Kidney CLC-K Chloride Channels Show Differential Pharmacological Profiles Depending on the Heterologous Expression System

Abstract

The human chloride channels CLC-Ka and CLC-Kb play a pivotal role in kidney by controlling chloride and water absorption. Both channels require barttin as an accessory subunit for full activity. Mutations in CLC-Kb and barttin genes lead to severe renal salt loss and CLC-K polymorphisms are associated with hypertension. Therefore, novel therapeutic approaches targeting CLC-K/barttin might be helpful in treating these diseases. We have recently identified relevant molecular determinants that distinguish CLC-K activators from blockers using the X.laevis oocyte expression system. We recognized niflumic acid (NFA) as a powerful activator and phenyl-benzofuran carboxylic acid analogs as potent inhibitors. Here, by using these molecules as lead compounds and the whole cell configuration of the patch-clamp technique, we explored the pharmacological profile of CLC-K/barttin expressed in mammalian HEK-293 cells. Similarly to what observed in amphibian oocytes, benzofuran derivatives resulted as efficacious blockers of CLC-Ka mediated currents. A rational drug design allowed us also to ameliorate drug potency, finally identifying a newly synthesized benzofuran derivative (SRA-36) with an inhibitory affinity of 4 μM. Surprisingly, NFA failed to increase CLC-Ka currents, producing only an inhibitory effect in the 1-1000 μM range. In order to gain insight into this relevant difference between the two expression systems, we are currently evaluating the NFA effect on mutants of CLC-Ka that drastically affect NFA potentiation in oocytes. Besides identifying new therapeutic drugs, our results could shed light on structural determinants of CLC-Ks channels activity. (MIUR- COFIN-2009; Telethon GGP08064, GGP10101, GGP12008).