G551D is a major disease-associated gating mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP- and phosphorylation-dependent chloride channel. G551D causes severe CF disease by disrupting ATP-dependent channel opening, however, whether G551D affects phosphorylation-dependent channel activation is unclear. Here, we use macro patch recording and Ussing chamber approaches to demonstrate that G551D impacts on phosphorylation-dependent activation of CFTR and PKA-mediated phosphorylation regulates the interaction between x-loop in NBD2 and cytosolic loop (CL) 1. We show that G551D not only disrupts ATP-dependent channel opening, but also impairs phosphorylation-dependent channel activation by largely reducing PKA sensitivity consistent with the reciprocal relationship between channel opening/gating, ligand binding, and phosphorylation. Furthermore, we identified two novel GOF mutations: D1341R in the x-loop near the ABC signature motif in NBD2 and D173R in CL1, each of which strongly increased PKA sensitivity both in the WT background and when introduced into G551D-CFTR. When D1341R was combined with a second GOF mutation (e.g., K978C in CL3), we find that the double GOF mutation maximally increased G551D channel activity such that VX-770 had no further effect. We further show that a double charge-reversal mutation of D1341R/D173R-CFTR exhibited similar PKA sensitivity when compared to WT-CFTR. Together, our results suggest that charge repulsion between D173 and D1341 of WT-CFTR normally inhibits channel activation at low PKA activity by reducing PKA sensitivity and negative allostery by the G551D is coupled to reduced PKA sensitivity of CFTR that can be restored by second GOF mutations.

中文翻译：

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G551D 突变会损害 CFTR 通道的蛋白激酶 A (PKA) 依赖性激活，该通道可通过新型功能增益 (GOF) 突变恢复。

G551D 是囊性纤维化跨膜电导调节器 (CFTR) 蛋白（一种依赖于 ATP 和磷酸化的氯通道）中与疾病相关的主要门控突变。G551D 通过破坏 ATP 依赖性通道的开放导致严重的 CF 疾病，然而，G551D 是否影响磷酸化依赖性通道激活尚不清楚。在这里，我们使用宏贴片记录和 Ussing 室方法来证明 G551D 对 CFTR 的磷酸化依赖性激活的影响和 PKA 介导的磷酸化调节 NBD2 中的 x 环与胞质环 (CL) 1 之间的相互作用。我们表明 G551D 不不仅会破坏 ATP 依赖性通道的开放，而且还会通过大大降低 PKA 敏感性来削弱磷酸化依赖性通道的激活，这与通道开放/门控、配体结合之间的相互关系一致，和磷酸化。此外，我们发现了两个新的 GOF 突变：NBD2 中靠近 ABC 特征基序的 x 环中的 D1341R 和 CL1 中的 D173R，每个突变在 WT 背景中和引入 G551D-CFTR 时都显着增加了 PKA 敏感性。当 D1341R 与第二个 GOF 突变（例如，CL3 中的 K978C）结合时，我们发现双 GOF 突变最大程度地增加了 G551D 通道活性，因此 VX-770 没有进一步的影响。我们进一步表明，与 WT-CFTR 相比，D1341R/D173R-CFTR 的双电荷反转突变表现出相似的 PKA 敏感性。一起，