Members of the ABCC family of the ABC superfamily are generally exporters of a broad range of substrates with both hydrophobic and hydrophilic properties, but also include the sulfonyl urea receptors that regulate potassium channels and CFTR which is itself a chloride channel and may regulate other ion channels.
To examine the means by which the ABC transporter modular organization is utilized to produce a finely regulated ion channel, we have studied its single channel gating properties and its ability to bind, hydrolyse and trap purine nucleotides. Since the pores of most known ion channels are formed by assemblies of multiple subunits, we have also assessed whether the CFTR chloride channel has a quaternary structure. Biochemical experiments in which two differentially epitope tagged CFTR species were solubilized from membranes with a range of mild detergents indicated that they migrated as monomers on velocity gradient centrifugation, were not co-immunoprecipitated by antibodies recognizing either of the epitopes and were not appreciably cross-linked by a variety of chemical cross-linkers, although CFTR was cross-linked to other proteins. Functional experiments in which CFTR mutants with very different unitary conductances were co-expressed failed to detect channels with intermediate conductance values. Hence contrary to some other proposals, we suggest that as with members of the major ClC chloride channel family, residues from a single CFTR polypeptide chain form an ion pore. It is clear that ATP hydrolysis, believed to drive solute translocation by ABC transporters, is not energetically coupled to chloride permeation through CFTR. However, several models of CFTR gating have proposed that opening and closing are driven by ATP hydrolysis at the first (NBD1) and second (NBD2) nucleotide binding domains, respectively. We found that while the energy of activation of the opening transition is ~100 kJ mol-1, that of closing is ~10 kJ mol-1, similar to that of diffusion in water. Therefore channel closing is not dependent on the input of free energy from ATP hydrolysis at any site. We have also found that channel opening can also occur in the absence of ATP hydrolysis. Nevertheless CFTR does bind and hydrolyse ATP and in the presence of vanadate traps the hydrolysis product, ADP at each NBD. Substitution of Walker A lysine residues in either NBD of CFTR prevents hydrolysis and trapping only at that NBD and not at the other, indicating greater independence of the domains than in some other ABC proteins such as P-glycoprotein. In the absence of vanadate, ADP dissociates much more readily from NBD2 than NBD1. The non-hydrolysable ATP analogue, AMP-PNP, which prolongs channel opening in the presence of ATP, interacts with high affinity at NBD1.
These data and others are consistent with channel opening due to nucleotide binding and closing on dissociation either before or after hydrolysis. ATP serves as a readily available cytoplasmic ligand; its hydrolysis provides efficient reversibility of channel opening. Under normal conditions phosphorylation of the R-domain by protein kinase A is obligatory for the NBD controlled gating cycle as it enables transduction of the conformational impact of nucleotide binding and transition state formation to the pore. This phosphorylation does not alter the affinity of the NBDs for nucleotides.
This work was supported by the NIDDK of the NIH.
ABC Transporters: Molecular Structure and Physiological RolesABC Transporters: Molecular Structure and Physiological RolesABC Transporters: Molecular Structure and Physiological Roleso the pore. This phosphorylation does not alter the affinity of the NBDs for nucleotides.
This work was supported by the NIDDK of the NIH.