Spontaneous Openings of CFTR are Coupled to Dimer Formation of its Nucleotide Binding Domains

Abstract

CFTR pore opening is coupled to dimer formation of its ATP bound nucleotide binding domains (NBDs) and channel closing to disruption of this dimer following the hydrolysis of one of the nucleotides. In addition to such ATP driven openings CFTR infrequently opens also in the absence of any nucleotides. The molecular mechanism behind this latter “spontaneous” activity is unknown because its open probability is far lower than required for reliable kinetic analysis.Previous work showed that transmembrane domain mutations P355A (1) and K978C (2) significantly and additively increase the spontaneous open probability of CFTR. Here we studied spontaneous gating of P355A/K978C CFTR, which is amenable to kinetic analysis, and constructed thermodynamic mutant cycles to test whether such pore openings are coupled to NBD dimer formation. For this purpose, we examined gating-related changes in energetic coupling in our background construct between residues R555 and T1246 on opposing sides of the NBD dimer interface, employing the same two mutations (R555K and T1246N) that were used in the past to demonstrate coupling of pore opening to NBD dimer formation in the presence of ATP (3).We found similar open probabilities and opening rates for background and double mutant (R555K/T1246N) channels, whereas these parameters were significantly reduced for the two single mutants (R555K and T1246N). In contrast, all four constructs displayed similar closing rates. Thus, CFTR residues R555 and T1246 become energetically coupled in the open state (ΔΔGopen-closed=-2.16±0.58 kT), and this interaction is already present in the transition state for opening (ΔΔGopening=-2.10±0.56 kT; ΔΔGclosing=0.45±0.36 kT).Our results suggest that even ATP-free spontaneous pore openings of CFTR are coupled to dimerization of its NBDs.(1) J Biol Chem. 289:19942-19957.(2) PNAS 107:3888-3893.(3) Nature 433:876-880.