ANALYSIS OF AMINO-ACID-RESIDUES IN THE H5-H6 TRANSMEMBRANE AND EXTRACELLULAR DOMAINS OF NA,K-ATPASE ALPHA-SUBUNIT IDENTIFIES THREONINE-797 AS A DETERMINANT OF OUABAIN SENSITIVITY
ANALYSIS OF AMINO-ACID-RESIDUES IN THE H5-H6 TRANSMEMBRANE AND EXTRACELLULAR DOMAINS OF NA,K-ATPASE ALPHA-SUBUNIT IDENTIFIES THREONINE-797 AS A DETERMINANT OF OUABAIN SENSITIVITY
复制标题
DOI：
10.1021/bi00180a015
复制
发表时间：
1994-04-12
期刊：
影响因子：
2.9
通讯作者：
LINGREL, JB
中科院分区：
文献类型：
--
作者：
FENG, JN;LINGREL, JB
已经鉴定出Na，K-ATP酶α亚基的几个氨基酸残基改变哇巴因敏感性。这些残基位于α 1亚基的N-末端一半，表明分子的这一部分可能代表强心苷的结合位点。然而，并不是所有的细胞外和跨膜区域，包括H5-H6跨膜区域进行了研究。为了确定α亚基的该区域是否有助于哇巴因敏感性，将具有形成氢键潜力的氨基酸用丙氨酸（一种非氢键氨基酸）取代。在哇巴因敏感的HeLa细胞中表达编码含有这些单独氨基酸替换的酶的cDNA，并检查改变的酶赋予哇巴因抗性的能力。研究了19个氨基酸取代。T797A（Thr 797为Ala）是唯一使敏感的HeLa细胞对哇巴因产生抗性的取代。在该位置产生三个另外的取代（T797 V、T797 S和T797 D），以检查Thr 797的取代对哇巴因抑制Na，K-ATP酶活性的影响。T797V置换赋予哇巴因抗性，但T797S和T797D置换不赋予哇巴因抗性。表达T797A和T797V取代的哇巴因抗性细胞系表现出Na，K-ATP酶活性，其对哇巴因的抗性是内源性HeLa或绵羊酶的60和70倍。在氨基酸797处不存在羟基可能是在该位置具有取代的酶的灵敏度降低的原因。进一步分析T797置换对强心苷抑制Na，K-ATP酶活性的作用，强心苷仅在抑制剂的特定部分变化。配对药物，哇巴苷元-哇巴因，二氢哇巴因-哇巴因和地高辛-洋地黄毒苷的I50比，对于野生型绵羊和取代的酶都是相似的，这表明T797不直接与糖部分，内酯环的不饱和键或类固醇环的C-12位的羟基相互作用。
Several amino acid residues of the a subunit of the Na,K-ATPase have been identified which alter ouabain sensitivity. These residues are located in the N-terminal half of the al subunit suggesting that this portion of the molecule may represent the binding site for cardiac glycosides. However, not all extracellular and transmembrane regions have been investigated, including the H5-H6 membrane-spanning region. To determine if this region of the alpha subunit contributes to ouabain sensitivity, amino acids which have the potential to form hydrogen bonds were substituted with alanine, a non-hydrogen-bonding amino acid. cDNAs encoding enzyme containing these individual amino acid replacements were expressed in ouabain-sensitive HeLa cells, and the ability of the altered enzymes to confer ouabain resistance was examined. Nineteen amino acid substitutions were investigated. T797A (Thr 797 to Ala) was the only substitution which conferred ouabain resistance to sensitive HeLa cells. Three additional substitutions at this position (T797V, T797S, and T797D) were generated in order to examine the effects of the replacements of Thr 797 on ouabain inhibition of Na,K-ATPase activity. The T797V substitution conferred ouabain resistance, but T797S and T797D substitutions did not. The ouabain-resistant cell lines expressing the T797A and T797V substitutions exhibited Na,K-ATPase activity that was 60 and 70 times more resistant to ouabain than the endogenous HeLa or sheep enzymes. The absence of a hydroxyl group at amino acid 797 may be responsible for the reduced sensitivity of the enzyme with substitutions at this position. The T797 substitutions were further analyzed for inhibition of Na,K-ATPase activity by cardiac glycosides which vary only in a specific portion of the inhibitor. The I50 ratios for the paired drugs, ouabagenin-ouabain, dihydroouabain-ouabain, and digoxin-digitoxin, were similar for both the wild-type sheep and substituted enzymes, suggesting that T797 does not interact directly with the sugar moiety, the unsaturated bond of the lactone ring, or the hydroxyl group at position C-12 of the steroid ring.