Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel activated by PKA phosphorylation on the regulatory (R) domain. Phosphorylation at several R domain residues stimulates ATP-dependent channel openings and closings, termed channel gating. To explore the protein segment responsible for channel potentiation and PKA-dependent activation, deletion mutations were constructed by removing 1 to 3 protein segments of the R domain, including residues 708-759 (ΔR708-759), R760-783 and R784-835, each of which contains one or two PKA phosphorylation sites. Deletion of R708-759 or R760-783 had little effect on CFTR gating, whereas all mutations lacking R784-835 reduced CFTR activity by decreasing the mean burst duration (MBD) and increasing the interburst interval (IBI). The data suggest that R784-835 plays a major role in stimulating CFTR gating. For ATP-associated regulation, ∆R784-835 had minor impact on gating potentiation by 2'dATP, CaATP and pyrophosphate. Interestingly, introducing a phosphorylated peptide matching R809-835 shortened the IBI of ΔR708-835-CFTR. Consistently, ΔR815-835, but not ΔR784-814, enhanced IBI, whereas both reduced MBD. These data suggest that entire R784-835 is required for stabilizing the open state of CFTR; however, R815-835 through interactions with the channel is dominant for enhancing the opening rate. Of note, PKA markedly decreased the IBI of ΔR708-783-CFTR. Conversely, the IBI of ΔR708-814-CFTR was short and PKA-independent. These data reveal that for stimulating CFTR gating, PKA phosphorylation may relieve R784-814-mediated auto-inhibition that prevents IBI shortening by R815-835 This mechanism may elucidate how the R domain potentiates channel gating and may unveil CFTR stimulation by other protein kinases.

中文翻译：

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PKA磷酸化可通过减轻调节域的刺激性C末端上的自抑制作用来增强CFTR门控。

囊性纤维化跨膜电导调节剂（CFTR）是由调节（R）域上的PKA磷酸化激活的氯离子通道。多个R结构域残基的磷酸化刺激了ATP依赖性通道的打开和关闭，称为通道门控。为探索负责通道增强和PKA依赖性激活的蛋白片段，通过去除R结构域的1-3个蛋白片段（包括残基708-759（ΔR708-759），R760-783和R784-835）来构建缺失突变，每个含有一个或两个PKA磷酸化位点。R708-759或R760-783的缺失对CFTR门控几乎没有影响，而所有缺少R784-835的突变均通过减少平均猝发持续时间（MBD）和增加突发间隔（IBI）降低CFTR活性。数据表明，R784-835在刺激CFTR门控中起主要作用。对于ATP相关的调节，∆R784-835对2'dATP，CaATP和焦磷酸盐的门控电位影响较小。有趣的是，引入匹配R809-835的磷酸化肽可缩短ΔR708-835-CFTR的IBI。一致地，ΔR815-835而非IR784-814增强了IBI，而两者均降低了MBD。这些数据表明，要稳定CFTR的打开状态，就需要整个R784-835。但是，R815-835通过与通道的交互作用对于提高打开速率起着主导作用。值得注意的是，PKA明显降低了ΔR708-783-CFTR的IBI。相反，ΔR708-814-CFTR的IBI较短且独立于PKA。这些数据表明，为了刺激CFTR门控，