Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel.
Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR's opening-closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N(3)-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N(3)-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1-NBD2 interface. The open state of CFTR has been shown to represent a two-ATP-bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a "partial NBD dimer" state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and "partial" separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR's NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.
Tsai
Ming-Feng
MF
Department of Medical Pharmacology and Physiology and 2 Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 65211, USA.
Li
Min
M
Hwang
Tzyh-Chang
TC
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000502:physiology*; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000502:physiology*; D055724:Electrophysiological Phenomena; D005260:Female; D015640:Ion Channel Gating / Q000502:physiology; D008958:Models, Molecular; D009711:Nucleotides / Q000502:physiology; D010053:Ovary / Q000166:cytology; D018408:Patch-Clamp Techniques
D018528:ATP-Binding Cassette Transporters; D009711:Nucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate