷的识别
DOI：
10.1007/s00424-008-0521-6
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
P. Linsdell
中科院分区：
文献类型：
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作者：
Jing‐Jun Zhou;M. Fatehi;P. Linsdell
我们使用定点诱变和功能分析来鉴定囊性纤维化跨膜传导调节剂（CFTR） Cl -通道中与细胞外阴离子相互作用的带正电的氨基酸残基。CFTR第一胞外环（ECL）的两个带正电的精氨酸残基R104和R117，以及第六跨膜区赖氨酸残基K335的突变，导致电流电压关系向内整流，单通道电导降低。这些作用取决于取代侧链的电荷和Cl -浓度，这表明这些正电荷通常会将细胞外的Cl -离子集中在孔的外口附近。侧链电荷依赖效应是通过将这些氨基酸突变为半胱氨酸，然后用带电的半胱氨酸反应试剂进行共价修饰来原位操纵电荷来模拟的，从而确认这些侧链在孔外前庭内的位置。R104C和R117C的状态无关修饰表明，这些残基位于孔的最外层。我们认为ECL1有助于CFTR孔外前庭，该区域带正电的氨基酸侧链吸引Cl -离子进入孔中。相比之下，我们发现没有证据表明其他ecl中的固定正电荷有助于孔的渗透特性。
We have used site-directed mutagenesis and functional analysis to identify positively charged amino acid residues in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel that interact with extracellular anions. Mutation of two positively charged arginine residues in the first extracellular loop (ECL) of CFTR, R104, and R117, as well as lysine residue K335 in the sixth transmembrane region, leads to inward rectification of the current–voltage relationship and decreased single channel conductance. These effects are dependent on the charge of the substituted side chain and on the Cl− concentration, suggesting that these positive charges normally act to concentrate extracellular Cl− ions near the outer mouth of the pore. Side chain charge-dependent effects are mimicked by manipulating charge in situ by mutating these amino acids to cysteine followed by covalent modification with charged cysteine-reactive reagents, confirming the location of these side chains within the pore outer vestibule. State-independent modification of R104C and R117C suggests that these residues are located at the outermost part of the pore. We suggest that ECL1 contributes to the CFTR pore external vestibule and that positively charged amino acid side chains in this region act to attract Cl− ions into the pore. In contrast, we find no evidence that fixed positive charges in other ECLs contribute to the permeation properties of the pore.