Gong Xiandi, Linsdell Paul
Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada.
J Physiol. 2003 Jun 1;549(Pt 2):387-97. doi: 10.1113/jphysiol.2002.038232. Epub 2003 Apr 4.
Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is blocked by highly lyotropic permeant anions which bind tightly within the pore. Here we show that several different substitutions of a positively charged amino acid residue, arginine R334, in the putative outer mouth of the CFTR pore, greatly reduce the block caused by lyotropic Au(CN)2- ions applied to the intracellular side of the channel. Fixed positive charge at this site appears to play a role in Au(CN)2- binding, as judged by multiple substitutions of differently charged amino acid side chains and also by the pH dependence of block conferred by the R334H mutant. However, non-charge-dependent effects also appear to contribute to Au(CN)2- binding. Mutation of R334 also disrupts the apparent electrostatic interaction between intracellular Au(CN)2- ions and extracellular permeant anions, an interaction which normally acts to relieve channel block. All six mutations studied at R334 significantly weakened this interaction, suggesting that arginine possesses a unique ability to coordinate ion-ion interactions at this site in the pore. Our results suggest that lyotropic anions bind tightly to a site in the outer mouth of the CFTR pore that involves interaction with a fixed positive charge. Binding to this site is also involved in coordination of multiple permeant anions within the pore, suggesting that anion binding in the outer mouth of the pore is an important aspect in the normal anion permeation mechanism.
氯离子通过囊性纤维化跨膜传导调节因子（CFTR）氯离子通道的渗透被高度离液序列高的渗透性阴离子所阻断，这些阴离子紧密结合在孔道内。在此我们表明，在CFTR孔道假定的外口处，带正电荷的氨基酸残基精氨酸R334的几种不同取代，极大地降低了施加于通道胞内侧的离液序列高的Au(CN)₂⁻离子所引起的阻断作用。从不同电荷氨基酸侧链的多重取代以及R334H突变体所赋予的阻断作用的pH依赖性判断，该位点的固定正电荷似乎在Au(CN)₂⁻结合中起作用。然而，非电荷依赖性效应似乎也对Au(CN)₂⁻结合有贡献。R334的突变还破坏了胞内Au(CN)₂⁻离子与胞外渗透性阴离子之间明显的静电相互作用，这种相互作用通常起到缓解通道阻断的作用。在R334处研究的所有六个突变都显著削弱了这种相互作用，表明精氨酸在孔道的该位点具有协调离子 - 离子相互作用的独特能力。我们的结果表明，离液序列高的阴离子紧密结合在CFTR孔道外口的一个位点，该位点涉及与固定正电荷的相互作用。与该位点的结合也参与了孔道内多个渗透性阴离子的协同作用，这表明孔道外口的阴离子结合是正常阴离子渗透机制的一个重要方面。
