Dahl Neera K, Jiang Lianwei, Chernova Marina N, Stuart-Tilley Alan K, Shmukler Boris E, Alper Seth L
Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 2003 Nov 7;278(45):44949-58. doi: 10.1074/jbc.M308660200. Epub 2003 Aug 21.
Cl-/HCO3- exchange activity mediated by the AE1 anion exchanger is reduced by carbonic anhydrase II (CA2) inhibition or by prevention of CA2 binding to the AE1 C-terminal cytoplasmic tail. This type of AE1 inhibition is thought to represent reduced metabolic channeling of HCO3- to the intracellular HCO3- binding site of AE1. To test the hypothesis that CA2 binding might itself allosterically activate AE1 in Xenopus oocytes, we compared Cl-/Cl- and Cl-/HCO3- exchange activities of AE1 polypeptides with truncation and missense mutations in the C-terminal tail. The distal renal tubular acidosis-associated AE1 901X mutant exhibited both Cl-/Cl- and Cl-/HCO3- exchange activities. In contrast, AE1 896X, 891X, and AE1 missense mutants in the CA2 binding site were inactive as Cl-/HCO3- exchangers despite exhibiting normal Cl-/Cl- exchange activities. Co-expression of CA2 enhanced wild-type AE1-mediated Cl-/HCO3- exchange, but not Cl-/Cl- exchange. CA2 co-expression could not rescue Cl-/HCO3- exchange activity in AE1 mutants selectively impaired in Cl-/HCO3- exchange. However, co-expression of transport-incompetent AE1 mutants with intact CA2 binding sites completely rescued Cl-/HCO3- exchange by an AE1 missense mutant devoid of CA2 binding, with activity further enhanced by CA2 co-expression. The same transport-incompetent AE1 mutants failed to rescue Cl-/HCO3- exchange by the AE1 truncation mutant 896X, despite preservation of the latter's core CA2 binding site. These data increase the minimal extent of a functionally defined CA2 binding site in AE1. The inter-protomeric rescue of HCO3- transport within the AE1 dimer shows functional proximity of the C-terminal cytoplasmic tail of one protomer to the anion translocation pathway in the adjacent protomer within the AE1 heterodimer. The data strongly support the hypothesis that an intact transbilayer anion translocation pathway is completely contained within an AE1 monomer.
碳酸酐酶II（CA2）抑制或阻止CA2与AE1 C末端胞质尾结合可降低由AE1阴离子交换器介导的Cl⁻/HCO₃⁻交换活性。这种类型的AE1抑制被认为代表了HCO₃⁻向AE1细胞内HCO₃⁻结合位点的代谢通道减少。为了验证CA2结合本身可能在非洲爪蟾卵母细胞中变构激活AE1的假说，我们比较了C末端尾有截短和错义突变的AE1多肽的Cl⁻/Cl⁻和Cl⁻/HCO₃⁻交换活性。远端肾小管酸中毒相关的AE1 901X突变体表现出Cl⁻/Cl⁻和Cl⁻/HCO₃⁻交换活性。相比之下，CA2结合位点的AE1 896X、891X和AE1错义突变体作为Cl⁻/HCO₃⁻交换器无活性，尽管它们表现出正常的Cl⁻/Cl⁻交换活性。CA2的共表达增强了野生型AE1介导的Cl⁻/HCO₃⁻交换，但未增强Cl⁻/Cl⁻交换。CA2共表达不能挽救在Cl⁻/HCO₃⁻交换中选择性受损的AE1突变体的Cl⁻/HCO₃⁻交换活性。然而，具有完整CA2结合位点的无转运能力的AE1突变体与缺乏CA2结合的AE1错义突变体共表达完全挽救了Cl⁻/HCO₃⁻交换，CA2共表达进一步增强了活性。相同的无转运能力的AE1突变体未能挽救AE1截短突变体896X的Cl⁻/HCO₃⁻交换，尽管后者保留了其核心CA2结合位点。这些数据增加了AE1中功能定义的CA2结合位点的最小范围。AE1二聚体内HCO₃⁻转运的原聚体间挽救显示一个原聚体的C末端胞质尾与AE1异二聚体中相邻原聚体的阴离子转运途径在功能上接近。数据有力地支持了完整的跨膜阴离子转运途径完全包含在AE1单体中的假说。
