Random mutagenesis of the sheep Na,K-ATPase alpha 1 subunit generating the ouabain-resistant mutant L793P
Random mutagenesis of the sheep Na,K-ATPase alpha 1 subunit generating the ouabain-resistant mutant L793P
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DOI：
10.1074/jbc.271.27.15879
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发表时间：
1996-07-05
影响因子：
4.8
通讯作者：
Price, EM
中科院分区：
文献类型：
--
作者：
Burns, EL;Nicholas, RA;Price, EM
采用聚合酶链式反应随机诱变羊Na，K-ATPase α亚基691-946氨基酸的cDNA磁带，将诱变磁带替换全长cDNA中的野生型区域，将突变体池转染到HeLa细胞中，在0.5 mu M - ouabain中选择产生抗性细胞后，利用聚合酶链式反应从稳定的转染体基因组DNA中扩增诱变磁带。对聚合酶链反应产物进行序列分析，发现有I729V、L793P和K836R三个氨基酸取代，随后的定点诱变实验表明，只有L793P对抗性有重要作用，为了阐明L793在抑制瓦巴因中的作用，在该位置制备了额外的突变。在HeLa细胞中构建并表达了L793A和L793I突变体，只有L793A在瓦巴因的选择中存活下来，这表明抗性不是由于亮氨酸被脯氨酸特异性取代而产生的。为了探索耐药机制，我们将L793P突变体对钠和钾的表观亲和力与野生型HeLa泵进行了比较，虽然对钠的表观亲和力相当，但突变体对钾的表观亲和力高出1倍，这表明L793P对乌阿巴因不敏感的机制是由于酶的区域可能包括K+结合位点的扰动。
The polymerase chain reaction was used to randomly mutagenize a cDNA cassette encoding amino acids 691-946 of the sheep Na,K-ATPase alpha subunit, The mutagenized cassettes were used to replace the wild type region in the full-length cDNA, and pools of mutants were transfected into HeLa cells, After the generation of resistant cells via selection in 0.5 mu M ouabain, polymerase chain reaction was used to amplify the mutagenized cassette from the genomic DNA of the stable transfectants, Sequence analysis of the polymerase chain reaction product revealed three amino acid substitutions: I729V, L793P, and K836R, Subsequent site-directed mutagenesis experiments showed that only L793P was important for resistance, To elucidate the role of L793 in ouabain inhibition, additional mutations at this position were prepared. L793A and L793I mutants were constructed and expressed in HeLa cells, Only L793A survived selection using ouabain, which suggested that resistance is not due to the specific substitution of leucine with proline. To explore the mechanism of resistance, apparent affinities of the L793P mutant for sodium and potassium were compared to the wild-type HeLa pump, Although the apparent affinities were comparable for sodium, the mutant had a a-fold higher apparent affinity for potassium, This suggests that the mechanism of ouabain insensitivity of L793P is due to a perturbation in the region of the enzyme that may include the K+ binding site.