Mechanism of ClC-7 transporter inhibition by the lysosomal phospholipid PI(3,5)P2.

Abstract

Lysosomes process cellular waste and play a central role in nutrient sensing and metabolism regulation. To function properly, lysosomes must maintain a highly acidic lumen at around pH 4.5; this is achieved by an ATP-driven proton pump and a suite of ion channels and transporters that provide counterion pathways to dissipate the charge buildup. One such transporter is ClC-7, a slowly activating, voltage dependent chloride/proton antiporter that has been implicated in lysosome acidification. We recently reported that the lysosome-specific phosphoinositide PI(3,5)P2 modulates lysosomal pH by inhibiting transport through ClC-7. However, the mechanism of this inhibition is unclear. Here we investigate the mechanism of ClC-7 inhibition by PI(3,5)P2. A recent ClC-7 structure reveals that a bound phosphoinositide contributes to a network of interactions that includes contributions from residues on both the transmembrane and cytosolic domains of ClC-7. We used electrophysiology experiments to examine the effects of mutations that disrupt these interactions. These mutations result in gain of function and loss of PI(3,5)P2 inhibition of ClC-7. These results suggest a model in which PI(3,5)P2-mediated hydrogen bonds cause tight association between the ClC-7 cytosolic and transmembrane domains, which in turn result in inhibition of transport. In this model, the inactive, cytosolic domain-bound state is promoted by PI(3,5)P2, and the active, cytosolic domain-unbound state is promoted by gain-of-function mutations that disrupt the interaction network.