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

Abstract

Abstract. CLC-K chloride channels are expressed in the kidney, where they play a pivotal role in the mechanisms of urine concentration and Na ϩ reabsorption. The identification of barttin as an essential ␤-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 M 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 Ϫ channel blockers (9-anthracenecarboxylic acid,

In-vivo administration of CLC-K kidney chloride channels inhibitors increases water diuresis in rats

“…Here we demonstrate that renal CLC-K chloride channels may represent novel drug targets for diuretic and antihypertensive effects. In previous structureactivity and mutagenesis studies of chloride channels in vitro, we have identified benzofuran derivatives (MT-189 and RT-93) as specific ligands able to block CLC-K channels activity with a K D value less than 10 mmol/l [22][23][24]. Here we report that acute in-vivo administration of such newly synthesized molecules resulted in significant diuretic effects and lowered SBP in normotensive rats.…”

“…The key structural features required for ligands with blocking or activating properties on heterologously expressed CLC-K has been recently characterized [22][23][24]; interestingly, benzofuran derivatives seem to act as new high-affinity blockers for these chloride channels types [24]. Among them, derivative MT-189 ( Fig.…”

Role of kidney chloride channels in health and disease

“…The importance of ClC-K/barttin channels is illustrated by human genetic diseases and by transgenic mouse models. Co-expression of barttin with ClC-K1 or ClC-K2 in Xenopous laevis oocytes resulted in an increase in current amplitude, a difference in calcium sensitivity and enhanced ClC-K abundance in the plasma membrane [12,13]. Mutations in the gene encoding barttin affect both ClC-Ka and ClC-Kb and cause type-III Bartter's syndrome in kidney (Fig.…”

“…Some ClC channels (ClC-0, -1 and -2) have measurable currents when expressed alone in heterologous systems, while others such as ClC-K channels require an associated protein [11]. The related channels ClC-Ka and ClC-Kb are the only channels known to require a β-subunit (barttin) for functional expression [11][12][13]. The associated subunit barttin is also crucial for efficient trafficking of ClC-K proteins to plasma membrane [13].…”

Novel diuretic targets

“…Of relevance to the kidney, Pusch and colleagues (48) have systematically characterized and developed small-molecule inhibitors and activators of the ClC-K channels. In a series of elegant studies, they evolved the low-affinity (mM) ClC-1 inhibitor p-chlorophenoxy-propionic acid (CPP) (5, 112) into a low-micromolar inhibitor of ClC-Ka, MT-189 (80,83,84,108) (Fig. 3; see below).…”