Regulation of ClC-3 Cl-/H+ Transport and “Gating” Transients by Chloride Pathway Residues and External Protons

We investigated ClC-3 ion transport properties by employing a “ClC-5/3” plasmid, consisting of the ClC-5 N-terminal (M1-A46) linked to the core ClC-3 protein. This ClC-3 protein is redirected to the plasma membrane of HEK cells. Functionally it exhibits rapidly activated, outwardly rectifying sustained ion currents (ISS) representing coupled Cl-/H+ transport, and prominent “on/off” transient gating charge (Q) movements, as recently described for a ClC-3 mutant (Guzman et al, 2013, ACS Chem Neurosci). Interpreting Q to represent movements of unprotonated (ie, incompletely cycling) “Gluext”/ E224, ClC-3 exhibits low transport efficiency compared to ClC-5 or ClC-4, which exhibit larger ISS and much smaller Q. Replacement of external Cl- with SCN- increases ClC-3 currents by 3-4 fold, and reduces H+ coupling by ∼90%. Removal of a conserved tyrosine (Y630S, V) positioned at the intersection of the Cl-/H+ pathways (Accardi et al, 2006, JMB) greatly increases currents and decreases Q. For Y630S, H+ coupling is reduced by ∼50%; SCN substitution decreases rather than increases current, and eliminates H+ transport. An M568A mutant moderately impacts transport, increasing currents and decreasing Q by <50%. Reduction of external pH (pH 6 or 5) weakly inhibits ClC-3 ISS, but markedly reduces Q, and shifts the Q(V) relationship toward more positive voltages. These results are consistent with external protons inhibiting cycling by neutralization of Gluext. An endogenous Cl- current activated at pH5 (Iacid) has slow activation kinetics and lacks gating transients, is inhibited by 60-120 microM phloretin, and is decreased by ClC-3 expression. Thus, Iacid is readily distinguished from expressed ClC-3 transport. These data indicate that ClC-3 H+ coupling and transport is influenced by anion interaction, and by external protons, as suggested for the bacterial ClCec-1.