2,3-butanedione monoxime affects cystic fibrosis transmembrane conductance regulator channel function through phosphorylation-dependent and phosphorylation-independent mechanisms:: The role of bilayer material properties.
2,3-butanedione monoxime affects cystic fibrosis transmembrane conductance regulator channel function through phosphorylation-dependent and phosphorylation-independent mechanisms:: The role of bilayer material properties.
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DOI：
10.1124/mol.106.026070
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发表时间：
2006-12-01
影响因子：
3.6
通讯作者：
Andersen, Olaf S.
中科院分区：
文献类型：
--
作者：
Artigas, Pablo;Al'Aref, Subhi J.;Andersen, Olaf S.
2,3 - 丁二酮单肟（BDM）被广泛认为是一种化学磷酸酶。因此，我们研究了BDM对囊性纤维化跨膜传导调节因子（CFTR）Cl⁻通道的影响，该通道以一种复杂的方式受磷酸化调节。在豚鼠心室肌细胞中，福司柯林激活的全细胞CFTR电流对细胞外20 mM BDM呈现双相反应：先是快速的约2倍的电流激活，随后是较慢的（时间常数约为20秒）抑制（至对照的约20%）。用磷酸酶抑制剂微囊藻毒素进行细胞内透析可消除抑制反应，这表明内源性磷酸酶参与其中。在表达人上皮CFTR的非洲爪蟾卵母细胞中进一步研究了BDM诱导的激活。半最大BDM激活浓度（K₀.₅）具有状态依赖性，对于高度磷酸化的通道约为2 mM，对于部分磷酸化的通道约为20 mM，这表明是一种受调节的受体机制。由于BDM以相似的K₀.₅值调节许多不同的膜蛋白，我们测试了BDM是否可以通过改变脂质双层特性而非通过BDM与蛋白质的直接相互作用来改变蛋白质功能。我们使用不同长度的短杆菌肽通道（不同的通道 - 双层疏水错配）作为双层刚度的报告分子，发现BDM增加了通道出现率和寿命（降低了双层刚度）。在20 mM BDM时，出现率增加约4倍（对于较长的、每个单体15个残基的通道）至约10倍（对于较短的、每个单体13个残基的通道）；寿命独立于通道长度增加约50%。因此，BDM降低了双层变形的能量成本，这种效应可能是BDM对CFTR和其他膜蛋白影响的基础；K₀.₅的状态依赖性变化与这种双层介导的机制是一致的。
2,3-Butanedione monoxime (BDM) is widely believed to act as a chemical phosphatase. We therefore examined the effects of BDM on the cystic fibrosis transmembrane regulator ( CFTR) Cl- channel, which is regulated by phosphorylation in a complex manner. In guinea pig ventricular myocytes, forskolin-activated whole-cell CFTR currents responded biphasically to external 20 mM BDM: a rapid similar to 2-fold current activation was followed by a slower (tau similar to 20 s) inhibition (to similar to 20% of control). The inhibitory response was abolished by intracellular dialysis with the phosphatase inhibitor microcystin, suggesting involvement of endogenous phosphatases. The BDM-induced activation was studied further in Xenopus laevis oocytes expressing human epithelial CFTR. The concentration for half-maximal BDM activation (K-0.5) was state-dependent, similar to 2 mM for highly and similar to 20 mM for partially phosphorylated channels, suggesting a modulated receptor mechanism. Because BDM modulates many different membrane proteins with similar K-0.5 values, we tested whether BDM could alter protein function by altering lipid bilayer properties rather than by direct BDM-protein interactions. Using gramicidin channels of different lengths ( different channel-bilayer hydrophobic mismatch) as reporters of bilayer stiffness, we found that BDM increases channel appearance rates and lifetimes (reduces bilayer stiffness). At 20 mM BDM, the appearance rates increase similar to 4-fold (for the longer, 15 residues/monomer, channels) to similar to 10-fold (for the shorter, 13 residues/monomer channels); the lifetimes increase similar to 50% independently of channel length. BDM thus reduces the energetic cost of bilayer deformation, an effect that may underlie the effects of BDM on CFTR and other membrane proteins; the state- dependent changes in K-0.5 are consistent with such a bilayer-mediated mechanism.