Istituto di Biofisica, CNR, Via De Marini 6, I-16149 Genova, Italy.
Istituto di Biofisica, CNR, Via De Marini 6, I-16149 Genova, Italy.
J Biol Chem. 2011 Dec 23;286(51):44134-44144. doi: 10.1074/jbc.M111.272815. Epub 2011 Sep 15.
Mammalian CLC proteins comprise both Cl- channels and Cl-/H+ antiporters that carry out fundamental physiological tasks by transporting Cl- across plasma membrane and intracellular compartments. The NO3- over Cl- preference of a plant CLC transporter has been pinpointed to a conserved serine residue located at Scen and it is generally assumed that the other two binding sites of CLCs, Sext and Sin, do not substantially contribute to anion selectivity. Here we show for the Cl-/H+ antiporter CLC-5 that the conserved and extracellularly exposed Lys210 residue is critical to determine the anion specificity for transport activity. In particular, mutations that neutralize or invert the charge at this position reverse the NO3- over Cl- preference of WT CLC-5 at a concentration of 100 mm, but do not modify the coupling stoichiometry with H+. The importance of the electrical charge is shown by chemical modification of K210C with positively charged cysteine-reactive compounds that reintroduce the WT preference for Cl-. At saturating extracellular anion concentrations, neutralization of Lys210 is of little impact on the anion preference, suggesting an important role of Lys210 on the association rate of extracellular anions to Sext.
哺乳动物 CLC 蛋白既包括 Cl-通道，也包括 Cl-/H+反向转运体，通过跨质膜和细胞内隔室转运 Cl-，执行基本的生理任务。植物 CLC 转运体对 NO3-的转运偏好超过 Cl-，这一特性已被确定与 Scen 处的一个保守丝氨酸残基有关，一般认为 CLC 另外两个结合位点 Sext 和 Sin 不会对阴离子选择性有实质性贡献。在这里，我们展示 Cl-/H+反向转运体 CLC-5 中，保守且位于细胞外的 Lys210 残基对于决定转运活性的阴离子特异性至关重要。特别是，该位置的电荷中和或反转突变会在 100mM 的浓度下反转 WT CLC-5 对 NO3-的转运偏好，但不会改变与 H+的偶联计量比。通过用带正电荷的半胱氨酸反应性化合物对 K210C 进行化学修饰，重新引入 WT 对 Cl-的偏好，证明了电荷的重要性。在外源阴离子浓度饱和的情况下，Lys210 的中和对阴离子偏好几乎没有影响，这表明 Lys210 在 Sext 上的外源阴离子结合速率中起重要作用。
