High affinity ATP/ADP analogues as new tools for studying CFTR gating
High affinity ATP/ADP analogues as new tools for studying CFTR gating
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DOI：
10.1113/jphysiol.2005.095083
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发表时间：
2005-12-01
影响因子：
5.5
通讯作者：
Hwang, TC
中科院分区：
文献类型：
--
作者：
Zhou, Z;Wang, XH;Hwang, TC
先前使用不可水解的ATP类似物和水解缺陷型囊性纤维化跨膜传导调节因子（CFTR）突变体的研究表明，ATP水解先于通道关闭。我们最近的数据表明，ATP结合在调节闭合速率方面也很重要。后一种假说预测，具有更高结合亲和力的ATP类似物应该比ATP更稳定开放状态。在这里，我们探讨了使用N-6-修饰的ATP/ADP类似物作为CFTR门控的高亲和力配体的可能性，因为这些类似物已被证明比其他ATP结合蛋白中的天然ATP/ADP更有效。在所测试的三种N-6-修饰的ATP类似物中，N-6-（2-苯乙基）-ATP（P-ATP）是最有效的，K-1/2为1.6 +/- 0.4 μ M（比ATP有效50倍以上）。在P-ATP存在下的最大开放概率（P-o）比ATP高30%，表明P-ATP也具有比ATP更高的功效。单通道动力学分析表明，随着[P-ATP]的增加，开放率增加，而关闭率下降。这两个动力学参数对[P-ATP]的变化具有不同的敏感性，这一事实表明涉及两个不同的ATP结合位点，一个高亲和力位点调节通道关闭，一个低亲和力位点控制通道打开。当通过突变核苷酸结合结构域2（NBD 2）步行者B谷氨酸（即E1371）或通过使用不可水解的ATP类似物AMP-PNP来消除ATP水解时，P-ATP对开放状态的稳定性的影响更加明显。类似的策略，开发具有修饰的腺嘌呤环的核苷酸类似物可能是有价值的CFTR门控的未来研究。
Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N-6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N-6-modified ATP analogues tested, N-6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K-1/2 of 1.6 +/- 0.4 mu M (> 50-fold more potent than ATP). The maximal open probability (P-o) in the presence of P-ATP was similar to 30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating.