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 cystic fibrosis (CF) disease by disrupting ATP-dependent channel opening; however, whether G551D affects phosphorylation-dependent channel activation is unclear. Here, we use macropatch recording and Ussing chamber approaches to demonstrate that G551D impacts on phosphorylation-dependent activation of CFTR, and PKA-mediated phosphorylation regulates the interaction between the x-loop in nucleotide-binding domain 2 (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 ATP-binding cassette signature motif in NBD2 and D173R in CL1, each of which strongly increased PKA sensitivity both in the wild-type (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 chargereversal mutation of D1341R/D173R-CFTR exhibited similar PKA sensitivity when compared with 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.