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

The ClC-3 2Cl^- /1H⁺ exchanger modulates endosome pH and Cl^- concentration. We investigated the relationships between ClC-3-mediated ion transport (steady-state transport current, I_SS ), gating charge (Q) and cytoplasmic alkalization. ClC-3 transport is functionally unidirectional. ClC-5 and ClC-3 display indistinguishable exchange ratios, but ClC-3 cycling is less "efficient", as reflected by a large Q/I_SS . An M531A mutation predicted to increase water-wire stability and cytoplasmic proton supply improves efficiency. Protonation (pH 5.0) of the outer glutamate gate (Glu_ext ; E224) reduces Q, inhibits transport, and weakens coupling. Removal of the central tyrosine anion gate (Y572S) greatly increases uncoupled anion current. Tyrosine -OH removal (Y572F) alters anion selectivity and impairs coupling. E224 and Y572 act as anion barriers, and contribute to gating. The Y572 side chain and -OH regulate Q movement kinetics and voltage dependence. E224 and Y572 interact to create a "closed" inner gate conformation that maintains coupling during cycling.

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.