Structural and ionic determinants of 5-nitro-2-(3-phenylpropylamino)-benzoic acid block of the CFTR chloride channel

1 The goals of this study were to identify the structural components requir ed for arylaminobenzoate block of the cystic fibrosis transmembrane conduct ance regulator (CFTR) chloride channel and to determine the involvement of two positively charged amino acid residues, found within the channel, in dr ug binding. 2 Wild-type and mutant CFTR chloride channels were expressed in Xenopus ooc ytes and CFTR currents measured using the two microelectrode voltage clamp. Block of the wild-type CFTR current by 5-nitro-2-(3-phenylpropylamino)-ben zoate (NPPB) occurred in a voltage-dependent manner with preferential inhib ition of the inward currents (K-d = 166 mu M at -90 mV). 3 Removal of the phenyl ring from the aliphatic chain of NPPB, with the com pound 2-butylamino-5-nitrobenzoic acid, caused only a small change in CFTR inhibition (K-d = 243 mu M), while addition of an extra phenyl ring at this position (5-nitro-2-(3,3-diphenylpropylamino)-benzoic acid) increased drug potency (K-d = 58 mu M). In contrast, removal of the benzoate ring (2-amin o-4-phenylbutyric acid) or the 5-nitro group (2-(3-phenylpropylamino)-benzo ic acid) of NPPB severely limited drug block of the wild-type channel. 4 NPPB inhibition of CFTR currents in oocytes expressing the mutants K335E and R347E also occurred in a voltage-dependent manner. However, the k(d)s f or NPPB block were increased to 371 and 1573 mu M, for the K335E and R347E mutants, respectively. 5 NPPB block of the inward wild-type CFTR current was reduced in the presen ce of 10 mM of the permeant anion SCN-. 6 These studies present the first step in the development of high affinity probes to the CFTR channel.