Investigations of pharmacologic properties of the renal CLC-K1 chloride channel co-expressed with barttin by the use of 2-(p-chlorophenoxy)propionic acid derivatives and other structurally unrelated chloride channels blockers

CLC-K chloride channels are expressed in the kidney, where they play a pivotal role in the mechanisms of urine concentration and Na+ reabsorption. The identification of barttin as an essential beta-subunit of CLC-K channels allowed performance of a pharmacologic characterization of wild-type CLC-K1 expressed in Xenopus oocytes. To this end, a series of 2-(p-chlorophenoxy)propionic acid (CPP) derivatives were screened using the two-microelectrode voltage-clamp technique. Several chemical modifications regarding the phenoxy group of the side chain (elimination of the oxygen atom or of methylenic groups, substitutions of the chlorine atom) did not alter the drug blocking activity, with five different derivatives showing a similar potency. Among these, a derivative of CPP carrying a benzyl group on the chiral center in the place of the methyl group represented the minimal structure for blocking CLC-K1. It inhibited the channel from the extracellular side with an affinity in the 150 muM range. The blocking potency of this compound is fourfold increased by lowering the extracellular chloride concentration, suggesting that the drug interacts with the channel pore. Concomitantly, the effect of some "classical" Cl- channel blockers (9-anthracenecarboxylic acid, 2-(phenylamino)benzoic acid, iminodibenzoic acid, niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt, and 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid disodium salt) was screened. 4,4'-Diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt was the only one capable of blocking CLC-K1 with a potency similar to the CPP derivative, although in an irreversible manner. The newly identified substances provide a useful tool to investigate the biophysical and physiologic role of these renal channels and a starting point for the development of therapeutic drugs with diuretic action.