Mechanism of block of single protopores of the Torpedo chloride channel ClC-0 by 2-(p-chlorophenoxy)butyric acid (CPB)

We investigated in detail the mechanism of inhibition by the S(-) enantiomer of 2-(p-chlorophenoxy)butyric acid (CPB) of the Torpedo Cl- channel, ClC-0. The substance has been previously shown to inhibit the homologous skeletal muscle channel, CLC-1. ClC-0 is a homodimer with probably two independently gated protopores that are conductive only if an additional common gate is open. As a simplification, we used a mutant of ClC-0 (C212S) that has the common gate "locked open" (Lin, Y.W., C.W. Lin, and T.Y. Chen. 1999. J. Gen. Physiol. 114:1-12). CPB inhibits C212S currents only when applied to the cytoplasmic side, and single-channel recordings at voltages (V) between -120 and -80 mV demonstrate that it acts independently on individual protopores by introducing a long-lived nonconductive state with no effect on the conductance and little effect on the lifetime of the open state. Steady-state macroscopic currents at -140 mV are half-inhibited by ∼0.5 mM CPB, but the inhibition decreases with V and vanishes for V ≥ 40 mV. Relaxations of CPB inhibition after voltage steps are seen in the current responses as an additional exponential component that is much slower than the gating of drug-free protopores. For V ≤ -40 mV, where a significant inhibition is observable for the CPB concentrations used in this study (≤10 mM), the concentration dependence of its onset kinetics is consistent with CPB binding according to a bimolecular reaction. At all voltages, only the openings of drug-free protopores appear to contribute significantly to the current observed at any time. Lowering internal Cl- hastens significantly the apparent "on" rate, sug- gesting that internal Cl- antagonizes CPB binding to closed pores. Vice versa, lowering external Cl- reduces the apparent rate of CPB dissociation from open pores. We studied also the point mutant K519E (in the context of the C212S mutant) that has altered conduction properties and slower single protopore gating kinetics. In experiments with CPB, the mutant exhibited drastically slowed recovery from CPB inhibition. In addition, in contrast to WT (i.e., C212S), the mutant K519E showed also a significant CPB inhibition at positive voltages (≥60 mV) with an IC50 of ∼30-40 mM. Altogether, these findings support a model for the mechanism of CPB inhibition in which the drug competes with Cl- for binding to a site of the pore where it blocks permeation. CPB binds preferentially to closed channels, and thereby also strongly alters the gating of the single protopore. Since the affinity of CPB for open WT pores is extremely low, we cannot decide in this case if it acts also as an open pore blocker. However, the experiments with the mutant K519E strongly support this interpretation. CPB block may become a useful tool to study the pore of ClC channels. As a first application, our results provide additional evidence for a double-barreled structure of ClC-0 and ClC-1.