Distinct ClC-6 and ClC-7 Cl - sensitivities provide insight into ClC-7's role in lysosomal Cl - homeostasis.

ClC-6 and ClC-7 are closely related, intracellular Cl - /H + antiporters belonging to the CLC family of channels and transporters. They localize to acidic late endosomes and lysosomes and probably function in ionic homeostasis of these contiguous compartments. ClC-7 transport function requires association with the accessory protein Ostm1, whereas ClC-6 transport does not. To elucidate their roles in endo-lysosomes, we measured Cl - - and pH-dependences of over-expressed wild-type ClC-6 and ClC-7, as well as disease-associated mutants, using high-resolution recording protocols. Lowering extracellular Cl - (corresponding to luminal Cl - in endo-lysosomes) reduced ClC-6 currents, whereas it increased transport activity of ClC-7/Ostm1. Low extracellular Cl - activated ClC-7/Ostm 1 under acidic extracellular conditions, as well as under conditions of low intracellular chloride. Activation is conserved in ClC-7 Y713C , a variant displaying disrupted PI(3,5)P 2 inhibition. Detailed biophysical analysis of disease-associated ClC-6 and ClC-7 gain-of-function (GoF) variants, ClC-6 Y553C and ClC-7 Y713C , and the ClC-7 Y577C and ClC-6 Y781C correlates, identified additional functional nuances distinguishing ClC-6 and ClC-7. ClC-7 Y577C recapitulated GoF produced by ClC-6 Y553C . ClC-6 Y781C displayed transport activation qualitatively similar to ClC-7 Y713C , although current density did not differ from that of wild-type ClC-6. Finally, rClC-7 R760Q , homologous to hClC-7 R762Q , an osteopetrosis variant with fast gating kinetics, appeared indifferent to extracellular Cl - , identifying altered Cl - sensitivity as a plausible mechanism underlying disease. Collectively, the present studies underscore the distinct roles of ClC-6 and ClC-7 within the context of their respective localization to late endosomes and lysosomes. In particular, we suggest the atypical inhibition of ClC-7 by luminal Cl - serves to limit excessive intraluminal Cl - accumulation. KEY POINTS: ClC-6 and ClC-7 are late endosomal and lysosomal 2 Cl - /1 H + exchangers, respectively. When targeted to the plasma membrane, both activate slowly at positive voltages. ClC-6 activity is decreased in low extracellular (i.e. luminal) chloride, whereas ClC-7 is activated by low luminal chloride, even at acidic pH. The functional gain-of-function phenotypes of the ClC-6 and ClC-7 disease mutations ClC-6 Y553C and ClC-7 Y715C are maintained when introduced in their respective homologues, ClC-7 Y577C and ClC-6 Y781C , with all mutations retaining chloride dependence of the respective wild type (WT). An osteopetrosis mutation of ClC-7 displaying fast gating kinetics (R762Q) was less sensitive to extracellular chloride compared to WT. The opposing substrate dependences of ClC-6 and ClC-7 Cl - / H + exchangers point to non-overlapping physiological functions, leading us to propose that inhibition of ClC-7 by luminal chloride and protons serves to prevent osmotic stress imposed by hyper-accumulation of chloride.