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首页> 外文期刊>American Journal of Physiology >Dibasic phosphorylation sites in the R domain of CFTR have stimulatory and inhibitory effects on channel activation.
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Dibasic phosphorylation sites in the R domain of CFTR have stimulatory and inhibitory effects on channel activation.

机译：CFTR R域中的二元磷酸化位点对通道激活具有刺激和抑制作用。

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摘要

To better understand the mechanisms by which PKA-dependent phosphorylation regulates CFTR channel activity, we have assayed open probabilities (P(o)), mean open time, and mean closed time for a series of CFTR constructs with mutations at PKA phosphorylation sites in the regulatory (R) domain. Forskolin-stimulated channel activity was recorded in cell-attached and inside-out excised patches from transiently transfected Chinese hamster ovary cells. Wild-type CFTR and constructs with a single Ser-to-Ala mutation as well as octa (Ser-to-Ala mutations at 8 sites) and constructs with one or two Ala-to-Ser mutations were studied. In cell-attached patches, Ser-to-Ala mutations at amino acids 700, 795, and 813 decreased P(o), whereas Ser-to-Ala mutations at 737 and 768 increased P(o). In general, differences in P(o) were due to differences in mean closed time. For selected constructs with either high or low values of P(o), channel activity was measured in excised patches. With 1 mM ATP, P(o) was similar to thatobserved in cell-attached patches, but with 10 mM ATP, all constructs tested showed elevated P(o) values. ATP-dependent increases in P(o) were due to reductions in mean closed time. These results indicate that R-domain phosphorylation affects ATP binding and not the subsequent steps of hydrolysis and channel opening. A model was developed whereby R-domain phosphorylation, in a site-dependent manner, alters equilibrium between forms of CFTR with low and high affinities for ATP.
机译：为了更好地理解PKA依赖性磷酸化调节CFTR通道活性的机制，我们分析了一系列CFTR构建体的开放概率（P（o）），平均开放时间和平均封闭时间，这些CFTR构建体在PKA磷酸化位点发生了突变。监管（R）域。在从瞬时转染的中国仓鼠卵巢细胞的细胞附着和由内而外的切除斑块中记录了受佛司可林刺激的通道活性。研究了野生型CFTR和具有单个Ser-to-Ala突变以及八位（8个位点的Ser-to-Ala突变）的构建体以及具有一个或两个Ala-Ser突变的构建体。在细胞附着的补丁中，氨基酸700、795和813的Ser-to-Ala突变降低了P（o），而737和768的Ser-to-Ala突变则提高了P（o）。通常，P（o）的差异是由于平均关闭时间的差异引起的。对于具有高或低P（o）值的选定构建体，在切除的小片中测量通道活性。使用1 mM ATP，P（o）类似于在细胞贴片中观察到的P（o），但是使用10 mM ATP，测试的所有构建物均显示升高的P（o）值。 ATP依赖的P（o）增加是由于平均关闭时间减少所致。这些结果表明，R结构域的磷酸化影响ATP的结合，而不影响水解和通道打开的后续步骤。开发了一种模型，其中R结构域的磷酸化以位点依赖性方式改变了具有低和高亲和力的ATP的CFTR形式之间的平衡。

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