Abstract
The CFTR channel is an essential mediator of electrolyte transport across epithelial tissues. CFTR opening is promoted by ATP binding and dimerization of its two nucleotide binding domains (NBDs). Phosphorylation of its R domain (e.g. by PKA) is also required for channel activity. The CFTR structure is unsolved but homology models of the CFTR closed and open states have been produced based on the crystal structures of evolutionarily related ABC transporters. These models predict the formation of a tetrahelix bundle of intracellular loops (ICLs) during channel opening. Here we provide evidence that residues E267 in ICL2 and K1060 in ICL4 electrostatically interact at the interface of this predicted bundle to promote CFTR opening. Mutations or a thiol modifier that introduced like charges at these two positions substantially inhibited ATP-dependent channel opening. ATP-dependent activity was rescued by introducing a second site gain of function (GOF) mutation that was previously shown to promote ATP-dependent and ATP-independent opening (K978C). Conversely, the ATP-independent activity of the K978C GOF mutant was inhibited by charge- reversal mutations at positions 267 or 1060 either in the presence or absence of NBD2. The latter result indicates that this electrostatic interaction also promotes unliganded channel opening in the absence of ATP binding and NBD dimerization. Charge-reversal mutations at either position markedly reduced the PKA sensitivity of channel activation implying strong allosteric coupling between bundle formation and R domain phosphorylation. These findings support important roles of the tetrahelix bundle and the E267-K1060 electrostatic interaction in phosphorylation-dependent CFTR gating.
Highlights
ATP binding promotes CFTR channel opening by an unknown mechanism
Shown in yellow are residues E267 in ICL2 and K1060 in ICL4 in the CFTR open state model, which locate to the base of the predicted helix bundle near the nucleotide binding domains (NBDs) coupling helices
Combining E267 and K1060 Charge-reversal Mutations with a Gain of Function (GOF) Mutation Reveals the Importance of the Tetrahelix Bundle in Mediating Channel Opening in the Absence of ATP Binding or NBD Dimerization—Previously we reported a class of gain of function (GOF) mutation in ICL3 near the base of TM9 that increases both ATP-dependent and ATP-independent channel activity (e.g. K978C [12])
Summary
ATP binding promotes CFTR channel opening by an unknown mechanism. Results: Introducing like charges at positions 267 and 1060 in the opposing cytosolic loops that couple the nucleotide binding domains to the pore inhibits channel opening. Given the aforementioned homology between CFTR and the bacterial exporters, structural models of the open and closed states have been produced using inward-facing (MsbA) and outward-facing (Sav1866) crystal structures as templates, respectively (20 –24) Certain aspects of these models have been verified experimentally; notably, the orientation of the predicted NBD dimer interface and the nature of the coupling helices that link the NBDs to the cytosolic extensions of the TMs [10, 20]. A major structural feature of the outward-facing structures of the homodimeric bacterial exporters is a tetrahelix bundle along the symmetry axis that appears to link ATP-induced NBD dimerization to conformational changes in the TMs [17, 18, 25, 26] This helix bundle is created by the close apposition of the cytosolic extensions of TMs 3 and 4 across the subunit interface and extends cytosolically to where the ICLs bind to the NBDs via coupling helices. Perturbing the bundle interface markedly reduced the PKA sensitivity of CFTR channel activation, a result we interpret as evidence of strong allosteric coupling between R domain phosphorylation and bundle formation
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