Relationship between nucleotide binding and ion channel gating in cystic fibrosis transmembrane conductance regulator.
We have employed rate-equilibrium free energy relationship (REFER) analysis to characterize the dynamic events involved in the allosteric regulation of cystic fibrosis transmembrane conductance regulator (CFTR) function. A wide range of different hydrolysable and poorly hydrolysable nucleoside triphosphates were used to elucidate the role of ATP hydrolysis in CFTR function. The linearity of the REFER plots and Phi values near unity for all ligands tested implies that CFTR channel gating is a reversible thermally driven process with all structural reorganization in the binding site(s) completed prior to channel opening. This is consistent with the requirement for nucleotide binding for channel opening. However, the channel structural transition from the open to the closed state occurs independently of any events in the binding sites. Similar results were obtained on substitution of amino acids at coupling joints between both nucleotide binding domains (NBD) and cytoplasmic loops (CL) in opposite halves of the protein, indicating that any structural reorganization there also had occurred in the channel closed state. The fact that fractional Phi values were not observed in either of these distant sites suggests that there may not be a deterministic 'lever-arm' mechanism acting between nucleotide binding sites and the channel gate. These findings favour a stochastic coupling between binding and gating in which all structural transitions are thermally driven processes. We speculate that increase of channel open state probability is due to reduction of the number of the closed state configurations available after physical interaction between ligand bound NBDs and the channel.
Aleksandrov
Andrei A
AA
Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC 27599, USA. aleksand@email.unc.edu
Cui
Liying
L
Riordan
John R
JR
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism; D004735:Energy Transfer / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D008024:Ligands; D009154:Mutation / Q000502:physiology; D009711:Nucleotides / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D008024:Ligands; D009711:Nucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017868:Cyclic AMP-Dependent Protein Kinases