Membrane Protein Disease Research Group, Department of Physiology, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Alberta, Canada.
Am J Physiol Cell Physiol. 2011 Aug;301(2):C336-46. doi: 10.1152/ajpcell.00005.2011. Epub 2011 May 4.
Anion exchanger 1 (AE1) is the plasma membrane Cl(-)/HCO(3)(-) exchanger of erythrocytes. Carbonic anhydrases (CA) provide substrate for AE1 by catalyzing the reaction, H(2)O + CO(2) ↔ HCO(3)(-) + H(+). The physical complex of CAII with AE1 has been proposed to maximize anion exchange activity. To examine the effect of CAII catalysis on AE1 transport rate, we fused either CAII-wild type or catalytically inactive CAII-V143Y to the cytoplasmic COOH terminus of AE1 to form AE1.CAII and AE1.CAII-V143Y, respectively. When expressed in transfected human embryonic kidney 293 cells, AE1.CAII had a similar Cl(-)/HCO(3)(-) exchange activity to AE1 alone, as assessed by the flux of H(+) equivalents (87 ± 4% vs. AE1) or rate of change of intracellular Cl(-) concentration (93 ± 4% vs. AE1), suggesting that CAII does not activate AE1. In contrast, AE1.CAII-V143Y displayed transport rates for H(+) equivalents and Cl(-) of 55 ± 2% and of 40 ± 2%, versus AE1. Fusion of CAII to AE1 therefore reduces anion transport activity, but this reduction is compensated for during Cl(-)/HCO(3)(-) exchange by the presence of catalytically active CAII. Overexpression of free CAII-V143Y acts in a dominant negative manner to reduce AE1-mediated HCO(3)(-) transport by displacement of endogenous CAII-wild type from its binding site on AE1. To examine whether AE1.CAII bound endogenous CAII, we coexpressed CAII-V143Y along with AE1 or AE1.CAII. The bicarbonate transport activity of AE1 was inhibited by CAII-V143Y, whereas the activity of AE1.CAII was unaffected by CAII-V143Y, suggesting impaired transport activity upon displacement of functional CAII from AE1 but not AE1.CAII. Taken together, these data suggest that association of functional CAII with AE1 increases Cl(-)/HCO(3)(-) exchange activity, consistent with the HCO(3)(-) transport metabolon model.
阴离子交换蛋白 1（AE1）是红细胞质膜 Cl(-)/HCO(3)(-)交换蛋白。碳酸酐酶（CA）通过催化反应 H(2)O + CO(2) ↔ HCO(3)(-) + H(+)为 AE1 提供底物。CAII 与 AE1 的物理复合物被提出以最大限度地提高阴离子交换活性。为了研究 CAII 催化对 AE1 转运速率的影响，我们将 CAII-野生型或催化失活的 CAII-V143Y 分别融合到 AE1 的细胞质 COOH 末端，形成 AE1.CAII 和 AE1.CAII-V143Y。当在转染的人胚肾 293 细胞中表达时，AE1.CAII 的 Cl(-)/HCO(3)(-)交换活性与单独的 AE1 相似，通过 H(+)当量的通量（87 ± 4%与 AE1 相比）或细胞内 Cl(-)浓度变化率（93 ± 4%与 AE1 相比）评估，表明 CAII 不会激活 AE1。相比之下，AE1.CAII-V143Y 的 H(+)当量和 Cl(-)转运速率分别为 55 ± 2%和 40 ± 2%，与 AE1 相比。因此，CAII 与 AE1 的融合降低了阴离子转运活性，但在 Cl(-)/HCO(3)(-)交换过程中，由于存在催化活性 CAII，这种降低得到了补偿。游离 CAII-V143Y 的过表达以显性负性方式起作用，通过从 AE1 上的结合位点置换内源性 CAII-野生型来减少 AE1 介导的 HCO(3)(-)转运。为了检查 AE1.CAII 是否结合内源性 CAII，我们共表达 CAII-V143Y 与 AE1 或 AE1.CAII。AE1 的碳酸氢盐转运活性被 CAII-V143Y 抑制，而 AE1.CAII 的活性不受 CAII-V143Y 影响，表明功能性 CAII 从 AE1 上置换会导致转运活性受损，但 AE1.CAII 不会。综上所述，这些数据表明，功能性 CAII 与 AE1 的结合增加了 Cl(-)/HCO(3)(-)交换活性，与 HCO(3)(-)转运代谢物模型一致。
