title: Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation.
abstract: Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of                      the ATP-binding cassette (ABC) protein superfamily. Unlike most other ABC                      proteins that function as active transporters, CFTR is an ATP-gated chloride                      channel. The opening of CFTR's gate is associated with ATP-induced                      dimerization of its two nucleotide-binding domains (NBD1 and NBD2), whereas gate                      closure is facilitated by ATP hydrolysis-triggered partial separation of the                      NBDs. This generally held theme of CFTR gating-a strict coupling between                      the ATP hydrolysis cycle and the gating cycle-is put to the test by our                      recent finding of a short-lived, post-hydrolytic state that can bind ATP and                      reenter the ATP-induced original open state. We accidentally found a mutant CFTR                      channel that exhibits two distinct open conductance states, the smaller O1 state                      and the larger O2 state. In the presence of ATP, the transition between the two                      states follows a preferred O1→O2 order, a telltale sign of a violation of                      microscopic reversibility, hence demanding an external energy input likely from                      ATP hydrolysis, as such preferred gating transition was abolished in a                      hydrolysis-deficient mutant. Interestingly, we also observed a considerable                      amount of opening events that contain more than one O1→O2 transition,                      indicating that more than one ATP molecule may be hydrolyzed within an opening                      burst. We thus conclude a nonintegral stoichiometry between the gating cycle and                      ATP consumption. Our results lead to a six-state gating model conforming to the                      classical allosteric mechanism: both NBDs and transmembrane domains hold a                      certain degree of autonomy, whereas the conformational change in one domain will                      facilitate the conformational change in the other domain.
authors.affiliation: Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211, USA.
authors.forename: Kang-Yang
authors.initials: KY
authors.lastname: Jih
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Sohma
authors.forename: Tzyh-Chang
authors.initials: TC
authors.lastname: Hwang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002712:Chlorides / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006868:Hydrolysis; D015640:Ion Channel Gating; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary
chemical_list: D002712:Chlorides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate