Deficient HCO3- transport in an AE1 mutant with normal Cl- transport can be rescued by carbonic anhydrase II presented on an adjacent AE1 protomer
Deficient HCO3- transport in an AE1 mutant with normal Cl- transport can be rescued by carbonic anhydrase II presented on an adjacent AE1 protomer
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DOI：
10.1074/jbc.m308660200
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发表时间：
2003-11-07
影响因子：
4.8
通讯作者：
Alper, SL
中科院分区：
文献类型：
--
作者：
Dahl, NK;Jiang, LW;Alper, SL
通过碳酸酐酶II（CA 2）抑制或通过阻止CA 2与AE 1 C-末端胞质尾结合，降低AE 1阴离子交换剂介导的Cl-/HCO 3-交换活性。这种类型的AE 1抑制被认为代表减少代谢通道的HCO 3-的细胞内HCO 3-结合位点的AE 1。为了检验这一假设，即CA 2结合本身可能会变构激活AE 1在非洲爪蟾卵母细胞，我们比较了Cl-/Cl-和Cl-/HCO 3-交换活动的AE 1多肽的截断和错义突变的C-末端尾巴。远端肾小管酸中毒相关的AE 1 901 X突变体表现出Cl-/Cl-和Cl-/HCO 3-交换活性。相反，AE 1 896 X，891 X，和AE 1错义突变体的CA 2结合位点是无活性的Cl-/HCO 3-交换，尽管表现出正常的Cl-/Cl-交换活动。CA 2的共表达增强了野生型AE 1介导的Cl-/HCO 3-交换，但不增强Cl-/Cl-交换。CA 2共表达不能拯救AE 1突变体中Cl-/HCO 3-交换活性选择性受损的Cl-/HCO 3-交换。然而，共表达的运输能力不强的AE 1突变体与完整的CA 2结合位点完全拯救了Cl-/HCO 3-交换的AE 1错义突变体缺乏CA 2的结合，与活性进一步增强CA 2的共表达。相同的运输能力不强的AE 1突变体未能拯救由AE 1截断突变体896 X的Cl-/HCO 3-交换，尽管后者的核心CA 2结合位点的保存。这些数据增加了AE 1中功能定义的CA 2结合位点的最小程度。AE 1二聚体内HCO 3转运的原聚体间拯救显示了一个原聚体的C-末端胞质尾区与AE 1异二聚体内相邻原聚体中的阴离子易位途径的功能接近。这些数据强烈支持的假设，一个完整的transbilayer阴离子易位途径是完全包含在AE 1单体。
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.