Activation and inhibition of kidney CLC-K chloride channels by fenamates

CLC-K Cl(-) channels are selectively expressed in kidney and ear, where they are pivotal for salt homeostasis, and loss-of-function mutations of CLC-Kb produce Bartter's syndrome type III. The only ligand known for CLC-K channels is a derivative of the 2-p-chlorophenoxypropionic acid (CPP), 3-phenyl-CPP, which blocks CLC-Ka, but not CLC-Kb. Here we show that in addition to this blocking site, CLC-K channels bear an activating binding site that controls channel opening. Using the voltage-clamp technique on channels expressed in Xenopus laevis oocytes, we found that niflumic acid (NFA) increases CLC-Ka and CLC-Kb currents in the 10 to 1000 microM range. Flufenamic acid (FFA) derivatives or high doses of NFA produced instead an inhibitory effect on CLC-Ka, but not on CLC-Kb, and on blocker-insensitive CLC-Ka mutants, indicating that the activating binding site is distinct from the blocker site. Evaluation of the sensitivity of CLC-Ka to derivatives of NFA and FFA together with a modeling study of these ligands allow us to conclude that one major characteristic of activating compounds is the coplanarity of the two rings of the molecules, whereas block requires a noncoplanar configuration. These molecules provide a starting point for identification of diuretics or drugs useful in the treatment of Bartter's syndrome.