Modulation of ClC-3 gating and proton/anion exchange by internal and external protons and the anion selectivity filter

We utilized plasma membrane-localized ClC-3 to investigate relationships between steady-state transport current (I-SS), gating charge (Q) movement, and cytoplasmic alkalization rate. ClC-3 exhibited lower transport efficiency than ClC-5, as reflected by a larger Q/I-SS ratio, but an indistinguishable Cl-/H+ coupling ratio. External SCN- reduced H+ transport rate and uncoupled anion/H+ exchange by 80-90%. Removal of the external gating glutamate (Glu(ext)) (E224A mutation) reduced Q and abolished H+ transport. We hypothesized that Methionine 531 (M531) impedes water wire H+ transfer from the cytoplasm to E224. Accordingly, an M531A mutation decreased the Q/I-SS ratio by 50% and enhanced H+ transport. External protons (pH 5.0) inhibited I-SS and markedly reduced Q while shifting the Q-voltage (V) relationship positively. The Cl-/H+ coupling ratio at pH 5.0 was significantly increased, consistent with externally protonated Glu(ext) adopting an outward/open position. Internal anion gate removal (Y572S) dramatically increased I-SS and impaired coupling, without slowing H+ transport rate. Loss of both gates (Y572S/E224A) resulted in a large open pore conductance. Y572F (removing only the phenolic hydroxide) and Y572S shortened Q duration similarly, resulting in faster Q kinetics at all voltages. These data reveal a complex relationship between Q and ion transport. Q/I-SS must be assessed together with coupling ratio to properly interpret efficiency. Coupling and transport rate are influenced by the anion, internal proton supply and external protons. Y572 regulates H+ coupling as well as anion selectivity, and interacts directly with E224. Disruption of this closed gate conformation by internal protons may represent a critical step in the ClC-3 transport cycle.