8-CYCLOPENTYL-1,3-DIPROPYLXANTHINE AND OTHER XANTHINES DIFFERENTIALLYBIND TO THE WILD-TYPE AND DELTA-F508 MUTANT FIRST NUCLEOTIDE-BINDING FOLD (NBF-1) DOMAINS OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR

Cystic fibrosis is an autosomal recessive disorder affecting chloride transport in pancreas, lung, and other tissues, which is caused by mut ations in the cystic fibrosis transmembrane conductance regulator (CFT R). Certain alkyl xanthines such as CPX (8-cyclopentyl-1,3-dipropylxan thine) stimulate Cl- efflux from cells bearing the Delta F508 genotype common to most cases of cystic fibrosis. We have hypothesized that th e CFTR molecule itself might be the site for CPX action, perhaps in th e region of the first nucleotide binding fold (NBF-1) domain. Therefor e, to test this hypothesis directly we have used a rapid membrane filt ration assay to measure the kinetics of association and dissociation o f [H-3]CPX to both recombinant NBF-1 and recombinant NBF-1 bearing the Delta F508 mutation. We report that [H-3]CPX binds with higher affini ty to the Delta F508-NBF-1 of CFTR (K-d = 1.0 nM) than to the wild-typ e NBF-1 of CFTR (K-d = 17.0 nM). These K-d values were calculated from direct measurements of the association and dissociation rate constant s. The rate constants for the dissociation reaction of the wild-type N BF-1 and Delta F508-NBF-1 of CFTR were not different from each other. However, the corresponding rate constants for the association reaction were k(+1) (NBF-1) = 4.7 +/- 0.9 x 10(4) M-1 s(-1) and k(+1) (Delta F 508-NBF-1) = 1.6 +/- 0.3 x 10(5) M-1 s(-1), respectively. These K-d va lues were corroborated by equilibrium-binding experiments, which gave very similar values. We have also measured the relative displacement o f various xanthines from both wild-type NBF-1 and Delta F508-NBF-1, in anticipation. that the order of potencies for binding might parallel the action of the different xanthines on CF cells. For wild-type NBF-1 , the rank order was DA-CPX > DAX > CPX > caffeine > adenosine much gr eater than IBMX > 2-thioCPX. For Delta F508-NBF-1, the rank order was DAX > CPX > caffeine > DA-CPX > adenosine much greater than IBMX > 2-t hioCPX. These relative potencies show close parallels with previously observed relative potencies of these drugs on CF cells, and thus lend strong support to the hypothesis that the mechanism of action on CF ce lls may involve direct interaction with the CFTR molecule itself.