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

Abstract

SUMMARY 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 the first specific small-molecule inhibitor directed against a CLC antiporter (ClC-ec1). This compound, 4,4′-octanamidostilbene-2,2′-disulfonate (OADS), inhibits ClC-ec1 with low micromolar affinity and has no specific effect on a CLC channel (ClC-1). Inhibition of ClC-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. The discovery of this compound will empower research to elucidate differences between antiporter and channel mechanisms and to develop treatments for CLC-mediated disorders.

Identification of a proton-chloride antiporter (EriC) by Himar1 transposon mutagenesis in Lactobacillus reuteri and its role in histamine production

“…In the EriC-deficient mutant, regulation of intracellular anions and pH may also be altered and result in an indirect effect upon hdc gene cluster expression. The role of L. reuteri EriC in transmembrane potential maintenance could be further characterized by comparing intracellular pH (using 2,7-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (Molenaar et al 1991)) and membrane potential (with 3,3′-dipropylthiodicarbocyanine (Sip et al 1990)) between those of WT 6475 and the EriC-deficient mutant in the presence or absence of the recently discovered CIC-specific inhibitor, 4,4′-octanamidostilbene-2,2′-disulfonate (OADS) (Howery et al 2012). …”

Integral Membrane Proteins and Bilayer Proteomics

“…High-throughput, top-down proteomics of membrane fractions will require significant developments in separations technologies, possibly involving two dimensional chromatography 68 , or multi-dimensional mixed electrophoresis/chromatography systems 74 . Membrane proteins were detected in the latter analysis that identified over 1000 total proteins in ‘discovery’ mode, though it should be noted that classical features of traditional top-down analyses were compromised, resulting in some identifications without intact mass measurements, and a false discovery rate of 5%.…”

Novel diuretic targets

“…Nevertheless, recent progress demonstrates the potential for development of novel ClC-specific compounds. Compounds specific to the ClC-2 channel and to the ClC-ec1 antiporter have recently been described (59,137). Of relevance to the kidney, Pusch and colleagues (48) have systematically characterized and developed small-molecule inhibitors and activators of the ClC-K channels.…”