A Designed Inhibitor of a CLC Antiporter Blocks Function through a Unique Binding Mode

The lack of small-molecule inhibitors for anion-selective transporters and channels has impeded our understanding of the complex mechanisms that underlie ion passage. The ubiquitous CLC Chloride Channel family represents a unique target for biophysical and biochemical studies because its distinctive protein fold supports both passive chloride channels and secondary-active chloride-proton transporters. Here, we describe the synthesis and characterization of a specific small-molecule inhibitor directed against a CLC antiporter (CIC-ec1). This compound, 4,4'-octanamidostilbene-2,2'-disulfonate (OADS), inhibits CIC-ec1 with low micromolar affinity and has no specific effect on a CLC channel (CIC-1). Inhibition of CIC-ec1 occurs by binding to two distinct intracellular sites. The location of these sites and the lipid dependence of inhibition suggest potential mechanisms of action. This compound will empower research to elucidate differences between antiporter and channel mechanisms and to develop treatments for CLC-mediated disorders.