Fluoride-dependent interruption of the transport cycle of a CLC Cl−/H+ antiporter

Cl(-)/H(+) antiporters of the CLC superfamily transport anions across biological membranes in varied physiological contexts. These proteins are weakly selective among anions commonly studied, including Cl(-), Br(-), I(-), NO3(-) and SCN(-), but they seem to be very selective against F(-). The recent discovery of a new CLC clade of F(-)/H(+) antiporters, which are highly selective for F(-) over Cl(-), led us to investigate the mechanism of Cl(-)-over-F(-) selectivity by a CLC Cl(-)/H(+) antiporter, CLC-ec1. By subjecting purified CLC-ec1 to anion transport measurements, electrophysiological recording, equilibrium ligand-binding studies and X-ray crystallography, we show that F(-) binds in the Cl(-) transport pathway with affinity similar to Cl(-) but stalls the transport cycle. Examination of various mutant antiporters implies a 'lock-down' mechanism of F(-) inhibition, in which F(-), by virtue of its unique hydrogen-bonding chemistry, greatly retards a proton-linked conformational change essential for the transport cycle of CLC-ec1.