Cystic fibrosis is the most common life-threatening inherited disease in Caucasian populations. The protein whose dysfunction causes cystic fibrosis, CFTR, belongs to the superfamily of ABC transporters, which couple hydrolytic cycles at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most of these are transporters, which harness the chemical potential of ATP to move very diverse compounds across membranes, often against steep electrochemical potential gradients. However, CFTR, alone, functions as an ion channel providing a permeation pathway for anions to cross the membrane, driven by their electrochemical potential difference. It is now emerging that the CFTR channel employs the molecular mechanism of its transporter relatives in its own, individual way. As in other ABC proteins, ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with two complete, composite catalytic sites now buried at the interface (site 1 and site 2). ATP hydrolysis and γ-phosphate dissociation at site 2, with loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where they result in opening (Vergani et al, 2005) and closing (Csanády et al, 2010), respectively, of the ion-permeation pathway. At no time point along the reaction coordinate of an active transporter can there be a pore open to both sides of the membrane since this would allow dissipation of hard-won electrochemical gradients (Läuger 1980). Because in a channel ions move down their electrochemical gradient, and not against it, access to the permeation pathway need not be controlled by two gates. Since in crystals of homologous proteins, which have the NBDs in a tight dimer conformation, the TMDs have an “outward-facing” conformation (Dawson & Locher 2006; Aller et al, 2009) it is simplest to imagine that CFTR’s present working gate might be equivalent to a transporter’s external gate. The formation of a tight NBD dimer would flip the TMDs into an outward facing conformation in which the structures homologous to a transporter’s internal gate no longer prevent anion flow between the diffusion pathway and the cytosol. Hydrolysis of the ATP bound at site 2, by triggering NBD dimer dissociation, would flip the TMDs to an inward facing conformation in which a functional external gate acts as an effective barrier between the external solution and the permeation pathway. To investigate steps within the allosteric coupling mechanism linking the composite sites in the NBDs to the channel gates in CFTR, we used the information contained in the evolutionary record to select target sites for mutagenesis, concentrating on the NBD/TMD interface. Multiple sequence alignments grouping thousands of ABC protein sequences are available. If two positions are energetically coupled, changes in side-chain distribution at one position are likely to occur in concert with changes at the other. Algorithms have been developed to detect such “coevolution” between the two positions in multiple sequence alignments (Fleishman et al. 2004; Fodor and Aldrich 2004). We constructed an alignment containing only transporters with an NBD asymmetry close to CFTR’s and implemented 5 different correlation algorithms. We accepted only pairs scoring in the top 0.5%, and selected targets for mutagenesis which were detected by more than one algorithm and were separated by less than 15Å on homology models of CFTR. The formalism of double mutant cycles was applied to test for interaction between coevolving side chains. In a generalized double mutant cycle, the WT protein, two single mutants, and the double mutant form the four corners of a thermodynamic cycle. If the two residues do not interact, the effects of the single mutations should be independent and hence additive, i.e. the effects of mutating one site will not depend on whether the mutation was done in a WT or mutant (at the other site) background and mutation-linked changes in ΔG on parallel sides of the cycle are equal (coupling energy, ΔΔGint = 0). Any difference (ΔΔGint ≠ 0) signifies, and (to some extent) quantifies, energetic coupling between the two residues. Using excised, inside-out patch-clamp recording on CFTR, several kinetic parameters were measured and used to characterize WT and mutants in terms of the ΔG between two states. In most cases tested, no significant energetic coupling was detected. In one case the coupling between the two target sites was seen to change upon channel opening, consistent with the formation of a molecular contact between them which was not present in the closed state.