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Correspondence to László Csanády: csanady{at}puskin.sote.hu; or David C. Gadsby: gadsby{at}rockefeller.edu
CFTR is the only ABC (ATP-binding cassette) ATPase known to be an ion channel. Studies of CFTR channel function, feasible with single-molecule resolution, therefore provide a unique glimpse of ABC transporter mechanism. CFTR channel opening and closing (after regulatory-domain phosphorylation) follows an irreversible cycle, driven by ATP binding/hydrolysis at the nucleotide-binding domains (NBD1, NBD2). Recent work suggests that formation of an NBD1/NBD2 dimer drives channel opening, and disruption of the dimer after ATP hydrolysis drives closure, but how NBD events are translated into gate movements is unclear. To elucidate conformational properties of channels on their way to opening or closing, we performed non-equilibrium thermodynamic analysis. Human CFTR channel currents were recorded at temperatures from 15 to 35°C in inside-out patches excised from Xenopus oocytes. Activation enthalpies(
H
) were determined from Eyring plots. H was 117 ± 6 and 69 ± 4 kJ/mol, respectively, for opening and closure of partially phosphorylated, and 96 ± 6 and 73 ± 5 kJ/mol for opening and closure of highly phosphorylated wild-type (WT) channels. H for reversal of the channel opening step, estimated from closure of ATP hydrolysis–deficient NBD2 mutant K1250R and K1250A channels, and from unlocking of WT channels locked open with ATP+AMPPNP, was 43 ± 2, 39 ± 4, and 37 ± 6 kJ/mol, respectively. Calculated upper estimates of activation free energies yielded minimum estimates of activation entropies (S), allowing reconstruction of the thermodynamic profile of gating, which was qualitatively similar for partially and highly phosphorylated CFTR. S appears large for opening but small for normal closure. The large H and S (TS 
41 kJ/mol) for opening suggest that the transition state is a strained channel molecule in which the NBDs have already dimerized, while the pore is still closed. The small S for normal closure is appropriate for cleavage of a single bond (ATP's beta-gamma phosphate bond), and suggests that this transition state does not require large-scale protein motion and hence precedes rehydration (disruption) of the dimer interface.
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