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

Abstract

AbstractClC‐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‐7Y713C, a variant displaying disrupted PI(3,5)P2 inhibition. Detailed biophysical analysis of disease‐associated ClC‐6 and ClC‐7 gain‐of‐function (GoF) variants, ClC‐6Y553C and ClC‐7Y713C, and the ClC‐7Y577C and ClC‐6Y781C correlates, identified additional functional nuances distinguishing ClC‐6 and ClC‐7. ClC‐7Y577C recapitulated GoF produced by ClC‐6Y553C. ClC‐6Y781C displayed transport activation qualitatively similar to ClC‐7Y713C, although current density did not differ from that of wild‐type ClC‐6. Finally, rClC‐7R760Q, homologous to hClC‐7R762Q, 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. imageKey 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‐6Y553C and ClC‐7Y715C are maintained when introduced in their respective homologues, ClC‐7Y577C and ClC‐6Y781C, 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.