Pharmacological characterization of chloride channels belonging to the ClC family by the use of chiral clofibric acid derivatives

The enantiomers of 2-(p-chlorophenoxy)propionic acid (CPP) and of its analogs with substitutions on the asymmetric carbon atom were tested on human ClC-1 channel, the skeletal muscle chloride channel, after heterologous expression in Xenopus laevis oocytes, to gain insight in the mechanism of action of these stereoselective modulators of macroscopic chloride conductance (gCl) of rat striated fibers. By means of two microelectrode voltage clamp recordings, we found that S(-)-CPP shifted the activation curve of the ClC-1 currents toward more positive potentials and decreased the residual conductance at negative membrane potential; both effects probably account for the decrease of gCl at resting potential of native muscle fibers. Experiments on expressed Torpedo marmorata ClC-0 channels and a mutant lacking the slow gate suggest that S(-)-CPP could act on the fast gate of the single protochannels constituting the double-barreled structure of ClC-0 and ClC-1. The effect of S(-)-CPP on ClC-1 was markedly increased at low external pH (pH = 6), possibly for enhanced diffusion through the membrane (i.e., because the compound was effective only when applied to the cytoplasmic side during patch clamp recordings). The R(+)-isomer had little effect at concentrations as high as 1 mM. The CPP analogs with an ethyl, a phenyl, or an n-propyl group in place of the methyl group on the asymmetric center showed a scale of potency and a stereoselective behavior on ClC-1 similar to that observed for blocking gCl in native muscle fibers. The tested compounds were selective toward the ClC-1 channel. In fact, they were almost ineffective on an N-terminal deletion mutant of ClC-2 that is volume- and pH-independent while they blocked wild-type ClC-2 currents only at high concentrations and independently of pH and drug configuration, suggesting a different mechanism of action compared with ClC-1. No effects were observed on ClC-5 that shows less than 30% homology with ClC-1. Thus, CPP-like compounds may be useful both to gain insight into biophysical properties of ClC-1 and for searching tissue-specific therapeutic agents.