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

Abstract

Key points: 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, ISS), 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/ISS. An M531A mutation predicted to increase water-wire stability and cytoplasmic proton supply improves efficiency. Protonation (pH 5.0) of the outer glutamate gate (Gluext; 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. Abstract: We utilized plasma membrane-localized ClC-3 to investigate relationships between steady-state transport current (ISS), gating charge (Q) movement, and cytoplasmic alkalization rate. ClC-3 exhibited lower transport efficiency than ClC-5, as reflected by a larger Q/ISS 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 (“Gluext”) (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/ISS ratio by 50% and enhanced H+ transport. External protons (pH 5.0) inhibited ISS 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 Gluext adopting an outward/open position. Internal “anion gate” removal (Y572S) dramatically increased ISS 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/ISS 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.