Jennings M L
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555, USA.
J Gen Physiol. 1995 Jan;105(1):21-47. doi: 10.1085/jgp.105.1.21.
One of the modes of action of the red blood cell anion transport protein is the electrically silent net exchange of 1 Cl- for 1 SO4= and 1 H+. Net SO4(=)-Cl- exchange is accelerated by low pH or by conversion of the side chain of glutamate 681 into an alcohol by treatment of intact cells with Woodward's reagent K (WRK) and BH4-. The studies described here were performed to characterize the electrical properties of net SO4(=)-Cl- exchange in cells modified with WRK/BH4-. The SO4= conductance measured in 100 mM SO4= medium is smaller in modified cells than in control cells. However, the efflux of [35S] SO4= into a 150-mM KCl medium is 80-fold larger in modified cells than in control cells and is inhibited 99% by 10 microM H2DIDS. No detectable H+ flux is associated with SO4(=)-Cl- exchange in modified cells. In the presence of gramicidin to increase the cation permeability, the stoichiometry of SO4(=)-Cl- exchange is not distinguishable from 1:1. In modified cells loaded with SO4=, the valinomycin-mediated efflux of 86Rb+ into an Na-gluconate medium is immediately stimulated by the addition of 5 mM extracellular Cl-. Therefore, SO4(=)-Cl- exchange in modified cells causes an outward movement of negative charge, as expected for an obligatory 1:1 SO4(=)-Cl- exchange. This is the first example of an obligatory, electrogenic exchange process in band 3 and demonstrates that the coupling between influx and efflux does not require that the overall exchange be electrically neutral. The effects of membrane potential on SO4(=)-SO4= exchange and SO4(=)-Cl- exchange in modified cells are consistent with a model in which nearly a full net positive charge moves inward through the transmembrane field during the inward Cl- translocation event, and a small net negative charge moves with SO4= during the SO4= translocation event. This result suggests that, in normal cells, the negative charge on Glu 681 traverses most of the transmembrane electric field, accompanied by Cl- and the equivalent of two protein-bound positive charges.
红细胞阴离子转运蛋白的作用模式之一是1个Cl-与1个SO4=和1个H+进行电沉默净交换。低pH值或用伍德沃德试剂K（WRK）和BH4-处理完整细胞，使谷氨酸681侧链转化为醇，均可加速SO4(=)-Cl-净交换。本文所述研究旨在表征经WRK/BH4-修饰的细胞中SO4(=)-Cl-净交换的电学性质。在100 mM SO4=培养基中测得的SO4=电导率，修饰细胞低于对照细胞。然而，在修饰细胞中，[35S] SO4=向150 mM KCl培养基中的外流比对照细胞大80倍，并被10 μM H2DIDS抑制99%。在修饰细胞中，未检测到与SO4(=)-Cl-交换相关的H+通量。在存在短杆菌肽以增加阳离子通透性的情况下，SO4(=)-Cl-交换的化学计量比与1:1无明显差异。在加载了SO4=的修饰细胞中，加入5 mM细胞外Cl-可立即刺激缬氨霉素介导的86Rb+向葡萄糖酸钠培养基中的外流。因此，修饰细胞中的SO4(=)-Cl-交换导致负电荷向外移动，这与强制性1:1 SO4(=)-Cl-交换预期一致。这是带3中强制性电生交换过程的首个例子，表明流入和流出之间的偶联并不要求整体交换是电中性的。膜电位对修饰细胞中SO4(=)-SO4=交换和SO4(=)-Cl-交换的影响与以下模型一致：在向内的Cl-转运过程中，几乎一个完整的净正电荷通过跨膜电场向内移动，而在SO4=转运过程中，一个小的净负电荷与SO4=一起移动。这一结果表明，在正常细胞中，Glu 681上的负电荷穿过大部分跨膜电场，伴随着Cl-和相当于两个蛋白质结合正电荷的物质。
