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最常见的囊性纤维化相关突变会破坏 CFTR 核苷酸结合结构域的二聚体状态。

The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR.

机构信息

Dalton Cardiovascular Research Center, University of Missouri, 134 Research Park Drive, Columbia, MO 65211, USA.

出版信息

J Physiol. 2011 Jun 1;589(Pt 11):2719-31. doi: 10.1113/jphysiol.2010.202861. Epub 2011 Apr 11.

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP binding cassette (ABC) superfamily. The deletion of the phenylalanine 508 (ΔF508-CFTR) is the most common mutation among cystic fibrosis (CF) patients. The mutant channels present a severe trafficking defect, and the few channels that reach the plasma membrane are functionally impaired. Interestingly, an ATP analogue, N6-(2-phenylethyl)-2′-deoxy-ATP (P-dATP), can increase the open probability (Po) to ∼0.7, implying that the gating defect of ΔF508 may involve the ligand binding domains, such as interfering with the formation or separation of the dimeric states of the nucleotide-binding domains (NBDs). To test this hypothesis, we employed two approaches developed for gauging the stability of the NBD dimeric states using the patch-clamp technique. We measured the locked-open time induced by pyrophosphate (PPi), which reflects the stability of the full NBD dimer state, and the ligand exchange time for ATP/N6-(2-phenylethyl)-ATP (P-ATP), which measures the stability of the partial NBD dimer state wherein the head of NBD1 and the tail of NBD2 remain associated. We found that both the PPi-induced locked-open time and the ATP/P-ATP ligand exchange time of ΔF508-CFTR channels are dramatically shortened, suggesting that the ΔF508 mutation destabilizes the full and partial NBD dimer states. We also tested if mutations that have been shown to improve trafficking of ΔF508-CFTR, namely the solubilizing mutation F494N/Q637R and ΔRI (deletion of the regulatory insertion), exert any effects on these newly identified functional defects associated with ΔF508-CFTR. Our results indicate that although these mutations increase the membrane expression and function of ΔF508-CFTR, they have limited impact on the stability of both full and partial NBD dimeric states for ΔF508 channels. The structure-function insights gained from this mechanism may provide clues for future drug design.

摘要

囊性纤维化跨膜电导调节因子(CFTR)是一种氯离子通道,属于 ATP 结合盒(ABC)超家族。在囊性纤维化(CF)患者中,最常见的突变是苯丙氨酸 508 缺失(ΔF508-CFTR)。突变通道存在严重的运输缺陷,到达质膜的少数通道功能受损。有趣的是,一种 ATP 类似物,N6-(2-苯乙基)-2′-脱氧-ATP(P-dATP),可以将开放概率(Po)增加到约 0.7,这表明 ΔF508 的门控缺陷可能涉及配体结合域,例如干扰核苷酸结合域(NBD)二聚体状态的形成或分离。为了验证这一假说,我们采用了两种方法,利用膜片钳技术来衡量 NBD 二聚体状态的稳定性。我们测量了焦磷酸(PPi)诱导的锁定开放时间,这反映了完整 NBD 二聚体状态的稳定性,以及 ATP/N6-(2-苯乙基)-ATP(P-ATP)的配体交换时间,这衡量了 NBD1 头部和 NBD2 尾部保持关联的部分 NBD 二聚体状态的稳定性。我们发现,ΔF508-CFTR 通道的 PPi 诱导的锁定开放时间和 ATP/P-ATP 配体交换时间都显著缩短,这表明 ΔF508 突变使完整和部分 NBD 二聚体状态不稳定。我们还测试了已被证明可改善 ΔF508-CFTR 运输的突变,即增溶突变 F494N/Q637R 和 ΔRI(调节插入缺失),是否对与 ΔF508-CFTR 相关的这些新发现的功能缺陷有任何影响。我们的结果表明,尽管这些突变增加了 ΔF508-CFTR 的膜表达和功能,但它们对 ΔF508 通道的完整和部分 NBD 二聚体状态的稳定性影响有限。从这种机制中获得的结构-功能见解可能为未来的药物设计提供线索。

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本文引用的文献

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Potentiation of disease-associated cystic fibrosis transmembrane conductance regulator mutants by hydrolyzable ATP analogs.
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