Regulation of the gating of cystic fibrosis transmembrane conductance regulator C1 channels by phosphorylation and ATP hydrolysis.

Opening of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels requires their phosphorylation by protein kinase A followed by exposure to ATP. We examined the interaction between nucleotides and phosphorylated CFTR channels by recording currents in intact cardiac myocytes and in excised patches. We found that, although the hydrolysis-resistant ATP analogue 5'-adenosine(beta,gamma- imino)triphosphate (AMP-PNP) cannot open phosphorylated CFTR channels, it can cause channels opened by ATP to remain open for many minutes. This suggests that ATP action at one site on CFTR is a prerequisite for AMP-PNP action at a second site. However, this action of AMP-PNP is restricted to highly phosphorylated CFTR channels, which, in the presence of ATP, display a relatively high open probability, but is not seen in partially phosphorylated CFTR channels, which have a low open probability in the presence of ATP. Our findings argue that incremental phosphorylation differentially regulates the interactions between nucleotides and the two nucleotide binding domains of CFTR. The nature of those interactions suggests that ATP hydrolysis at one nucleotide binding domain controls channel opening and ATP hydrolysis at the other regulates channel closing. IMAGES:

囊性纤维化跨膜传导调节因子（cystic fibrosis transmembrane conductance regulator, CFTR）氯离子通道的开放，需先经蛋白激酶A（protein kinase A, PKA）介导磷酸化，随后需暴露于三磷酸腺苷（adenosine triphosphate, ATP）环境中。本研究通过记录完整心肌细胞及离体膜片的离子电流，探究了核苷酸与磷酸化CFTR通道之间的相互作用。研究发现，尽管抗水解ATP类似物5'-腺苷(β,γ-亚氨基)三磷酸（5'-adenosine(β,γ-imino)triphosphate, AMP-PNP）无法直接激活磷酸化CFTR通道，却可使ATP诱导开放的通道维持开放状态长达数分钟。这表明，CFTR上某一位点的ATP结合与作用，是AMP-PNP在另一结合位点发挥功能的必要前提。然而，AMP-PNP的这一作用仅局限于高度磷酸化的CFTR通道：这类通道在ATP存在时展现出相对较高的开放概率；而在ATP存在时开放概率较低的部分磷酸化CFTR通道中，则未观察到此现象。本研究结果表明，逐级磷酸化会差异化调控核苷酸与CFTR的两个核苷酸结合结构域之间的相互作用。此类相互作用的特性提示，其中一个核苷酸结合结构域的ATP水解过程负责调控通道开放，而另一个结构域的ATP水解则负责调控通道关闭。图像：